JP2019537619A - Method for treating cancer with bavituximab based on β2-glycoprotein 1 levels, and assays therefor - Google Patents

Abstract

Disclosed are surprising new methods and kits for identifying and treating cancer patients treatable with PS-targeted antibodies, particularly for identifying and treating cancer patients using bavituximab and combination therapy with bavituximab. The methods and kits indicate that pre-treatment blood levels of β2-glycoprotein 1 (β2GPI), particularly functional β2GPI, in a defined range serve as an accurate predictor of patients with better therapeutic outcome. Based on surprising findings. [Selection diagram] FIG.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a co-pending provisional application No. 62 / 507,580 filed May 17, 2017, and provisional application No. 62/480, filed April 3, 2017. No. 994, provisional application No. 62 / 406,727 filed on October 11, 2016 (October 10, 2016 is a federal holiday in the United States) and provisional application filed on Sep. 27, 2016 No. 62 / 400,589, the entire specification, claims, drawings and sequence of these applications is incorporated herein by reference without waiver.

  The present invention relates to the field of biomarkers, and in particular, pre-treatment levels β2-glycoprotein 1 (β2GPI), particularly as an indicator for predicting a successful response to a therapy using a PS-targeting antibody such as bavituximab. It relates to the use of functional β2GPI.

  In fighting all diseases, including cancer and viral infections, a functional immune system is an important part of a successful treatment. Therefore, significant research has been done into immunotherapy, including the field of immunooncology (IO), which is currently recognized as a strategy for treating cancer. Recently, new targets and compounds that manipulate the immune response have been studied by researchers and clinicians. For example, while IO agents targeting programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have already been approved as therapeutics for some advanced malignancies, Compounds that interact with other IO targets are currently under development.

  Nevertheless, even these new immunotherapies are only effective for certain patients. Thus, given the variability in response to both old and new immunotherapies, and the desire to maximize clinical benefit, biomarkers that can predict therapeutic outcomes, including IO therapy, , Is still needed. Some researchers have focused on using therapeutic targets to develop personalized predictive approaches, while others have found that by using measurements of binding and downstream signaling molecules We are seeking alternatives for patient selection and identification.

  Recently, a membrane phospholipid, phosphatidylserine (PS), has been identified as a unique and highly immunosuppressive molecule that acts as an upstream immune checkpoint that regulates the host immune response. This means that PS plays an important role in a variety of diseases, including cancer and viral infections, and opens up a new field of immunotherapy in the form of PS-targeted antibodies that block PS.

  The primary PS targeting antibody is bavituximab, a mouse-human chimeric monoclonal antibody (mAb) derived from a mouse mAb called 3G4 (Ran et al., 2005; Huang et al., 2005; U.S. Patent No. 7,247,303). issue). 3G4 and Bavituximab are part of a family of mouse, chimeric, and fully human antibodies that target PS in a β2-glycoprotein 1 (β2GPI) -dependent manner. That is, bavituximab and related PS targeting antibodies bind to PS in the presence of β2GPI such that they form a high affinity antibody-β2GPI-PS complex (Luster et al., 2006). Numerous imaging studies (Jennewein et al., 2008; Marconescu & Thorpe, 2008; Saha et al., 2010; Stafford & Thorpe, 2011; Zhao, including the measurement and prediction of response to therapy (Gong et al., 2013; Stafford et al., 2013). Operationally, these β2GPI-dependent PS targeting antibodies are specific for PS in vivo, as specifically indicated by Zhang et al., 2014; and Zhou et al., 2014; US Patent No. 7,790,860). It is.

  Bavituximab is not only active against a wide range of diseases in which PS is a marker, particularly cancer and viral infections, but also inhibits intracellular parasites such as the protozoan parasite Leishmania amazonensis (Wanderley et al., 2013), and plague and tularemia, respectively. The activity against infectious diseases of intracellular bacterial pathogens (Lonsdale et al., 2011) such as causing Yersinia pestis and Francisella tularensis has also been demonstrated. For viral infections, PS-targeting antibodies such as bavituximab have been shown to inhibit viral replication, reduce viral load in organs, and increase survival (Soares et al., 2008; Moody et al., 2010). U.S. Patent No. 7,906,115). The anticancer activity of bavituximab and related PS-targeting antibodies has been demonstrated in a large number of preclinical studies and clinical trials, in which the effect blocks tumor vascular and blocks PS immunosuppressive signaling (Ran et al., 2005; U.S. Patent No. 7,572,448; DeRose et al., 2011).

  The anti-tumor effects of PS-targeting antibodies, such as bavituximab, combine these antibodies with agents or conditions that increase the exposure of PS in the tumor microenvironment, for example, through the use of radiation and / or co-administration of chemotherapy. In some cases (US Pat. No. 7,422,738; US Pat. No. 8,486,391; US Pat. No. 7,572,448). For example, when treating the breast tumor with the use of the bavituximab family of PS targeting antibodies in combination with docetaxel (Huang et al., 2005), or with the treatment of pancreatic tumor with gemcitabine (Beck et al., 2006), radiation To treat lung cancer (He et al., 2007) and brain cancer, glioblastoma (He et al., 2009) in combination with prostate cancer in combination with docetaxel to re-activate antitumor immunity (Yin et al., 2013), and when treating hepatocellular carcinoma in combination with sorafenib (Cheng et al., 2016), improved antitumor efficacy has been demonstrated preclinically. Shown preclinically for the treatment of melanoma (Freimark et al., 2016) and triple negative breast cancer (Gray et al., 2016a) in combination with a checkpoint inhibitor in the form of an antibody to CTLA-4 or PD-1. As such, the use of PS-targeting antibodies such as Bavituximab in combination therapy with other IO agents also results in enhanced anti-tumor effects.

  Bavituximab has also been evaluated in clinical studies in more than 800 patients, most of which were treated with combination therapy. These clinical trials include patients with viral infections such as chronic hepatitis C virus (HCV) and human immunodeficiency virus (HIV), as well as lung, breast, liver (hepatocellular carcinoma, HCC), pancreas, colorectal , And patients with several tumor types, including kidney (renal cell carcinoma, RCC). Babituximab in combination with paclitaxel in patients with HER2-negative metastatic breast cancer (Chalasani et al., 2015), in combination with paclitaxel-carboplatin in non-small cell lung cancer (NSCLC) (Digumarti et al., 2014), and in combination with sorafenib in hepatocellular carcinoma ( Cheng et al., 2016) and clinical trials combined with docetaxel in previously treated advanced non-squamous NSCLC (Gerber et al., 2016) report promising antitumor effects.

  Overall, the results of Phase I and Phase II clinical studies demonstrated a clinically significant therapeutic effect of Bavituximab. Nevertheless, there is still a need for effective methods for optimizing treatment with PS-targeted antibodies such as bavituximab, as no biomarkers related to bavituximab therapy exist yet. Attempts to address the lack of relevant biomarker data have been hampered by the unique properties of the bavituximab antibody and its physiological / pathological target, PS. Therefore, there is a need for improved patient screening methods that allow for optimization of treatment. Identifying one or more circulating biomarkers for bavituximab treatment is a particularly important advance, which will provide a minimally invasive test to select patients and improve treatment outcomes.

  The present invention provides new biomarker methods, compositions, kits, and assays for optimizing treatment with PS-targeting antibodies, such as bavituximab, and similar antibodies, eg, 1N11, thereby reducing the prior art. Address the foregoing and other needs. The invention particularly relates to the use of pre-treatment levels of β2-glycoprotein 1 (β2GPI), most preferably functional β2GPI, as an indicator or “biomarker” for predicting response to a therapy using bavituximab.

  These surprising new methods, compositions, kits, and assays are useful for identifying and treating patients treatable with a β2GPI-dependent PS-targeting antibody, particularly cancer patients for treatment with a treatment regimen containing bavituximab or combination therapy. Most preferably, it provides for the identification and treatment of patients with cancer using bavituximab (a first anticancer agent) and at least a second or third anticancer agent, all of which have a defined range of functional β2GPI treatments This is done by selecting patients with pre-blood levels. A “functional” β2GPI is a β2GPI that binds to both PS and a β2GPI-dependent PS targeting antibody, preferably bavituximab.

  In a most preferred embodiment, the present invention relates to selecting, identifying, diagnosing, and preferably treating a patient based on pre-treatment levels of functional β2GPI of 200 μg / ml or greater.

  The present invention provides a method for treating a disease in which PS is a marker (particularly cancer) in a human patient, the method comprising administering to the patient a PS targeting antibody, preferably bavituximab. Has a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or more. Preferably, a PS targeting antibody, such as bavituximab, referred to herein as a "first therapeutic agent" and a "first anticancer agent" is administered to a patient along with at least a second therapeutic or anticancer agent.

The present invention also provides a method for treating a disease in which PS is a marker (particularly cancer) in a human patient,
(A) measuring the concentration of functional β2GPI in a pre-treatment blood sample obtained from the patient;
(B) administering a PS targeting antibody, preferably bavituximab, to a patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or greater. Preferably, a PS targeting antibody such as bavituximab is administered to the patient along with at least a second therapeutic or anti-cancer agent.

The present invention provides a method for treating a disease in which PS is a marker (particularly cancer) in a human patient,
(A) obtaining a pre-treatment blood sample from the patient;
(B) measuring the concentration of functional β2GPI in the pre-treatment blood sample;
(C) administering a PS targeting antibody, preferably bavituximab, to a patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or greater. Preferably, a PS targeting antibody such as bavituximab is administered to the patient along with at least a second therapeutic or anti-cancer agent.

A method for identifying a human patient, preferably a human cancer patient, that can be treated with a PS targeting antibody, preferably with bavituximab,
(A) measuring the concentration of functional β2GPI in a pre-treatment blood sample obtained from the patient;
(B) if the concentration of functional β2GPI in the pre-treatment blood sample is greater than or equal to 200 μg / ml, identifying the patient as being treatable with a PS-targeting antibody, preferably bavituximab;
(C) administering a PS targeting antibody, preferably bavituximab, to a patient having a pre-treatment blood level of functional β2GPI of 200 μg / ml or greater. Preferably, (b) the patient is identified as being treatable with bavituximab and at least a second therapeutic or anti-cancer agent, and (c) bavituximab and at least a second therapeutic or anti-cancer agent are administered to the patient.

A method for identifying and treating a human patient, preferably a human cancer patient, treatable with a PS targeting antibody, preferably bavituximab, comprising:
(A) obtaining a pre-treatment blood sample from the patient;
(B) measuring the concentration of functional β2GPI in the pre-treatment blood sample;
(C) if the concentration of functional β2GPI in the pre-treatment blood sample is greater than or equal to 200 μg / ml, identifying the patient as being treatable with a PS-targeting antibody, preferably with bavituximab;
(D) administering a PS-targeting antibody, preferably bavituximab, to a patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or greater. Preferably, (c) the patient is identified as being treatable with bavituximab and at least a second therapeutic or anti-cancer agent, and (d) bavituximab and at least a second therapeutic or anti-cancer agent are administered to the patient.

  Another embodiment of the present invention is a method of diagnosing a patient, preferably a cancer patient, treatable with a PS targeting antibody, preferably bavituximab, wherein the method comprises determining the concentration of functional β2GPI in a blood sample of the patient. If the blood concentration of functional β2GPI is greater than or equal to 200 μg / ml, including measuring, the patient is determined to be treatable with a PS-targeting antibody, preferably bavituximab. Preferably, if the blood level of functional β2GPI is greater than or equal to 200 μg / ml, the patient is diagnosed as being treatable with bavituximab and at least a second therapeutic or anti-cancer agent.

  A further embodiment of the present invention is a PS targeting antibody, preferably bavituximab, for use in a method of treating a patient, preferably a method of treating cancer in a patient, wherein the functional β2GPI is a blood sample of the patient. Present at a concentration of 200 μg / ml or more. Preferably, this relates to bavituximab for use in a method of treating such cancer, further comprising administering at least a second anti-cancer agent.

Yet another embodiment of the present invention is a PS targeting antibody, preferably bavituximab, for use in a method of treating a disease (preferably cancer) where PS is a marker, the method comprising:
(A) determining the concentration of functional β2GPI in a blood sample of a patient, preferably a cancer patient;
(B) when the blood concentration of the functional β2GPI is 200 μg / ml or more, administering a PS-targeting antibody, preferably bavituximab, to the patient. Preferably, there is provided bavituximab and at least a second therapeutic or anti-cancer agent for use in a method of treating cancer.

  In addition to the most preferred levels of β2GPI above 200 μg / ml, in certain preferred embodiments, the invention relates to selection of patients based on pre-treatment functional β2GPI in the range of 200-290 μg / ml. All figures and ranges within these levels are based on pre-treatment levels of functional β2GPI of 200 or 210 μg / ml or higher for each of the aforementioned methods and uses such as patient selection, identification, diagnosis, and preferably treatment. Included, the β2GPI may range from a low number of either 200 or 210 μg / ml to a high number of any one of 270, 280, 290, 300, 310, or 320 μg / ml. (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 μg / ml, etc.), 210-270, 210-280, 210-290, 200- 80 and 200 to 290 is presently preferred.

  In administering a PS-targeting antibody, preferably bavituximab, to a patient, preferably a human cancer patient, the antibody will be administered at a dose of about 1-10, 1-6, 3-6, or 1-3 mg / kg, most preferably It is administered at a dose of about 3 mg / kg.

  In each of the foregoing methods and uses, the present invention employs 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. Ovarian, gastric, liver, colorectal, breast, esophageal, brain (eg, glioma, glioblastoma), prostate, skin (melanoma), head and neck, kidney cancer It can be applied to the treatment of solid tumors such as bladder cancer, pancreatic cancer, or lung cancer, preferably non-small cell lung cancer (NSCLC) (including non-squamous NSCLC). The mechanism of binding of bavituximab in the complex of functional β2GPI and PS, and the overall mechanism of immune activation of bavituximab, is common to all bavituximab therapies. Thus, the present invention is directed to any therapeutic method that uses a PS-targeting antibody such as bavituximab, or a similar antibody (eg, 1N11), especially in combination therapy with chemotherapeutic agents, preferably immune tumor (IO) agents and the like. Applied to the selection of patients for.

  Suitable IO agents are activating immune checkpoints, such as CD28, OX40, and / or GITR, immune checkpoint antibodies, including agonist (activating) antibodies that bind to a receptor or molecule, and preferably PD-1 Antagonistic (blocking) antibodies that bind to an inhibitory immune checkpoint, receptor, or molecule such as 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 referred to herein as "immune checkpoint inhibitors" or "ICI." Preferred examples of immune checkpoint antibodies (or immune checkpoint inhibitors) are blocking antibodies to CTLA-4, PD-1, or PD-L1, such as ipilimumab, tremelimumab, nivolumab, pembrolizumab, durvalumab, and atezolizumab.

  The present invention specifically contemplates the selection, diagnosis, and treatment of patients with a PS targeting antibody, preferably bavituximab, and a second and third therapeutic or anti-cancer agent. For example, with a chemotherapeutic agent and an immune checkpoint antibody, or two immune checkpoint antibodies, including treatment with a PS targeting antibody, preferably bavituximab, treatment with the chemotherapeutic agent is followed by treatment with the immune checkpoint antibody.

In connection with this β2GPI biomarker technology and as having other research and clinical uses, the present invention also provides novel assay methods, compositions, and kits particularly adapted for the detection and quantification of functional β2GPI. provide. Accordingly, in both the foregoing methods and uses, and as a new assay, the present invention is a method of measuring functional β2GPI,
(A) optionally, coating a solid support such as an ELISA plate with PS to prepare a PS coated solid support or a PS coated ELISA plate (or using a pre-prepared PS coated solid support or an ELISA plate; May be)
(B) adding a PS-targeting antibody, preferably bavituximab, and a biological sample suspected of containing β2GPI to the PS-coated solid support, thereby converting the β2GPI in the sample to a PS-targeting antibody, preferably bavituximab. Co-incubating the antibody and sample under conditions effective to bind both to the solid support and the PS coated solid support;
(C) detecting binding of the PS targeting antibody, preferably bavituximab, and β2GPI to the PS-coated solid support, thereby measuring functional β2GPI in the sample.

  The biological sample suspected of containing β2GPI can be a blood sample, such as a plasma or serum sample. Other biological fluid samples suspected of containing β2GPI may be used, including cell supernatants and the like.

  Many binding format assays disclosed herein and known to those of skill in the art can be used. In a preferred embodiment, the PS targeting antibody, preferably bavituximab, binds to a detectable substance that itself produces a detectable signal, such that binding of the antibody and β2GPI to the PS-coated solid support is detectable. It is detected and measured by detecting and measuring such a signal. In another preferred embodiment, the PS targeting antibody, preferably bavituximab, is added to the PS coated solid support prior to the addition of a sample suspected of containing β2GPI (such as a blood sample).

  The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood with reference to one or more of these drawings, in conjunction with the detailed description of the specific embodiments presented herein. The U.S. patent or application file may contain drawings made in at least one color. Copies of this U.S. patent or patent application publication with color drawing (s) will be provided by the Patent Office upon request and payment of the necessary fee.

Inhibition of tumor growth in mice treated with the 3G4 antibody purified to homogeneity in appearance. FIG. 1A, established (0.6-0.7 cm in diameter, 140 mm ³ volume) human MDA-MB-435 breast cancer, growing in the mammary fat pad of SCID mice; FIG. 1B, in the mammary fat pad of SCID mice Growing, established (0.5-0.7 cm in diameter, 110 mm ³ in volume) human MDAMB-231 breast cancer; FIG. 1C, small meta-A fibrosarcoma growing subcutaneously in syngeneic BALB / c mice; and FIG. 1D , grown subcutaneously in SCID mice, large (diameter 0.8～1Cm, volume 370 mm ³⁾ human L540 Hodgkin tumors. Starting from the day indicated by the arrow, groups of 8-10 mice were injected intraperitoneally with 100 μg of 3G4 antibody (（, open circle) or control BBG3 antibody (●, solid circle). Thereafter, treatment was continued three times a week. 3G4 antibodies binding to PS coated microtiter plates are serum dependent. 2A, 3G4 antibodies were purified from cells grown in bovine serum-containing medium (培 地, SCM) or serum-free medium (■, SFM). Microtiter plates were coated with PS and blocked in 1% ovalbumin (OVA) from chicken egg white. Serial dilutions of 3G4 were performed in 10% fetal bovine serum (solid line, FBS) or 1% ovalbumin from chicken egg white (dashed line, OVA). FIG. 2B, microtiter plates were coated with PS and blocked in 1% OVA. Serial dilutions of 3G4 in SFM were performed in 10% serum from mouse (◆), rat (■), human (●), and bovine (▲) species as indicated. The 3G4 antibody binds to the plasma protein β2GPI. Microtiter plates were coated with human β2GPI purified from human plasma (hβ2GPI) and blocked in 1% OVA. Serial dilutions of commercially available mouse anti-human β2GPI (◆, α-β2GPI), SFM-derived 3G4 antibody (■, 3G4), and control mouse IgG (▲, control mIgG) were performed in 1% OVA. The 3G4 antibody binds to β2GPI at domain II. The wells of the microtiter plate were filled with recombinant full-length hβ2GPI (◆, domain IV) or without domain I (-X-, domain II-V), without domain I and II (■, domain III- V), without domain I, II, and III (▲, domain IV-V), and without domain I, II, III, and IV (●, domain V) coated with hβ2GPI peptide. Plates were blocked in 1% OVA and serial dilutions of 3G4 antibody from SFM were performed in 1% OVA. Both the ch3G4 antibody and β2GPI bind to cells with exposed PS. Adult bovine aortic endothelial (ABAE) cells were added to DMEM + 200 μM lysophosphatidylcholine (LPC) in 10% normal mouse serum (MS) plus (i) ch3G4 only, (ii) ch3G4 + hβ2GPI concomitant, or (iii) purified hβ2GPI For 30 minutes. The cells were then washed and incubated with (i) buffer only, (ii) buffer only, or (iii) ch3G4 for 30 minutes each. Finally, cells were washed, fixed, and stained with a fluorescent marker to detect ch3G4 binding. ch3G4 and hβ2GPI were used at a concentration of 2 μg / ml. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are for ch3G4 binding under condition (i) and were set to 1. The lipid binding domain of β2GPI mediates the binding of the ch3G4 antibody to cells with exposed PS. FIG. 6A, ABAE cells were incubated with (i) non-lipid bound form of β2GPI (nicked hβ2GPI) or (ii) intact hβ2GPI (“hβ2GPI”) in addition to the ch3G4 antibody. Incubations were performed for 30 minutes in the presence or absence of 200 μM LPC in DMEM + 10% MS. Thereafter, the cells were washed, fixed, and stained with a fluorescent marker to detect ch3G4 binding. The ch3G4 antibody, intact hβ2GPI, and nicked hβ2GPI were used at a concentration of 2 μg / ml. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are for condition (i) binding of ch3G4 without LPC, which was set to 1. FIG. 6B, wells of a microtiter plate were coated with intact hβ2GPI (triangles, hβ2GPI) or nicked hβ2GPI (squares, nicked) and blocked in 1% OVA. Serial dilutions of the ch3G4 antibody (black) or control mIgG (white) were performed in 1% OVA. Bivalentity is required for β2GPI-mediated 3G4 antibodies to bind to cells with exposed PS. FIG. 7A, ABAE cells were incubated with 20 nM 3G4, 3G4F (ab ′) ₂ , or 3G4 Fab ′ monomer for 30 minutes in the presence of 200 μM LPC in DMEM + 10% FBS. The cells were then washed, fixed, and stained with a fluorescent marker to detect binding of the 3G4 antibody or antibody fragment (3G4F (ab ') ₂ and 3G4Fab' are shown in FIG. 7A). The pixel area of antibody binding was quantified using MetaVue software. Values are for 3G4 binding in the absence of LPC and were set to 1. FIG. 7B, ABAE cells were incubated with 200 μM LPC, 40 nM purified hβ2GPI, 20 nM ch3G4, and a titer of 3G4 Fab ′ monomer in DMEM + 10% MS for 30 minutes. Thereafter, the cells were washed, fixed, and stained with a fluorescent marker to detect ch3G4 binding. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are for binding of ch3G4 without competing 3G4 Fab 'and were set to 100. The 3G4 and Bavituximab antibody families bind to PS in a β2GPI dependent manner. 3G4, Bavituximab, and related therapeutic PS targeting antibodies bind to β2GPI (at domain II) and in turn bind to PS via domain V. Usually, β2GPI is a monomer, binds weakly to PS, and dissociates rapidly. In the presence of a surface with exposed PS in the presence of a PS-targeting antibody such as 3G4 or Bavituximab, two molecules of β2GPI can bind to PS and be cross-linked by a bivalent antibody, resulting in a stable complex. A body is formed. The antibody-β2GPI complex dissociates from cells with exposed PS more than 1,000-fold more slowly (in the absence of PS-targeting antibody) than monomeric β2GPI. Low levels of β2GPI support the preclinical antitumor activity of the 3G4 antibody in vivo. FIG. 9A, assuming a 90% purity of the 3G4 antibody when produced from hybrid cells, mice treated with the 3G4 antibody produced 2 μg / ml bovine β2GPI at a molar ratio of β2GPI to antibody of 0.12. It contains. FIG. 9B, starting with 80% purity, mice treated with the 3G4 antibody contain 4 μg / ml bovine β2GPI at a molar ratio of β2GPI to antibody of 0.25. Low levels of β2GPI support binding of bavituximab to cells with exposed PS in vivo. ABAE cells were incubated with 200 μM LPC to induce PS exposure, 40 nM purified hβ2GPI, and increasing concentrations of DMEM + 10% mouse serum in ch3G4 antibody (babituximab) for 30 minutes. Thereafter, the cells were washed, fixed, and stained with a fluorescent marker to detect ch3G4 binding. The pixel area of ch3G4 binding was quantified using MetaVue software. Values are for 320 pM ch3G4 binding and were set to 1. Low levels of β2GPI support 2aG4 antibodies that bind to PS on the plate in vitro. Microtiter plates were coated with PS. Solutions containing an aliquot of the 2aG4 antibody were tested for binding to PS in the presence of increasing amounts of human β2GPI (in ovalbumin). The bound antibody was detected using TMB as substrate and HRP-conjugated anti-human IgG as secondary antibody, and the absorbance was read at a wavelength of 450 nm. Bavituximab binds to PS in vitro in various human sera. Microtiter plates were coated with PS. Six different individual human serum samples were obtained and nominally numbered 3 (◆), 4 (■), 9 (▲), 11 (-x-), 13 (-x-), and 19 Called (●) and diluted in PBS to obtain various% human serum up to 0.1%. 2 μg / ml Bavituximab-HRP was added to each% human serum solution. Those mixtures of Bavituximab-HRP in human serum were added to PS plates and allowed to bind. The plate was washed, TMB substrate was added, and the absorbance was read at a wavelength of 450 nm. Mean serum bavituximab concentration after administration to patients with refractory advanced cancer. Bavituximab was administered intravenously at 0.1 mg / kg (３５, open circle) or 0.3 mg / kg (□, open square) on days 0, 28, 35, and 42, and bavituximab was administered at 0, 7, 14, , And 28 days, 1 mg / kg (（, closed circle) or 3 mg / kg (■, closed square). On indicated days after dosing, the mean serum bavituximab concentration was determined. The lower limit of quantification was 0.1 μg / ml. Β2GPI levels in patients after administration of bavituximab. Patients infected with HCV were administered repeated doses of bavituximab at the doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg (day indicated by bold black arrow ↓). For each dose (1 mg / kg, ▲, upper (broken) line; 3 mg / kg, Δ, median line; 6 mg / kg, Δ, underline), serum β2GPI levels were determined on indicated days after treatment, and Compared to β2GPI levels. The 1N11 (PGN635) antibody binds to PS in a serum-dependent manner. The binding of 1N11 in scFv form to the plated PS and a mixture of phosphatidylcholine (PC) and sphingomyelin (SM) (PC / SM) was tested by ELISA. Polystyrene plates were coated with 10 μg / ml PS or an equal volume of a mixture of PC / SM, each dissolved in hexane. After the hexane was evaporated, 10% human serum (+ 10% serum) or 1% ovalbumin (+ 1% OV) in PBS was added and incubated for 1 hour. 20 μg / ml purified 1N11 scFv was added to the first of six wells for each antigen in either 10% human serum (+ 10% serum) or 1% ovalbumin (+ 1% OV) And titrated by 3-fold dilution. The remaining bound scFv was detected by HRP-conjugated anti-c-myc tag mouse monoclonal antibody (Invitrogen). Standard curve for functional β2GPI. Samples of known amounts of functional β2GPI were tested and plotted in the functional β2GPI assay described in Example XVI to obtain a standard curve. From the standard curve, the amount of functional β2GPI in a test sample, especially a diluted plasma or serum test sample, can be determined. Distribution of functional β2GPI levels (μg / ml) prior to treatment 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 β2GPI in all 592 evaluable patients; FIG. 17B, box plot of β2GPI from the same patient; FIG. 17C, of patients treated with bavituximab and docetaxel (294 evaluable patients) Distribution of functional β2GPI; and FIG. 17D, distribution of functional β2GPI in patients treated with placebo and docetaxel (298 evaluable patients). Kaplan-Meier survival curves for Phase III studies showing that NSCLC patients with functional β2GPI levels of 200 μg / ml and higher tend to have longer survival (mOS) when treated with bavituximab. FIG. 18A, in patients treated with bavituximab, those with a functional β2GPI of 200 μg / ml or more (“bavituximab: B2GP1 ≧ 200”, solid blue overline) were patients with β2GPI of less than 200 μg / ml (“bavituximab : B2GP1 <200 ", dashed line and blue underline), as opposed to prolonged survival. FIG. 18B, in patients with functional β2GPI greater than or equal to 200 μg / ml, patients treated with bavituximab and docetaxel (“bavituximab”, blue overline) treated with placebo and docetaxel (“placebo”, green underline) the same In contrast to patients with β2GPI levels (200 μg / ml or higher), there is a tendency to prolong survival. Comparison of β2GPI levels supporting the functional and anti-tumor activity of 3G4 antibody binding to PS in preclinical studies with β2GPI levels in patients in Phase III and other clinical trials. The distribution of pre-treatment functional β2GPI levels in the 592 evaluable patients in the Phase III study is the same as specified in FIG. 17A. Functional β2GPI levels of 200 μg / ml or higher (dark orange and light orange bars combined) were shown in NSCLC patients treated with bavituximab in phase III trials (FIGS. 18A and 18B) (and Examples XVIII and XX). , As well as for other NSCLC and pancreatic cancer patients treated with bavituximab), provides a propensity to prolong survival. Functional β2GPI levels in the range of 200 μg / ml to 290 μg / ml (dark orange) provide better statistically significant mOS for NSCLC patients treated with bavituximab in a phase III study (Table 14A). . Functional β2GPI levels greater than about 10 μg / ml (→, long green arrow) or greater than about 60 μg / ml (>, short green arrow) indicate functional binding and antitumor activity of bavituximab in preclinical studies Sufficient for activity (Example V). Distribution of pre-treatment functional β2GPI levels (μg / ml) in patients in a Phase II trial of bavituximab and docetaxel for treating NSCLC of Example XIII, reported in Example XVIII A. FIG. 20A, Distribution of functional β2GPI in a total of 119 evaluable patients; FIG. 20B, Distribution of functional β2GPI in patients treated with 3 mg / kg of bavituximab and docetaxel (40 evaluable patients); 20C, Distribution of functional β2GPI in pooled control patients (placebo or 1 mg / kg bavituximab) (79 evaluable patients). Kaplan-Meier survival curves from the Phase II study of Example XIII, showing that NSCLC patients with functional β2GPI levels greater than or equal to 200 μg / ml tend to have longer survival when treated with bavituximab. FIG. 21A. Among patients treated with 3 mg / kg of bavituximab, patients with functional β2GPI of 200 μg / ml or more (“bavituximab: B2GP1 ≧ 200”, blue upper line) have patients with β2GPI of less than 200 μg / ml ( In contrast to “Bavituximab: B2GP1 <200”, underlined in yellow), there is a tendency to prolong survival. FIG. 21B, in patients with functional β2GPI greater than or equal to 200 μg / ml, patients treated with 3 mg / kg of bavituximab (“bavituximab”, blue upper line) are patients in the combination control group (“bavituximab”, green lower line) In contrast, survival tends to be prolonged. FIG. 21C, patients with functional β2GPI of 200 μg / ml or more (“placebo: B2GP1 ≧ 200”, blue line) in patients in the combination control group were patients with β2GPI of less than 200 μg / ml (“placebo: B2GP1 <200 ”, yellow line). Distribution of pre-treatment functional β2GPI levels (μg / ml) in patients in a phase II trial of gemcitabine and bavituximab for treating pancreatic cancer of Example XII, reported in Example XVIII B. 3 shows the distribution of functional β2GPI in all 31 evaluable patients. Pancreatic cancer patients treated with gemcitabine and bavituximab had functional β2GPI levels greater than 200 μg / ml (“B2GP1 Kaplan-Meier survival curve from the Phase II study of Example XII, showing a tendency to prolong survival when present at ≧ 200 ”, blue top line). Distribution of pre-treatment functional β2GPI levels (μg / ml) in patients in a phase II trial of bavituximab and paclitaxel / carboplatin to treat NSCLC, reported in Example XVIII C. FIG. 24A, Distribution of functional β2GPI in a total of 84 evaluable patients; FIG. 24B, Distribution of functional β2GPI in patients treated with bavituximab and paclitaxel / carboplatin (44 evaluable patients); Distribution of functional β2GPI in paclitaxel / carboplatin control group patients (40 evaluable patients). Kaplan-Meier survival curves from Phase II trials reported in Example XVIII C, showing that NSCLC patients with functional β2GPI levels greater than 200 μg / ml tend to have longer survival when treated with bavituximab. FIG. 25A, in patients treated with bavituximab, patients with functional β2GPI greater than or equal to 200 μg / ml (“C / P + bavituximab: B2GP1 ≧ 200”, blue line above) have patients with β2GPI less than 200 μg / ml (“ C / P + Bavituximab: B2GP1 <200 ", underlined yellow) tends to prolong survival. FIG. 25B, in patients with functional β2GPI greater than or equal to 200 μg / ml, patients treated with bavituximab (“C / P + bavituximab”, blue upper line) are compared to patients in the control group (“C / P”, green underline) In contrast, survival tends to be prolonged. FIG. 25C, patients with functional β2GPI of 200 μg / ml or more (“C / P: B2GP1 ≧ 200”, blue line) in the control group of patients with β2GPI of less than 200 μg / ml (“C / P P: B2GP1 <200 ”, yellow line), the survival time tends to decrease. Patients treated with bavituximab and docetaxel followed by subsequent immunotherapy ("SACT-IO") (blue underline), as opposed to patients treated with docetaxel alone followed by subsequent immunotherapy (green underline) Kaplan-Meier survival curves showing having better mOS that are statistically significant. Treatment groups, mOS, and statistical analysis are tabulated in Table 16 for these survival curves (Example XIX). Kaplan shows that NSCLC patients with functional β2GPI levels above 200 μg / ml have a statistically significant better mOS when treated with bavituximab followed by subsequent immunotherapy (“SACT-IO”). Meyer survival curve. Among patients with functional β2GPI greater than or equal to 200 μg / ml, patients treated with bavituximab (“docetaxel + bavituximab”, blue line) are patients receiving SACT-IO (“with SACT IO”, solid line) and SACT-IO There is a tendency for survival to be prolonged in contrast to control patients ("docetaxel + placebo", green line), including patients who do not receive ("no SACT IO", dashed line).

  Today, there is an increasing emphasis on tailoring treatment to individual patients based on factors such as the patient's disease risk and / or expected response. This concept can be generally described as "personalized medicine." A deeper understanding of the different components that contribute to the efficacy of a particular treatment can provide a basis for stratifying patients, thereby improving treatment outcome for a continuous patient population. The present invention is an advance along such a line by providing new biomarkers for optimizing immunotherapy using PS-targeting antibodies such as Bavituximab.

A. Phosphatidylserine as a therapeutic target Phosphatidylserine (PS) is a highly immunosuppressive molecule that functions as an upstream immune checkpoint and regulates the host immune response. Thus, PS has been implicated in a variety of diseases, including cancer and viral infections. Thus, immunotherapeutics in the form of PS-targeted antibodies offer new treatment options for these diseases, including cancer.

  More specifically, in normal cells, PS is segregated into the inner leaflet of the plasma membrane, but in various pathologies, especially in cancer and viral infections, the outer leaflet of the plasma membrane within affected abnormal cells. It is externalized. In the context of cancer, some of the environmental stressors that cause externalization of PS are hypoxia / reoxygenation, oxidative stress, and exposure to certain cytokines. Externalization of PS also occurs under conditions of cell death and immunophagocytic clearance (Birge et al., 2016). The PS is then recognized and recognized by PS receptors on immune cells (eg, TIM3 and TIM4, BAI1, stabilin 2, and RAGE), optionally via one or more of several cross-linking proteins. When combined, PS induces and maintains immunosuppression. Within the tumor microenvironment, PS is exposed on the surface of tumor vascular endothelial cells, tumor cells, and tumor-derived exosomes, and overlapping immunosuppressive processes prevent the development of antitumor and inflammatory responses ing.

  Exposed PS promotes the recognition and clearance of dying cells, causes the release of immunosuppressive cytokines (eg, TGF-β and IL-10), and pro-inflammatory cytokines (eg, TNF-α and IL- A phagocytosis signal that inhibits the production of 12). PS also biases macrophages to the immunosuppressive M2 phenotype, inhibiting DC maturation and the ability of DC to present antigen, while stimulating DC and promoting T cell resistance Secretes immunosuppressive mediators. In summary, PS is a central factor in the induction and maintenance of an immunosuppressed tumor microenvironment.

B. PS-Targeting Antibodies Due to the tendency of PS exposure to promote tumor progression within the tumor microenvironment, PS-targeting antibodies are used to block the binding of PS to specific receptors on immune cells, An effective cancer therapy can be provided (Yin et al., 2013). As exemplified below, several such PS targeting antibodies have been developed as therapeutics. The group of "PS-targeted antibodies" includes PSs engineered in vitro, whether the antibodies bind directly to PS or require serum proteins to form a tight binding complex with PS. All antibodies that can bind and specifically localize and bind to PS that are exposed during disease conditions in vivo, particularly on tumor cells and tumor vessels, are included. Such "direct" and "indirect" PS targeting antibodies are described in more detail below.

B1. Early monoclonal antibodies produced to evaluate the preclinical potential Babitsukishimabu PS targeting antibody, 3G4, _IgG is 3 antibody called mAb (Example I; Ran et al., 2005; Huang et al., 2005). A sample of the hybrid cell line secreting the 3G4 antibody was deposited with the American Type Culture Collection (ATCC) and given ATCC accession number PTA4545. The availability of a deposited hybrid cell should not be construed as a license to practice the present invention under the authority of any government in violation of the rights granted in accordance with its patent laws.

  Bavituximab is a human chimeric version of the 3G4 mouse antibody in which the mouse variable (antigen binding) region is operably linked to a human antibody constant region (Examples III, C). Antibodies of the bavituximab family are described in detail in a number of U.S. patents, e.g., U.S. Patent Nos. 7,247,303 and 7,572,448, which have been deposited as ATCC PTA4545. The antibody can be reconstituted by preparing a human chimeric form of the antibody. Bavituximab is poorly immunogenic when administered to patients because a significant portion of the antibodies are of human origin.

  3G4 and Bavituximab antibodies bind strongly to anionic phospholipids, especially PS, in the presence of serum, but also bind to phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylglycerol (PG), and cardiolipin (CL). Strongly binds (Ran et al., 2005). Of these anionic phospholipids, PS is the most physiologically and pathologically relevant. 3G4 and Bavituximab show no detectable binding to neutral phospholipids, phosphatidylcholine (PC), sphingomyelin (SM), or phosphatidylethanolamine (PE), regardless of the presence of serum.

  Initially, 3G4 and bavituximab antibodies were thought to bind directly to PS, but it was later determined that PS binding was mediated by a serum protein identified as β2-glycoprotein 1 (β2GPI) (Example IV; Luster et al., 2006). Indeed, an enzyme-linked immunosorbent assay (ELISA) performed in the presence of β2GPI, including purified β2GPI and β2GPI provided simply by being present in 10% serum typically used for ELISA In 3G4 and Bavituximab bind strongly to PS.

  Β2GPI, 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. I have. β2GPI folds into these five identifiable domains as a tertiary structure and may have a closed circular structure or an open J-shape or hook structure. β2GPI is a positive site in domain V, its C-terminal domain, unless domain V is “nicked” at the Lys317 / Thr318 cleavage site, such as by cleavage with the enzyme plasmin, which disrupts PS binding. It binds to anionic phospholipids, especially PS, through the charged region (Hunt et al., 1993; Hunt & Krilis, 1994). 3G4 and Bavituximab antibodies bind to domain II of β2GPI. This enhances the safety of 3G4 and Babituximab as therapeutic antibodies, since certain other antibodies that bind to β2GPI have been implicated in pathology, but those antibodies bind to domain I of β2GPI. To do that.

  High affinity binding of 3G4 and Bavituximab antibodies to PS requires a bivalent interaction between the antibody and β2GPI (Example IV, FIG. 8). In the absence of such antibodies, β2GPI only binds with low affinity to anionic phospholipids, especially PS. This has been quantified in studies showing that 3G4 and bavituximab bind to PS in the presence of β2GPI as a high affinity complex and regulate β2GPI binding to PS from 1 μM to 1 nM.

  Β2GPI-dependent binding of 3G4 and Bavituximab antibodies to PS is depicted in FIG. The antibodies of the bavituximab family bind to domain II of β2GPI. As noted, because bavituximab binds to domain II of β2GPI, it is independent of side effects such as those associated with the antiphospholipid antibody syndrome in which antibodies that bind to domain I of β2GPI are present (de Laat et al. 2005; de Laat et al., 2006; Ioannou et al., 2007). The high affinity bivalent interaction of the antibody with β2GPI coordinates the resulting high affinity binding to PS, including when PS is externalized on the cell surface and cell membrane.

  3G4 and Bavituximab antibodies bind to β2GPI, but they are referred to as “PS-targeted antibodies” because they specifically localize and bind to PS that is exposed during disease states in vivo. Since PS is maintained inside healthy normal cells and is only exposed on the cell surface during disease states, antibody localization in vivo is not only specific for PS but also a disease in which PS is a marker. It is also specific for cancer, especially for viral infections and certain other pathologies.

  FIG. 8 also shows that ELISA is an accurate model of therapy because β2GPI-dependent antibodies that bind to PS are the same in vitro and in vivo. In particular, in ELISAs where the plate is coated with PS and the ELISA is performed in the presence of serum, antibodies such as 3G4, bavituximab can form a stable binding complex with PS. Thus, the ELISA assay mimics the in vivo condition during treatment, wherein PS is uniquely exposed only on cells in the tumor microenvironment or cells in the disease environment, such as virus-infected cells. Similar to the ELISA, when 3G4 and bavituximab antibodies encounter exposed PS, they can form stable binding complexes with β2GPI present in the blood. Irrespective of whether PS is on ELISA wells or on diseased cells, the antibody-β2GPI conjugate is more likely to bind to PS than monomeric β2GPI, ie β2GPI without a PS targeting antibody. It has an affinity of more than 2,000 times.

Direct PS binding antibodies such as B2.11.31 In addition to indirect PS binding antibodies such as Bavituximab or PS targeting antibodies, the entire family of PS targeting antibodies is an antibody that directly binds to PS, ie, a direct PS binding antibody or Includes direct PS targeting antibody. Such “direct PS binding antibodies” (or “direct PS targeting antibodies”) are not only functionally specific for PS, but also (like indirect binding antibodies) An antibody that targets and also binds to it, but does not require serum proteins such as β2GPI to form a tight binding complex with PS, even in in vitro binding assays.

  One particular example of such a direct PS binding antibody is the mouse monoclonal antibody designated 9D2 (Ran et al., 2002). The 9D2 antibody has been shown to localize tumor blood vessels and exert antitumor effects in vivo (Ran et al., 2002). Another example of a direct PS binding antibody is the fully human antibody (PGN632), which is called 11.31. The 11.31 antibody has also been shown to exert anti-tumor effects in vivo (eg, in mice bearing MDA-MB-435 breast cancer xenografts) and shows impressive anti-viral effects (Moody) Et al., 2010; U.S. Patent No. 7,455,833).

  Thus, direct PS binding antibodies are useful in treating various diseases where PS is a marker, particularly cancer and viral infections. However, biomarkers for optimizing treatment with such directly conjugated PS targeting antibodies typically do not depend on serum proteins such as β2GPI, as in the present invention, but on other factors Will do. Useful biomarkers for direct binding antibodies include immune biomarkers for PS targeting antibodies.

Other β2GPI-Dependent PS-Targeting Antibodies, such as B3.1N11 Preferred embodiments of the present invention relate to another part of the PS-targeting antibody family, namely indirect PS-binding antibodies. As used herein, an "indirect PS binding antibody" or "indirect PS targeting antibody" is functionally specific for PS, operably binds to PS in vitro, and Are antibodies that target and bind to, but require serum proteins to form a tight binding complex with PS. The invention particularly relates to indirect PS binding antibodies or indirect PS targeting antibodies, ie a subset of β2GPI dependent PS binding antibodies or β2GPI dependent PS targeting antibodies. As used herein, a “β2GPI-dependent PS binding antibody” or “β2GPI-dependent PS targeting antibody” is functionally specific for PS and operably binds to PS in vitro. An antibody that targets PS in vivo, ie, binds PS in vitro in an assay performed in the presence of serum containing β2GPI or purified serum, but forms a tight binding complex with PS Is an antibody that requires the serum protein β2GPI to do so. As mentioned above, examples of such antibodies include the mouse antibody 3G4 and the chimeric antibody bavituximab.

  Other currently preferred examples of β2GPI-dependent PS targeting antibodies are fully human antibodies, preferably 1N11 antibodies, designated 1N11 (PGN635) and 1G15. Several studies have been described using the 1N11 antibody, and mouse chimeric versions thereof, including imaging and therapy (Gong et al., 2013; Freimark et al., 2016; Gray et al., 2016a). FIG. 15 shows the PS binding characteristics of the 1N11 antibody. 1N11 was generated by phage display and selected using an assay for binding to PS only in the presence of serum (or only in the presence of β2GP1). As shown in FIG. 15, one of ordinary skill in the art can routinely perform such studies to prepare and isolate additional PS-targeting antibodies and β2GPI-dependent PS-targeting antibodies.

C. Extensive therapeutic experience As expected from the PS biology discussed above, signals from PS inhibit the ability of immune cells to recognize and fight tumors. Bavituximab and related antibodies counteract this PS-mediated immunosuppressive signaling by blocking the involvement of PS with its receptor and by sending alternative immune activation signals. Thus, PS-targeting antibodies have been shown to reverse the function 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 blockade of PS-mediated immunosuppression by reprogramming immune cells within the tumor microenvironment in a multi-local manner to support immune activation (Yin et al., 2013). ). Thus, bavituximab and related antibodies destroy immune tolerance in the tumor microenvironment. Antibody-mediated PS blockade reduces levels of bone marrow-derived immunosuppressive cells (MDSC), transforming growth factor-beta (TGFβ), and interleukin-10 (IL-10), interferon gamma (IFNγ), tumor necrosis factor -Increases the levels of pro-inflammatory cytokines such as alpha (TNFα) and interleukin-12 (IL-12). This PS blockade also re-biases MDSCs and tumor-associated macrophages (TAMs) from the predominant M2 to the predominant M1 phenotype, promotes dendritic cell (DC) maturation and activates cytotoxic T cells Induce strong adaptive anti-tumor T cell immunity (Yin et al., 2013).

  Bavituximab and related antibodies also activate innate immunity, ie, NK cells and M1 macrophages. Importantly, these antibodies also cause selective arrest of pre-existing tumor blood vessels that uniquely expose PS (Ran et al., 2005; US Pat. No. 7,572,448), and this activity has Includes antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by M1 macrophages and NK cells. Such destruction of tumor blood vessels results in destruction of tumor cells. These dual mechanisms of immunotherapy and vascular targeting, especially ADCC action, mean that bavituximab may be effective against tumors that are resistant to immune activation or conventional antiproliferative chemotherapy.

  As with other immunotherapy, the anti-tumor effects of PS-targeted antibodies are increased when used in combination therapy. One group of agents for use with bavituximab and related antibodies are those agents and / or conditions that increase the exposure of PS in the tumor microenvironment, such as radiation and / or chemotherapeutic agents (US Pat. No. 7,422). , 738, U.S. Patent No. 8,486,391, U.S. Patent No. 7,572,448). Thus, the enhancement of antitumor effects was demonstrated in the treatment of breast tumors (Huang et al., 2005) and prostate cancer (Yin et al., 2013) in combination with docetaxel, and in the treatment of pancreatic tumors in combination with gemcitabine (Beck et al., 2006). ), Treatment of lung cancer (He et al., 2007) and glioblastoma (He et al., 2009) in combination with radiation, progressive mammary gland in combination with PRIMA-1 reactivating mutant tumor suppressor p53 Treatment of tumors (Liang et al., 2011), Retargeting of adenovirus to tumor vasculature in combination with adenovirus (Hogg et al., 2011), Treatment of lung cancer recurrence after surgery with cisplatin ( Judy et al., 2012), as well as in the treatment of hepatocellular carcinoma in combination with sorafenib (Cheng et al., 2016).

  Another group of agents for use with PS targeting antibodies such as Bavituximab are other IO agents. Because PS is an upstream immune checkpoint, the mechanism of action of bavituximab is complementary to available therapeutics. Thus, in combination with other checkpoint inhibitors in the form of antibodies to CTLA-4, PD-1, and PD-L1, an impressive combination therapy has been demonstrated preclinically for antibodies of the bavituximab family. (Freimark et al., 2016; Gray et al., 2016a). Such anti-tumor activity, including increased survival, was due to increased intratumoral activated CD8 T cells, reduced M2 macrophages and MDSCs associated with PD-L1 expression, and spleen as compared to PD-1 blockade alone. Associated with an increase in tumor-reactive T cells within the tumor.

  Thus, preclinical results such as these confirm that PS targeting antibodies of the bavituximab family reverse PS-mediated immunosuppression and initiate therapeutically effective adaptive anti-tumor immunity. Thus, treatment with bavituximab in combination with blocking the downstream immune checkpoint results in robust and long-lasting anti-tumor immunity that significantly improves the outcome, duration, and level of response (Freimark et al., 2016; Gray et al., 2016a) ).

  Given the favorable safety profile of PS-targeting antibodies such as bavituximab, these antibodies may also be used in combination with radiation, chemotherapeutics ("chemoradiotherapy"), and / or immunotherapy, and The combination can be effectively used in a triple combination therapy including a triple combination with an immunotherapy agent. Recently, impressive results have been shown for triplicate combinations using antibodies targeting PS, PD-1 and LAG-3 (Gray et al., 2016b).

  Based on such preclinical data, bavituximab has been evaluated in clinical studies involving more than 800 patients, primarily in combination with approved treatments for other indications. Various antiviral and antitumor studies have shown therapeutic activity. Based on extensive preclinical studies and pharmacokinetic profiles in humans (see also Example VI; Gerber et al., 2011; Digumarti et al., 2014), a dose of 3 mg / kg administered intravenously (IV), bavituximab was It was determined and selected in most clinical studies in oncology, including patients with lung, breast, liver, pancreas, colorectal, and kidney cancer. Currently, for pavituximab, paclitaxel in patients with HER2-negative metastatic breast cancer (Chalasani et al., 2015), paclitaxel-carboplatin in advanced non-small cell lung cancer (NSCLC) (Digumarti et al., 2014), sorafenib in hepatocellular carcinoma (Cheng et al.). Promising clinical anti-tumor results have been published in combination with docetaxel (Gerber et al., 2016) in previously treated advanced non-squamous NSCLC.

  In summary, the results from Phase I and Phase II clinical studies demonstrated a clinically significant therapeutic effect of Bavituximab. Although there is now a considerable amount of research showing the successful treatment of various diseases using PS targeting antibodies, to date there are no known biomarkers for such treatments. Clinical experience with PS-targeted antibodies has been primarily based on β2GPI-dependent PS-targeted antibodies such as bavituximab. It is those antibodies that are most needed for biomarkers to optimize treatment. This would be an important development if one could identify one or more circulating biomarkers for the treatment of bavituximab, together with a sensitive and rapid method for quantifying such biomarker (s), Provide minimally invasive test (s) to facilitate patient selection and improve treatment outcome.

D. Biomarkers for PS-Targeted Antibodies In the field of cancer therapeutics, biomarkers play an increasingly important role in identifying certain patient characteristics that affect response to treatment. This has been historically seen for targeted cancer treatments and more recently for checkpoint inhibitors, including PD-1 and PD-L1 inhibitors.

  Important biomarkers for treatment with PS targeting antibodies, such as bavituximab, have been analyzed. As used herein, a "bavituximab biomarker" is used to improve the clinical benefit of treatment with a therapy comprising a PS targeting antibody, preferably bavituximab, as at least part of the therapy. A biomarker for use alone or as one of two or more biomarkers in selecting a patient or patient population. Thus, such a bavituximab biomarker comprising β2GPI can be used to identify a PS-targeted antibody (PBS-targeted antibody) comprising a combination therapy in which the patient, patient population, or subpopulation comprises a PS-targeted antibody (preferably, bavituximab) prior to treatment. (Preferably bavituximab) can be used in a method for predicting whether it is likely to benefit from treatments.

  In order to improve the clinical benefit of a treatment regimen containing bavituximab, multiple marker codes to identify the most appropriate patient population are also being considered. The first biomarker identified in these analyzes is β2GPI (sections E, F). Overall, the biomarker pattern identified is the bavituximab "sign" to guide clinical development and therapy.

  As part of the bavituximab biomarker, the bavituximab immune biomarker has been analyzed. Such data support the use of bavituximab to “prime” the immune system, ie, amplify the anti-tumor immune response. In this regard, it is now known that tumors can be placed on a scale from "hot" to "cold" depending on how deeply they are infiltrated by T cells and other immune cells. The level of immune invasion ("fever") reflects whether the immune system recognizes and participates in tumors. Patients with tumors that are "fever" have a better prognosis; tumors that are "cold" have a much higher chance of recurrence. Importantly, it has been determined that bavituximab has a positive effect on cold tumors and can make them easier to treat (with those containing other checkpoint inhibitors). Therefore, the bavituximab immunity biomarker is not only useful in selecting patients for bavituximab therapy, but also in identifying patients for treatment with intelligent drugs selected for bavituximab and combination therapy. Has additional uses.

D1. Samples For biomarkers other than β2GPI (Section E), any biological sample containing or suspected of containing one or more of the biomarkers (including feces, using the present invention) (Including any tissue sample or biopsy from an animal, subject, or patient). A clarified lysate from a biological tissue sample may be used. However, the present invention provides tests that are preferably used with natural bodily fluids and can therefore be performed on biological samples obtained using minimally invasive or non-invasive techniques, also called "liquid biopsies" I do. This is an advantage over more rigorous techniques such as biopsy, which typically take longer to obtain results and can pose a health risk to themselves.

  Examples of bodily fluids (biological fluids) that contain or are suspected of containing one or more biomarkers include blood, urine, ascites, brain and cerebrospinal fluid (CSF), sputum, saliva, Nasal discharge, bone marrow aspirate, synovial fluid or synovial fluid, aqueous humor, amniotic fluid, follicular fluid, earwax, breast milk (including colostrum), bronchoalveolar lavage fluid, semen, seminal plasma (including prostate fluid), Cowper's fluid or Pre-ejaculatory fluid, vaginal fluid (female ejaculate), sweat, tears, cystic fluid, pleural effusion and ascites fluid (peritoneal fluid) or lavage fluid, pericardial fluid, lymph fluid, plaque, bile, liver perfusate, interstitial fluid, menstrual fluid, menstrual fluid, Pus, sebum, vomiting, vaginal secretion, mucosal secretion, stool water, faecal fluid, pancreatic juice, washings from the paranasal sinuses, body cavity, broncho-pulmonary aspirate, or other Washing liquid is included. Biological samples can also include blastocyll cavity, umbilical cord blood, or maternal circulation, which can be of fetal or maternal origin.

  Examples of preferred body fluids to test are blood, urine, and ascites, particularly ascites from animals, subjects, or patients having or suspected of having ovarian cancer. If a urine sample is used, it is preferably for urinary, genitourinary, and genital cancers, such as, for example, ovarian, prostate, kidney, bladder, testis, urethra, and penis cancers. Used in connection. As with β2GPI, detection and quantification of one or more of the other biomarkers is preferably performed from a peripheral blood sample, preferably plasma, and most preferably serum.

D2. PS-Positive Exosomes In recent years, exosomes have attracted attention in connection with cancer. Exosomes are 40-50 to 100 nanometer (nm) sized membrane-derived vesicles that are constantly released by all cells in vivo and in vitro. Exosomes are biologically active molecular shuttles that play an important role in the exchange of information between cells and affect many physiological and pathological processes. In cancer, exosome function involves 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).

  Due to multiple intracellular fusion events involved in exosome formation, the luminal content and proteomic profile of exosomes released extracellularly mimic those of the source cells. Thus, tumor-derived exosomes ("tumor exosomes") have a profile that reflects the cancer cells from which they are derived. Indeed, the presence of cytosol (particularly nucleic acids) and plasma membrane components from the source cells indicates that circulating exosomes are readily available surrogates that reflect the properties of the parent cell for biomarker analysis. means.

  In contrast to exosomes from normal cells, tumor exosomes are characterized by having PS on their surface. Thus, PS-positive tumor exosomes can be used for cancer diagnosis. New and improved methods, compositions, and kits for diagnosing cancer by detecting and quantifying PS-positive tumor exosomes in body fluid samples using solid-phase assays have recently been reported. Such techniques are described in U.S. Patent Application No. 15 / 177,747 and PCT Patent Application No. PCT / US16 / 036629, each filed on June 9, 2016.

  Because PS is highly immunosuppressive, release of PS-positive tumor exosomes is another means by which tumors promote an immunosuppressive environment. Therefore, the level of PS-positive tumor exosomes before treatment has been proposed to be used as a predictive marker of response to therapy for any cancer treatment. Clearly, PS targeting antibodies need to bind PS within the disease microenvironment. Therefore, measuring the level of PS-positive tumor exosomes before treatment is particularly compelling for use as a predictive biomarker of response to therapy using PS-targeting antibodies such as bavituximab.

  Thus, methods such as those in US 15 / 177,747 and PCT Application No. PCT / US16 / 036629 can be used as part of the biomarker test of the present invention. Current quantification of β2GPI and / or their combination with other bavituximab biomarkers may be preferred in certain embodiments, for example, to enhance the sensitivity of the overall predictive analysis.

E. FIG. Β2GPI as a biomarker
Despite extensive data that negates it (eg, Example V), we have used pre-treatment levels of β2GPI as a biomarker, or as part of a panel of biomarkers, to produce bavituximab. And the possibility of predicting the therapeutic outcome of therapies using related antibodies.

  β2GPI is an abundant plasma (serum) glycoprotein that is found both free and related to lipoproteins. The DNA and amino acid sequences of β2GPI from various mammalian species, including mouse, rat, dog, cow, chimpanzee, and human, are known (Steinkasserer et al., 1991). For illustrative reference, the human β2GPI amino acid sequence is provided as accession number 1C1ZA. β2GPI is glycosylated and is routinely reported as a 50 kDa protein (Examples IV, A4, B3; McNeil et al., 1990, FIG. 3; Balasubramanian et al., 1998, FIG. 1; Luster et al., 2006, FIG. See also 1D). Although β2GPI has been studied for decades, the exact physiological role of β2GPI remains unknown (Prakasam & Thiagaranjan, 2012). Indeed, the apparently healthy life of β2GPI-deficient knockout mice indicates that its role is not important (Sheng et al., 2011).

  Surprisingly, pre-treatment blood levels of β2GPI, particularly functional β2GPI, have been determined to be effective biomarkers for predicting response to therapy using PS-targeting antibodies such as bavituximab. . Indeed, the level of “functional” β2GPI, which refers to β2GPI binding to both PS and PS targeting antibodies such as bavituximab, is useful alone as a biomarker for response to bavituximab.

  In embodiments of the present invention that use pre-treatment β2GPI levels alone as a biomarker of response to a PS-targeting antibody such as bavituximab, those β2GPI levels are both numerically defined and defined herein as “ It is measured in an assay that can detect “functional” β2GPI. However, in embodiments of the invention that use pre-treatment β2GPI levels as one of more than one or more biomarkers of response to a PS-targeting antibody, such as bavituximab, β2GPI levels may be less strictly numerically. It need not be defined and need not be measured alone in a functional β2GPI assay.

  Thus, β2GPI levels as part of a dual or multi-marker code for bavituximab-containing therapy can be “β2GPI high” versus “β2GPI low”, VeriStrat® good (VS good) and poor VS, and Similar to the description for “hot” or “cold” tumors. For treatment with a PS-targeting antibody such as bavituximab, patients with “β2GPI high” should be selected. In this context, the level of β2GPI that is “β2GPI high” is about 180, 190, 200, 210, 220, 230, 240, 250, or 260 μg / ml or more, preferably about 200 μg / ml or more β2GPI (total β2GPI , Or, preferably, functional β2GPI). Thus, “β2GPI high” is equal to about 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, or 320 μg / ml, β2GPI, (total β2GPI, or Preferably, it includes a pre-treatment level of any functional β2GPI.

  The present invention also provides biomarkers for a specific numerically defined amount and range of functional β2GPI, as measured in an assay capable of detecting functional β2GPI. In a most preferred embodiment, the invention relates to the selection and treatment of patients based on pre-treatment levels of functional β2GPI of 200 μg / ml or more (Example XVII, FIGS. 18A and 18B, Example XVIII, Example XX). This may include a pre-treatment level of functional β2GPI equal to about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, or 320 μg / ml.

As demonstrated by the data in Examples XVII, XVIII, and XX, in all clinical trials in which β2GPI data is available, bavituximab is completely effective in patients with functional β2GPI greater than 200 μg / ml. Improved survival time. The results are summarized in Table A, where the treatment column refers to bavituximab treatment of the listed indications in combination with the cited drugs. In addition, in patients with NSCLC treated with bavituximab and subsequent IO (Example XX), more than 200 μg / ml functional β2GPI was shown to have an advantage over the survival of bavituximab over placebo (mOS reached with bavituximab Whereas placebo was 12.3 months; p = 0.002, FIG. 27), but a comparison between patients treated with bavituximab in the β2GPI group of 200 or more and the β2GPI group of less than 200 showed that mOS The group has not yet been reached and cannot be done yet.

  As with widely applicable levels of β2GPI above 200 μg / ml, certain presently preferred embodiments of the invention are particularly useful for treating NSCLC, prior to treatment of functional β2GPI in the range of 200-290 μg / ml. For patient selection and treatment based on level (compare Example XVII, Table 14A and Table 14B). This also means that 200-270, 200-280, 200-290, 200-300, 200-310, 200-320, 210-270, 210-280, 210-290, 210-300, 210-310, and 210 Also including pre-treatment levels of functional β2GPI, such as within the range of 320 μg / ml, 210-270, 210-280, 210-290, 200-280, and 200-290 are presently preferred.

In a further embodiment, the invention provides a method wherein the low number is from about any one of about 190, 200, 210, or 220 μg / ml and the high number is about 260, 270, 280, 290, 300, 310, or It relates to the selection and treatment of patients based on pre-treatment levels of functional β2GPI in any range up to any one of 320 μg / ml. These ranges include:
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;
About 220-260, 220-270, 220-280, 220-290, 220-320, 220-310, and 220-320, among which about 210-270, 210-280, 210-290, The ranges from 200 to 280 and from 200 to 290 μg / ml are preferred.

  In addition to the most preferred and generally applicable selection and treatment of patients, based on pre-treatment levels of functional β2GPI of 200 μg / ml or more, the number or range of the above for any particular or combination treatment Regardless of which one or more of which is selected, the use of pre-treatment levels of β2GPI, preferably functional β2GPI, as a biomarker or as part of a panel of biomarkers, requires that PS be a marker. Of PS-targeted antibodies, either alone or preferably in any combination therapy, with the selection of patients with a variety of diseases, especially cancer and viral infections, but also infections of intracellular parasites Apply to those treatments that use.

F. Assays for β2GPI Since pre-treatment levels of β2GPI are biomarkers for bavituximab and related antibodies, the following guidance is provided for assays for β2GPI. The present invention also provides certain preferred assays for quantifying functional β2GPI (Section G).

F1. β2GPI Sample As a serum protein, β2GPI is ideal for detection in peripheral blood (plasma, serum) samples as described below. However, studies have suggested that β2GPI localizes to endothelial cells in vivo under various pathophysiological conditions involving PS (Agostinis et al., 2011). Thus, the entire range of biological samples (section D1) could potentially be used for β2GPI detection.

  Nevertheless, peripheral blood, plasma, and serum samples, whether total or functional β2GPI, are particularly preferred for detection and quantification of β2GPI (Section G). Whole blood may be used (red blood cells, white blood cells, platelets, proteins, and plasma). Preferably, plasma is used, which is the liquid remaining after sedimentation of red and white blood cells. Plasma, which contains fibrinogen and other clotting factors, tends to coagulate on standing. Plasma that is difficult to coagulate is available, which is preferred, and platelet-free plasma may be used. Most preferably, serum is used for the detection and quantification of β2GPI. Since serum has no coagulation factor and is mainly plasma without fibrinogen, the serum does not coagulate even when left. Animal and human sera are commonly used for diagnostic purposes, and preparation techniques are widely known. An exemplary method for preparing a serum sample for a β2GPI test is provided herein (Example XV, A).

  An advantage of the present invention is that the test can be performed directly on a biological sample, preferably blood, plasma or serum. Due to sensitivity, β2GPI can be easily detected without any prior enrichment or enrichment (but this is not excluded). The test sample, preferably a serum sample, can be fresh or previously frozen and then thawed. Example XV, Example XVI, Example XVII, and Example XVIII show that β2GPI is stable for long-term storage at -70 ° C. It is preferred to use industry standard techniques of freezing, storage and / or thawing, such as using a bass storage tube or vial and / or a protease inhibitor to limit overall proteolysis.

F2. Range of β2GPI Assays Regardless of whether it is “functional” β2GPI, the breadth of assays to measure β2GPI (ie, assays for “total” β2GPI) is that pre-treatment β2GPI levels are 2 times higher than those of bavituximab. Applicable for use with embodiments of the present invention used as the only one of one or more biomarkers. If the level of β2GPI is used alone as a biomarker for bavituximab, a “functional” β2GPI assay should be used, as described in Section G.

  Total β2GPI levels can be detected and preferably quantified using any one or more of a number of in vitro binding assays and kits known in the art. Suitable binding assays include, for example, immunoblots, western blots, dot blots, RIAs, immunohistochemistry, fluorescence activated cell sorting (FACS), immunoprecipitations, affinity chromatography, and the like. While typically a solid phase binding assay is preferred, various other methods for detecting β2GPI have been described in the literature and any of them may be used. For example, β2GPI levels can be accurately determined by radial immunodiffusion. In fact, radial immunodiffusion has been used for the quantification of β2GPI since the late 1960s and more modernly (eg, Balasubramanian et al., 1998). As well as Western blots, immunoelectrophoresis, and octalony double immunodiffusion (Takeuchi et al., 2000), isoelectric focusing (IEF) and subsequent immunoblots can also be used to quantify β2GPI ( Kamboh et al., 1988).

F3. Solid Phase β2GPI Binding Assays Numerous sensitive solid phase binding assays for β2GPI are known in the art, and total β2GPI is preferably detected and quantified using one or more of such assays. One preferred example of such an assay is as an enzyme-linked immunosorbent assay (ELISA). Various ELISAs specific to all β2GPI have been reported in the literature, including modified capture ELISAs (eg, Mehdi et al., 1999) and competitive ELISAs (eg, Balasubramanian et al., 1998). A number of commercially available kits are available for assaying total β2GPI, as well as commercially available anti-β2GPI antibodies, including those attached to diagnostic labels. Any such kit or antibody can be used to detect and quantify total β2GPI. For example, US Biological's anti-β2GPI antibody is used herein in a comparative assay (Examples XVI, A10, B2).

  In general terms, an ELISA for whole β2GPI uses one or more anti-β2GPI antibodies. Although antibody technology has advanced significantly, commercial kits and commercially available anti-β2GPI antibodies often use polyclonal anti-β2GPI antibodies, which are perfectly suitable for use in such embodiments. In an exemplary assay for total β2GPI, anti-β2GPI antibodies are adsorbed to a solid surface, such as a 96-well plastic plate, and incubated with a biological sample suspected of containing β2GPI (in this case, the “antigen”). . Bound β2GPI (antigen) binds a secondary binding agent that is directly or indirectly labeled with a detectable substance, ie, a substance that produces a detectable signal such as color or fluorescence that is detectable and quantifiable. Detect using. Preferably, the secondary binding agent is an anti-β2GPI antibody that is labeled with a detectable substance.

  Such an ELISA for total β2GPI is illustrated in Examples XVI, A10, B2, and many common components and steps such as solid supports and detectable substances are also described in the functional β2GPI of the present invention. The assay is described more fully below (Section G). Thus, unless it is clear that the particular reagents or steps apply only for use in a functional β2GPI assay, their application in an assay to detect total β2GPI is contemplated herein.

G. FIG. Preferred ELISA for functional β2GPI
A variety of commercially available assays and research tools are available for analyzing clinical test results for biomarkers to predict better results, but all are specifically applicable to PS-targeting antibodies such as bavituximab It was not known. Despite extensive preclinical modeling and significant pre-clinical experience, showing that low and / or varying levels of serum β2GPI do not significantly affect the treatment outcome of bavituximab (Example V, FIG. 19) Instead, an analysis of β2GPI levels in patients in a phase III study (Example XIV) was sought.

  However, no reliable quantitative β2GPI assay for specifically detecting β2GPI that can bind to PS, as opposed to total β2GPI, was available. Such assays are particularly useful for biomarkers because it is well known that a portion of β2GPI (“total” β2GPI) exists as a nicked β2GPI, which cannot bind to PS and therefore cannot mediate antibody binding at disease sites. Is needed for the most accurate measurements applied to Furthermore, there was a significant lack of any assay to specifically detect β2GPI, which could bind not only to PS, but also to PS targeting antibodies such as Bavituximab. This is, for example, the highest fidelity to exclude the possibility that β2GPI with other significant changes (especially mutations and / or nicks or truncations in or affecting domain II) had been detected. Of particular importance for the biomarker measurement of is that any such β2GPI changes reduce or abolish the formation of the antibody: β2GPI: PS complex required for therapeutic activity.

  Therefore, to perform optimal analysis of β2GPI concentration in patients treated (or to be treated) with a PS-targeting antibody such as bavituximab, including patients in a Phase III study (Example XIV), a first It was necessary to invent a new assay. The present application discloses such an advantageous assay specifically adapted for the purpose of detecting and quantifying the amount of functional (active) β2GPI in human blood samples such as plasma and serum, which assay comprises PS And the level of β2GPI that can bind to both PS and a PS targeting antibody such as bavituximab can be determined.

  By using such an assay for functional β2GPI, the present invention provides a method of pre-treatment with a defined level of use for a single biomarker of response to treatment with bavituximab and related PS targeting antibodies. Provides β2GPI. In particular, a functional β2GPI greater than or equal to 200 μg / ml (Example XVII, FIGS. 18A and 18B, Example XVIII, Example XX), as illustrated by a functional β2GPI in the range of 200-290 μg / ml, Broadly predict response to treatment with bavituximab (compare Example XVII, Table 14A and Table 14B). A preferred ELISA for functional β2GPI provided by the present invention is illustrated by the detailed teachings of Example XVI and is described more fully below.

G1. Assay Methods In general terms, solid-phase assays such as ELISA for functional β2GPI use both PS and a PS targeting antibody such as bavituximab, at least one of which is engineered on a solid support. Possible associations and / or at least one of them are directly or indirectly labeled with a detectable substance. All connection types can be used. For example, a PS targeting antibody may be adsorbed on a solid support and PS may be labeled with a detectable substance. Many lipids are known in the art, such as PS, labeled with a detectable substance, any of which may be used. However, for simplicity, the presently preferred embodiment is one in which the PS is adsorbed to a solid surface, such as a 96-well plastic plate. This means that a PS-targeted antibody such as bavituximab or 1N11 can be labeled with a detectable substance, which is preferably a direct label attached to the antibody.

  In these assays, a PS coated solid support, such as an ELISA plate (or well thereof), is incubated with a PS targeting antibody, such as bavituximab or 1N11, and a biological sample suspected of containing β2GPI. Conceptually, it is important that the PS targeting antibody, preferably bavituximab, and the β2GPI sample be “co-incubated” on a PS coated solid support or plate. Scientifically, β2GPI binds to PS via (intact) domain V, followed by plate-bound β2GPI domain II, or bavituximab binds to β2GPI domain II in solution. It can be either bound, followed by complexed bavituximab / β2GPI (with intact domain V) that binds to PS on the plate. Co-incubation encompasses all such binding mechanisms, since both binding events occur during the incubation period.

  “Co-incubating” a PS targeting antibody, preferably bavituximab, and a β2GPI sample on a PS coated support is performed under “effective binding conditions”, ie, when “β2GPI is the PS targeting antibody and PS in the sample. Means incubating together under conditions and for a time effective to allow binding to both of the coated supports. "Binding" means "specific binding", ie, conditions and times effective to permit binding that is not removed by ordinary washing. Thus, when using bavituximab, effective binding conditions allow binding of PS to the intact (nick-free) domain V of β2GPI and binding of bavituximab to domain II of β2GPI. When using a PS targeting antibody other than Bavituximab, effective binding conditions are binding of PS to the intact (nick-free) domain V of β2GPI and of the PS targeting antibody to a domain other than domain V to β2GPI. (Preferably to the hinge region joining domains I and II, and most preferably to β2GPI to domain II).

  As long as the PS targeting antibody, preferably bavituximab, and the β2GPI sample are co-incubated on a PS-coated solid support or plate, the assays of the invention include several different formats. For example, a PS targeting antibody and a sample suspected of containing β2GPI may be added substantially simultaneously to a PS coated support. Preferably, the PS targeting antibody and the sample suspected of containing β2GPI are added to the PS coated support continuously, ie, at time intervals.

In performing a sequential assay binding step, a PS-coated solid support, such as an ELISA well, is first incubated with a biological sample suspected of containing β2GPI, and then incubated with a PS-targeting antibody, preferably bavituximab. It may be incubated. The above assay can utilize a pre-prepared PS-coated support, in which case the assay comprises the following (or the following steps):
(A) PS coating a biological sample suspected of containing β2GPI under conditions effective to bind β2GPI in the biological sample to the PS coated support via the intact domain V of β2GPI. Adding to the support, thereby preparing a PS and β2GPI coated support;
(B) Binding the PS-targeting antibody to a PS and β2GPI coated support by binding the antibody to a β2GPI domain other than domain V, preferably binding the bavituximab to a PS and β2GPI coated support by binding the antibody to β2GPI domain II. Adding a PS targeting antibody, preferably bavituximab, to the PS and β2GPI coated support under conditions effective for binding;
(C) detecting the binding of the PS targeting antibody, preferably bavituximab, to the PS and β2GPI coated support, thereby measuring the functional β2GPI in the biological sample.

However, in performing the sequential assay binding step, the PS targeting antibody, preferably bavituximab, and most preferably the detectably labeled bavituximab is first applied to a PS-coated solid support and then contains β2GPI. Co-incubated with the suspected biological sample. Such an assay or sequence of steps is preferred for technical reasons, for example to avoid cross-contamination during pipetting. Such preferred assays include the following (or the following steps):
(A) coating a solid support with PS to prepare a PS coated support;
(B) adding a PS-targeting antibody, preferably bavituximab, and most preferably a detectably labeled bavituximab to the PS-coated support to prepare an antibody-covered PS-coated support;
(C) Under conditions effective to bind β2GPI in a biological sample to the antibody-coated PS-coated support, ie, to bind β2GPI to the PS-coated support via the intact domain V of β2GPI. Essentially without washing, by binding and by binding of β2GPI to a PS targeting antibody via a β2GPI domain other than domain V, preferably by binding of β2GPI to bavituximab via β2GPI domain II. Adding a biological sample suspected of containing to an antibody-coated PS coated support;
(D) detecting binding of a PS targeting antibody, preferably bavituximab (most preferably a detectably labeled bavituximab), and β2GPI to a PS-coated support, thereby providing functional β2GPI in a biological sample. Measuring.

The assay may also utilize a pre-prepared PS-coated support, in which case the assay comprises the following (or the following steps):
(A) adding a PS targeting antibody, preferably bavituximab, and most preferably a detectably labeled bavituximab, to the PS coated support to prepare an antibody covered PS coated support;
(B) under conditions effective to bind β2GPI in a biological sample to the antibody-coated PS-coated support, ie, to the β2GPI to the PS-coated support via the intact domain V of β2GPI. Essentially without washing, by binding and by binding of β2GPI to a PS targeting antibody via a β2GPI domain other than domain V, preferably by binding of β2GPI to bavituximab via β2GPI domain II. Adding a biological sample suspected of containing to an antibody-coated PS coated support;
(C) detecting binding of a PS targeting antibody, preferably bavituximab (most preferably a detectably labeled bavituximab), and β2GPI to a PS-coated support, thereby providing functional β2GPI in a biological sample. Measuring.

A PS targeting antibody, preferably bavituximab, and most preferably a detectably labeled bavituximab is first applied to a PS-coated solid support and then co-incubated with a biological sample suspected of containing β2GPI. Preferred assays with successive binding steps are briefly described below (or the following steps):
(A) coating a solid support with PS to prepare a PS coated support;
(B) Under conditions effective to bind β2GPI in a sample to both a PS-targeted antibody, preferably a bavituximab, and a PS-coated support, a PS-targeted antibody, preferably a bavituximab, and most preferably a detection Adding a possibly labeled bavituximab, as well as a biological sample suspected of containing β2GPI, to the PS coated support, preferably prior to the addition of the sample wherein the PS targeting antibody contains β2GPI. Adding to the PS coated support and co-incubating them together;
(C) detecting the binding of the PS targeting antibody and β2GPI to the PS coated support, thereby measuring the functional β2GPI in the sample.

Again, the assay may utilize a pre-prepared PS coated support, in which case the assay comprises the following (or following steps):
(A) Under conditions effective to bind β2GPI in a sample to both a PS-targeted antibody, preferably a bavituximab, and a PS-coated support, a PS-targeted antibody, preferably a bavituximab, and most preferably a detection Adding a possibly labeled bavituximab, as well as a biological sample suspected of containing β2GPI, to the PS coated support, preferably prior to the addition of the sample wherein the PS targeting antibody contains β2GPI. Adding to the PS coated support and co-incubating them together;
(B) detecting binding of the PS targeting antibody and β2GPI to the PS coated support, thereby measuring functional β2GPI in the sample.

  Bound PS targeting antibody and β2GPI (antigen) are detected using at least one secondary binding agent in the form of at least a PS targeting antibody, preferably in the form of bavituximab or 1N11, which comprises a detectable substance. Directly or indirectly. An unlabeled PS targeting antibody can be used in the context of a tertiary binding agent, preferably another antibody, which binds to the PS targeting antibody and is directly labeled with a detectable substance. Such tertiary binding antibodies are well known in the art, and specifically bind, for example, to the Fc portion of a PS targeting antibody.

  For simplicity, a presently preferred embodiment is one in which the PS targeting antibody, preferably bavituximab, is directly conjugated to a substance that is itself detectable. A detectable substance is a substance that produces a detectable signal that can be detected and measured or quantified, such as color or fluorescence. An exemplary detectable substance is the enzyme horseradish peroxidase (HRP), which cleaves the substrate 3,3'5,5'tetramethylbenzidine (TMB) and is detected and measured at 450 nm. Generate a colored signal. Typically, the amount of binding material measured from the signal is compared to the level of a "reference signal", such as a standard curve. If necessary, the standard curve may be duplicated in all assays.

  In all formats of these assays, the only β2GPI that is ultimately detected is β2GPI, which can bind to both PS and PS targeting antibodies, ie, β2GPI that is not totally removed by routine washing. It is. Thus, these assays are based on the most relevant form of clinical treatment for pre-treatment β2GPI, ie, the administered antibody, preferably bavituximab, and preferably a conjugate with PS exposed to the disease site within the tumor microenvironment. Is particularly suitable for detecting β2GPI that “works” to form Thus, the use of these assays is advantageous in selecting patients for improved treatment outcome with bavituximab therapy.

  The functional β2GPI assay provided by the present invention is also simple, reproducible, sensitive, cost-effective, and for use with biological samples, especially blood (serum and plasma) samples obtained by minimally invasive techniques. Ideal to do. The rapid nature of the assay offers the important advantage that biomarker tests can be performed quickly and therapeutic decisions can be made and performed in a timely manner. However, a new assay for quantifying functional β2GPI provided by the present invention is only for use in measuring β2GPI for use as a biomarker in therapeutics that use PS-targeted antibodies such as Bavituximab. Not limited. Because β2GPI is an important molecule in basic and directed preclinical and clinical research, these assays may be performed in any one or more of such embodiments (eg, knockout mice, or anti-phosphorus in humans). It can be used to measure functional β2GPI, such as in embodiments related to lipid-antibody syndrome (APS).

G2. Solid Supports The solid phase binding assays of the invention typically involve operatively associating a binding construct with a solid support or substrate (having at least one surface for coating or binding). . As used herein, a "binding construct" includes a construct that binds to a component useful for detecting a biomarker. In the context of β2GPI biomarkers, binding constructs include anti-β2GPI antibodies, PS, and PS targeting antibodies such as Bavituximab.

  Such solid supports or substrates include, for example, plates, beads, and fibers. In a preferred embodiment of the invention, the solid support or substrate is a multi-well plate, such as a standard 96-well plate. The solid support or substrate can be made from any suitable material, such as Sepharose, latex, glass, polystyrene, polyvinyl, nitrocellulose, silicon, silica, polydimethylsiloxane (PDMS). The binding construct is operably associated with the solid support or substrate by efficiently contacting at least one surface of the support or substrate with the binding construct. Preferably, the binding construct is immobilized on at least one surface of a solid support or substrate. The binding construct may also be printed on a coated glass slide and used in a biomarker array or microarray. Such microarrays can be prepared using both non-contact and contact printing, with contact printing being preferred.

G3. Detectable Substances Suitable detectable substances include, for example, enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, and urease. Horseradish peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which results in a soluble product detectable at 450 nm in the presence of hydrogen peroxide. Other convenient enzyme-linked systems include, for example, alkaline phosphatase detection systems, which can be used with the chromogenic substrate p-nitrophenyl phosphate to obtain a soluble product that is easily detectable at 405 nm. . Similarly, a β-galactosidase detection system can be used with the chromogenic substrate O-nitrophenyl-β-D-galactopyranoside (ONPG) to obtain a soluble product detectable at 410 nm, or a urease detection system. Can be used with a substrate such as urea-bromocresol purple.

  Further examples of detectable substances include chemiluminescent labels and labels for fluorescence detection. Useful fluorescent dyes include DAPI (4 ', 6-diamidino-2-phenylindole), fluorescein, Hoechst3325S, 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 may be used. Isotopes can also be useful in detection methods, and their moieties and assays are well known in the art.

The detectable substance produces a detectable signal, which is subsequently detected and preferably quantified. The detectable signal may be, for example, a spectrophotometer for detecting color from a chromogenic substrate, a radiation counter for detecting radiation (such as a gamma counter for ¹²⁵ I detection), or in the presence of light of a particular wavelength. Can be analyzed using a fluorometer for detecting fluorescence. When using an enzyme-linked assay, quantitative analysis of the detectable signal can be performed using a spectrophotometer.

G4. Kits The invention also provides kits based on a range of biomarkers, including diagnostic, prognostic, and predictive therapeutic kits. The biomarker kit will typically include one or more of the binding constructs useful for detecting the biomarkers taught herein. Kits related to β2GPI biomarkers will generally include at least a first β2GPI binding construct, such as an anti-β2GPI antibody, PS, and a PS targeting antibody (such as bavituximab).

  Other kits comprise a binding construct for biomarker detection and at least a first therapeutic agent for use in treating a selected patient, eg, a PS targeting antibody such as bavituximab or 1N11, or an immunoconjugate thereof. Including both. Such a kit may further comprise at least a second or third different therapeutic agent for use in combination therapy with a PS targeting antibody. For example, one or more chemotherapeutic, radiotherapeutic, antiangiogenic, immunotherapeutic, and / or antiviral agents.

  Generally, the kit will contain the components described in at least the first suitable container (or container means). The container generally includes at least one vial, test tube, flask, bottle, syringe, or other container or container means into which the desired drug is placed, preferably suitably aliquoted. Kits also typically include means for securely enclosing individual vials or the like for delivery, e.g., injection or blow molded plastic containers for holding and holding desired vials and other equipment. including.

  The components of the kit may be contained in either an aqueous medium or lyophilized form. When the reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. The solvent may also be provided in another container in the kit. Any therapeutic component is preferably in a pharmaceutically acceptable formulation or is ready to be reconstituted in situ. The kit may also include a means for administering the therapeutic agent to the animal or patient, such as one or more needles or syringes, or eye drops, pipettes, or injecting the formulation into the animal or applying to the affected area of the body. May contain other similar devices.

  The kit will preferably have a different container for each desired component or agent, especially for biomarker detection and diagnostic components. However, for use in combination therapy, the kit may comprise one container containing the two or more therapeutic agents pre-mixed in molar equivalent combinations or with one component over the other. May be included. The kit may include a fully conjugated form of the pre-labeled antibody, or a separate label moiety conjugated by the kit user, preferably with instructions for binding. For immunodetection, one or more components, such as PS, may already be bound to a solid support, such as a well in a microtiter plate.

  The kit will also preferably include written or electronic instructions for use in quantification, preclinical, clinical, and / or veterinary embodiments, including, for example, use in combination therapy. Would. Being based on biomarkers, the kit is preferably controlled with or without labels, such as suitably aliquoted biological compositions, so that they can be used to prepare a standard curve for the detection assay. Will further contain the agent.

G5. Chip and nanoassay formats Solid-phase and ELISA-type biomarker assays, including total and / or functional β2GPI, can be automated or robotized as needed to simultaneously detect signals from multiple samples Can be. Although not in the context of the present invention, various such assay formats have generally been used to detect and quantify biomarkers. For example, nanoplasmonic sensors and microfluidic devices called "chips" have been described and used for on-chip isolation, quantification, and characterization of circulating biomarkers in cancer patients. Thus, the present assay can be accomplished using such microfluidics, chips, nanotechnology, and other streamlined and automated assays, while still maintaining the specificity of the invention.

  In addition to predictive methods and biomarker-guided therapies, the present invention also provides computer-based hardware and tests. Such computer-based embodiments of the present invention include interfaces configured to read one or more laboratory biomarker tests, including those of full and / or functional β2GPI, and such biomarker tests And preferably a computer that analyzes the data from the data set and compares the analyzed data with established data sets (including test and control data sets). Computer-implemented embodiments of the present invention preferably include memory storage, output capabilities, and instructions configured to derive a therapy based on the output.

H. The present invention provides animals, subjects, and patients to provide biomarker methods, compositions, and kits for selecting animals and humans and optimizing treatment with PS-targeting antibodies, such as bavituximab. The following guidelines for (including human patients) apply to biomarker detection and treatment of selected populations.

H1. Animals, Subjects, and Patients Human selection and treatment is the most preferred embodiment because the present invention is most directly applicable to human subjects and patients. Nevertheless, the commonality and conservation of biomarkers across species means that the invention is applicable to non-human animals. Among animals, mammals are preferred, most preferably household pets, racehorses, and animals that produce food for human consumption either directly (eg, meat) or indirectly (eg, milk). Although it is a valuable animal of value, laboratory animals are also included. Thus, the present invention includes clinical, veterinary, and research applications. Thus, in addition to humans, the invention includes dogs, cats, horses, cows, pigs, wild boars, sheep, goats, buffalo, bison, llama, deer, elk, and other large animals, as well as calves and lambs Applies to their young animals and to mice, rats, rabbits, guinea pigs, primates (such as monkeys), and other laboratory animals.

H2. Antibody Dose A “therapeutically effective” amount or dose of a PS-targeting antibody, such as bavituximab, is determined when administered to an animal, preferably a human patient, in need of such treatment, including when administered as part of a combination therapy. An amount or dose that produces a beneficial therapeutic effect. For example, a therapeutically effective anti-cancer dose is an amount that produces a beneficial anti-cancer effect when administered to an animal with cancer, preferably a human patient, including when administered as part of a combination cancer therapy. Dose. A therapeutically effective antiviral dose is an amount that produces a beneficial antiviral effect when administered to an animal, preferably a human patient, having a viral infection or disease, including when administered as part of a combination viral therapy. Or dose.

  “Beneficial anti-cancer effect” refers to any consistently detectable anti-tumor and anti-tumor activity, including tumor vasculature thrombosis and / or destruction, tumor necrosis, tumor regression, and tumor remission (including until healing). Including cancer effects. Clinical measures of beneficial anticancer effects include, for example, improvement in 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, where applicable, progression-free survival (PFS), disease-free survival (DFS), and overall survival (OS) in individual patients, patient populations, and subpopulations (middle of overall survival) Values (including mOS)).

  “Beneficial antiviral effect” refers to any effect, including inhibition of viral infection, replication, maturation, reproduction, and release, and / or ongoing infection or spread of further cells (host cells) or tissues. Includes consistently detectable antiviral effects. Clinical measures of beneficial antiviral effects include, for example, early virological response, reduced viral load and clearance of the virus, and amelioration of symptoms caused by viral infections.

  It is understood that a beneficial therapeutic effect, particularly an anti-cancer effect, may not be therapeutic, especially in the medium or long term, but that it does not abolish the therapeutic benefit. In this regard, but also generally, the "beneficial" therapeutic, anti-cancer, and anti-viral effects also include relatively and / or moderate therapeutic effects, but improvements in any one or more safety measures. Accompanied by Another consideration for a "beneficial" therapeutic effect is that PS-targeting antibodies, such as bavituximab, may make the disease or tumor more amenable to therapeutic treatment, so that subsequent treatments may result in an overall improved effect. The fact is that you get.

  Therapeutically effective doses of a PS-targeting antibody such as bavituximab or 1N11 can now be readily determined using a wide range of data, including data from animal models, but are described in particular herein and in the literature. Based on clinical studies such as those published. Generally, an effective dose range for a PS targeting antibody such as bavituximab administered intravenously (IV) and quoted in mg / kg is from about 0.1 to about 13-15, preferably from about 0.1 to about 6 to 10, preferably about 0.3 to about 6, more preferably about 0.5 to about 6, more preferably about 1 to about 6, more preferably about 0.5 to about 3, or about 3 To about 6, more preferably about 1 to about 3. Exemplary effective doses of a PS targeting antibody such as bavituximab administered intravenously and quoted in mg / kg are about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 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 about 3 mg / kg.

  The currently preferred dose of 3 mg / kg of bavituximab administered intravenously (IV) for clinical treatment, especially for all oncological indications, together with extensive safety data, as well as extensive preclinical and clinical data And especially based on the pharmacokinetic profile in humans (Example VI). Nevertheless, a range of doses has been shown to be effective, including clinical antiviral activity at 0.3 mg / kg (Example VI). In addition, 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, such extra-high doses are not recommended because bavituximab transiently reduced β2GPI in systemic circulation.

  Thus, it is clear from the extensive data that a dose of 3 mg / kg is preferred, but not limiting, of the present invention. Thus, it is understood that further variations in activity or optimal dosage ranges and dosages are encompassed by the present invention, in light of the parameters and detailed guidelines set forth herein. Thus, it is understood that lower doses may be more appropriate in combination with certain agents, and that higher doses may still be acceptable, especially when treating usually fatal diseases.

  In administering a PS targeting antibody such as bavituximab, a pharmaceutically acceptable composition (according to FDA standards of sterility, pyrogenicity, purity, and general safety) is administered systemically to an animal or patient. Intravenous injection is generally preferred, and continuous infusion over several hours is most preferred.

  In addition to varying the dose itself, the dosage regimen can also be adapted to optimize the treatment strategy, as is well known to those skilled in the art. Some variation in dosage and treatment regimens may be required depending on the condition of the subject being treated. The attending physician (s) will be able to determine the appropriate treatment for the individual subject in light of the present disclosure. Such optimizations and adjustments are routinely performed in the art and by no means reflect an excessive amount of experimentation.

H3. Supplemental Therapy with β2GPI Total β2GPI or functionality in using pre-treatment β2GPI levels, either alone or as part of a multiple biomarker selection, as a biomarker of response to PS targeting antibodies (such as bavituximab) Regardless of whether β2GPI is measured, the method selects only a subset of patients for treatment.

  Thus, another embodiment of the present application is to restore any non-selected patients to therapeutic eligibility by co-administering β2GPI to those patients with a PS targeting antibody such as bavituximab. In this way, the entire population can be treated with PS targeting antibodies. For example, in selecting a patient for treatment based on a pre-treatment level of functional β2GPI of 200 μg / ml or more, in combination with a functional β2GPI sufficient to restore β2GPI levels to at least about 200 μg / ml. Co-administration of bavituximab can return patients with pre-treatment levels of functional β2GPI to 150 μg / ml to a treatable group. Samples are applied to any pre-treatment levels of β2GPI used for biomarker analysis.

I. The first (and most important) indication of treatment is cancer (section K), especially solid tumors, since PS targeting antibodies such as bavituximab specifically target PS And their metastases, but humoral tumors (such as leukemia) and Hodgkin lymphoma are also indications.

  In normal and healthy cells, PS is maintained inside the cell membrane and is not available for binding. Only diseased cells have PS exposed outside the cell membrane, especially those cells that are within the tumor microenvironment, but are dying, abnormal, improperly activated, infected, And the pathogenic organism itself. In cancer, PS exposure within the tumor microenvironment is "immunosuppressive," meaning that the body is unable to fight the cancer properly. By blocking PS, bavituximab counteracts PS-mediated immunosuppression and helps the body attack tumors.

  In cells that are within the tumor microenvironment, especially those that cover blood vessels in tumors (and in cells infected and in viruses), PS is a relatively stable marker, which makes it an ideal target for therapy. It means there is. In diseases where there is a lot of cell death, PS is also exposed to the outside of cells, which indicates that bavituximab can be used in a variety of diseases where increased or inappropriate cell death occurs (such conditions include, for example, , Including cancer and heart attacks), and especially also for "imaging" (ie, in vivo diagnosis) (see below for imaging).

  The major pathogen that externalizes PS to host cells is the virus (Section J). Indeed, the role of PS and PS receptor as enhancers of enveloped virus entry and infection is now well documented and applies to a wide range of viruses. Furthermore, the relationship between PS and virus is not limited to enveloped viruses, but extends to non-enveloped viruses. In particular, it is known that "PS lipid vesicles" released from virus-infected cells allow for efficient mass infection of enteroviruses (Chen et al., 2015).

  In addition to cancer and viral infections, a wide range of diseases and pathogenic infections push PS out of its internal location within healthy cells and expose it to the outside of the cell, which is a PS-targeting antibody such as bavituximab Can be localized to their cells and pathogens and exert a beneficial effect. Taken together, these are "diseases and disorders where PS is a marker."

  Notable diseases and disorders for which PS is a marker, other than cancer, viral and pathogenic infections, are those with (coagulable) prothrombotic blood vessels and those involved in abnormal angiogenesis And diseases involving abnormal vasculature (blood vessels). Angiogenesis is the process by which new blood vessels are formed from existing blood vessels, and the development of new blood vessels begins with the formation of endothelial blasts that require PS (Weihua et al., 2005). Abnormal angiogenesis has been implicated in many diseases, especially cancer. In view of their abnormal vasculature, PS-targeting antibodies such as bavituximab are useful for benign (as opposed to malignant) tumors such as benign prostatic hyperplasia (BPH), acoustic nerve tumors, neurofibromas, trachoma, Granulomas (including purulent granulomas and sarcoidosis (sarcoids)), meningioma, angiomas, hemangiomas, and hemangiomatosis, a systemic hemangioma, can be treated.

  Conditions directly related to abnormal vasculature that can be treated with PS-targeted antibodies such as bavituximab include restenosis after vascular angioplasty, venous occlusion, arterial occlusion, and obstructive carotid or occlusive disease. Including vascular restenosis (narrowing of blood vessels); Behcet's disease (also eye disease), polyarteritis nodosa (nodular panarteritis or PAN), and Wegener's granulomatosis (WG) or sarcoidosis (polyangiitis) Vasculitis (a disorder in which inflammation destroys blood vessels); arteriovenous malformations (AVM) and arteriovenous fistulas; epistaxis (nasal bleeding); vascular adhesions; No.

  PS-targeting antibodies, such as bavituximab, have been implicated in joint diseases such as rheumatoid arthritis and osteoarthritis, synovitis, hemophilic joints, and Paget's disease due to their association with abnormal vasculature; psoriasis Dermatitis, scleroderma (systemic sclerosis or CREST syndrome), elastic fibrous pseudoxanthoma (PXE, known as Gronnblad-Strandberg syndrome), rosacea, Stevens-Johnson syndrome or Stevens-Johnson disease ( PXE, rosacea, and Stevens-Johnson syndrome are also eye diseases) Skin diseases such as pemphigus, hypertrophic scars, and keloids; Osler-Weber (or Osler-Randue-Weber) syndrome or Osler-Weber ( Or Osler-Lundue-Weber disease (hereditary hemorrhagic) Treetops vascular dilatation, it can be treated clinically important diseases including also known) as HHT.

  A particularly important example of a disease involving an abnormal vasculature that is treated with a PS-targeting antibody such as bavituximab is an ocular neovascular disease. These diseases are characterized by the invasion of new blood vessels into structures of the eye, such as the retina, choroid, and / or cornea. They are the most common cause of blindness and are involved in about 20 eye diseases. The most common ocular neovascular diseases are (proliferative) diabetic retinopathy, macular degeneration, including age-related macular degeneration (AMD), retinopathy of prematurity (ROP or Terry syndrome, formerly posterior lens fibrosis) Disease, known as RLF), neovascular glaucoma, corneal transplant neovascularization, and post-corneal transplant rejection. Choroidal neovascularization (CNV) accounts for 90% of the cases of severe visual loss in patients with advanced AMD and has been effectively treated with PS targeting antibodies, including both direct and indirect PS targeting antibodies (Li et al., 2015).

  Other diseases associated with retinal / choroidal neovascularization that can be treated with PS-targeting antibodies such as bavituximab include syphilitic eye infections, mycobacterial eye infections, and / or other diseases that cause retinitis or choroiditis. Ocular infections of the eyes; uveitis including vititis and peripheral uveitis (iridocyclitis); Ehls disease, putative ocular histoplasmosis syndrome (POHS), best disease (yolk-like macular degeneration), Stargardt disease , Eye trauma, and post-laser complications.

  Additional diseases particularly associated with corneal neovascularization that can be treated with PS-targeted antibodies such as bavituximab include atopic keratitis, superior limbal keratoconjunctivitis, dry pterygium keratitis, and marginal epidermis detachment. All forms of keratoconjunctivitis, including keratitis (only the cornea is inflamed) and conjunctivitis (only the conjunctiva is inflamed); phylectenulosis; Mohren's ulcer; chemical burn, bacterial ulcer, fungal ulcer, herpes Infections, as well as ocular trauma and excessive wearing of contact lenses.

  Other eye diseases that can be treated with PS-targeting antibodies, such as bavituximab, whether scleritis, rubeosis (angiogenesis of the iris), angle neovascularization (NVA), and whether related to diabetes Diseases caused by abnormal growth of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy (PVR).

  Since the formation of endothelial blasts requires PS, the development of new blood vessels also requires PS (Weihua et al., 2005). This process is also involved in certain normal physiological events, especially wound healing and regeneration, and is important in ovulation and implantation of the blastula after fertilization. Therefore, prevention of this process using bavituximab will induce amenorrhea (absence of menstrual periods in women of reproductive age), block ovulation and / or prevent implantation by blastula, ie use as a contraceptive can do. In wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures, and adhesions are a frequent complication of surgery, which can lead to problems such as small bowel obstruction. These can also be treated with PS targeting antibodies such as Bavituximab.

  Chronic inflammation also involves abnormal and pathological vasculature. In particular, 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 tissue. Therefore, those diseases can also be treated with PS-targeting antibodies, such as bavituximab.

  Several other diseases and disorders are known where host cells expose PS and / or PS-positive extracellular microvesicles and exosomes have been demonstrated. For example, in sickle cell disease (also called sickle cell anemia) and sickle cell crisis, 30-40% of red blood cells age prematurely and are PS-positive ("sickle cell"), In healthy people this is only about 1%. PS-positive sickle cells remain circulating and adhere to the endothelium, and their exposed PS acts as a platform for the initiation of the coagulation cascade responsible for clot growth (Kennedy et al., 2015).

  PS is also expressed in atherosclerosis, and PS-positive extracellular microvesicles are released from atherosclerotic plaques (Mallat et al., 1999). Plaques formed within the lumen of blood vessels that are PS positive have also been shown to have angiogenic stimulatory activity. There is special evidence for the pathophysiological significance of markers of angiogenesis, such as VEGF, in the progression of human coronary atherosclerosis and in the recanalization process in obstructive coronary artery disease. Thus, PS-targeted antibodies, such as bavituximab, provide an effective treatment for atherosclerosis and occlusive coronary artery disease.

  Both type 1 and type 2 diabetic patients have PS-positive extracellular microvesicles, as indicated by annexin V positivity (Sabatier et al., 2002). In Alzheimer's disease, brain exosomes contain PS and the amyloid β-peptide (Aβ), the pathogen of the disease (Yuyama et al., 2012). PS and PS-positive extracellular microvesicles are also involved in sepsis (septic shock), which are markers and mediators of sepsis-induced microvascular dysfunction and immunosuppression (Souza et al., 2015).

  Antiphospholipid antibody syndrome (APS) and systemic lupus erythematosus (SLE or lupus), autoimmune disorders in which antibodies are produced against the body's own phospholipids, include abortion and thrombocytopenia (low platelet count) Associated with coagulation disorders. Thus, the antiphospholipid antibodies in these patients are pathogenic antibodies that cause thrombosis. However, PS targeting antibodies such as Bavituximab target PS without exhibiting any such side effects. Thus, bavituximab can also treat antiphospholipid antibody syndrome, related diseases, and complications thereof. In particular, bavituximab may antagonize or compete with pathogenic antibodies in APS patients, thus rejecting pathogenic antibodies from their phospholipid-protein targets in the body.

  Regarding pathogenic infectious diseases, for example, Leishmania amazonensis (Zandbergen et al., 2006; Wanderley et al., 2009; Wanderley et al., 2013), which is a parasitic protozoan that causes leishmaniasis, Plasmodium falciparum, E. sampa, and Pat. ), And intracellular parasites such as Trypanosoma cruzi, a parasite that causes trypanosomiasis (DaMatta et al., 2007), all result in PS exposure. Similarly, similar to Toxoplasma gondii, which causes toxoplasmosis (Seabra et al., 2004), Schistosoma, a parasitic flatworm that causes schistosomiasis, also exposes PS (van der Kleij et al., 2002).

  PS exposure has also been shown on the extracellular surface following infection with intracellular bacterial pathogens such as Yersinia pestis and Francisella tularensis causing plague and tularemia, respectively (Lonsdale et al., 2011). Listeria monocytogenes, which causes listeriosis, also promotes the release of membrane-derived vesicles with extracellular surface PS from infected host cells (Czuczman et al., 2014). Similarly, endothelial cells infected with Neisseria meningitidis, a pathogen causing meningitis, exhibit PS translocation to the cell surface (Schubert-Unkmair et al., 2007). Infection with Mycobacterium tuberculosis which replicates intracellularly in macrophages and causes tuberculosis (TB) has been linked to PS externalization in neutrophils of tuberculosis lesions (Francis et al., 2014). Similarly, Legionella pneumophila, a facultative intracellular parasite that causes legionellosis, induces PS externalization in human monocytes (Hagele et al., 1998).

  Thus, PS externalization common to the facultative intracellular parasites detailed above causes brucellosis, and other diseases such as Brucella and Salmonella, respectively, which cause diseases such as typhoid, paratyphoid, and food poisoning. Can occur for such pathogens. This has also been demonstrated for infections by obligate intracellular parasites such as Chlamydia spp. That cause sexually transmitted chlamydial infections, and PS externalization is important for pathogenesis and Shown on cells, endothelial cells, granulocyte cells, and monocyte cells (Goth & Stephens, 2001). Chlamydia trachomatis, which causes trachoma, can also be treated (see also above).

  Indeed, PS externalization on host cells is a currently recognized phenomenon that responds to infection with various bacteria and pathogens (Wandler et al., 2010). This further includes Helicobacter pylori, which invades gastric epithelial cells (Petersen & Krogfeld, 2003) and causes gastric ulcers. H. When pylori has direct contact with gastric epithelial cells, it induces PS externalization of the host plasma membrane to the outer lobule (Murata-Kamiya et al., 2010). PS is also present in Treponema pallidum, which causes syphilis. Bartonellosis, a bacterial infection found in South America, can be treated with bavituximab, especially when bartonellosis leads to a chronic phase characterized by the proliferation of vascular endothelial cells, which is evident in cancer treatment. This is because one of the indicated mechanisms of action of bavituximab is to destroy vascular endothelial cells.

  For in vivo diagnostics, PS-targeting antibodies such as bavituximab can be used to image any of the aforementioned diseases, disorders, and infections, and most preferably to image vascularized tumors (Jennewein et al., 2008; Marconescu & Thorpe, 2008; Saha et al., 2010; Staffford & Thorpe, 2011; Gong et al., 2013; Staffford et al., 2013; U.S. Patent No. 7,790,860). Bavituximab may also be used for deep vein thrombosis, pulmonary embolism, myocardial infarction, atrial fibrillation, cardiovascular material problems, stroke (cerebrovascular disorder (CVA) or cerebral vascular stroke (CVI), PS-targeting antibodies, such as bavituximab, can also be used for imaging of nearby vascular thrombosis, for example, in conditions such as abscesses, restenosis, arthritis, as well as arterial thrombosis, coronary thrombosis, venous thrombosis. And activated platelets in haemostatic disorders such as cerebral thrombosis.

  Thus, PS-targeting antibodies such as bavituximab are suitable for treating and / or diagnosing all of the above diseases and disorders for which PS is a proven marker.

J. Treatment of Viral Infections The primary pathogen that externalizes PS to host cells is the virus. The presence of PS has been demonstrated on the surface of the virus and cells infected with the virus, and / or includes the following: (Tables 2A and 2 of US Patent Application No. 14 / 634,607, filed February 27, 2015, and PCT Patent Application No. PCT / US15 / 18183, respectively). See also Table 2B). In addition, data has been presented demonstrating that such PS exposure on the virus and cells infected with the virus is not merely accidental, but has an important role in viral infections (US Patent Application See also Tables 2C and 2D of US Pat. No. 14,634,607, US Pat. No. 7,906,115, WO 2015 / 131153A1). This has been demonstrated both in vitro and in vivo by the use of PS targeting antibodies to inhibit infection by various viral families.

  The relationship between PS and viral infections is also now well documented in the literature (eg, US Pat. No. 7,906,115; Soares et al., 2008; Mercer and Helenius, 2008; Moody et al., 2010; Morizono et al., 2011; Meertens et al., 2012; Best, 2013; Bhattacharya et al., 2013; Jemielity et al., 2013; Moller-Tank & Maury, 2014; Birge et al., 2016). This includes the role of PS and PS receptor as enhancers of enveloped virus entry and infection (see, eg, Table 1 of Moller-Tank & Maury, 2014). The relationship between PS, viral infections, and extracellular microvesicles such as exosomes has also become increasingly clear in recent years (Mecks & Raab-Traub, 2011; Sims et al., 2014) and has been applied to a wide range of viruses. (E.g., Walker et al., 2009; Mecks et al., 2010; Izquierdo-Ueros et al., 2010; Mecks & Raab-Traub, 2011).

  Furthermore, the relationship between PS and virus is not limited to enveloped viruses, but extends to non-enveloped viruses (Clayson et al., 1989; Chen et al., 2015). In particular, the cover of a Cell paper by Chen et al., 2015, which shows "PS lipid vesicles" (essentially exosomes) and attaches data indicating that PS vesicles allow efficient mass infection of enteroviruses. Please refer to FIG. Without being bound by a particular mechanism, the following rationale explains that PS is involved in infections by both enveloped and non-enveloped viruses.

  All viruses regulate the timed excretion of mature virions from host cells to ensure successful infection of new host cells. Envelope viruses utilize the host cell plasma membrane to embed viral proteins that mediate efficient entry of progeny virions to the next host cell. PS is found outside of cells infected with the virus before virus release, and enveloped viruses incorporate PS into the viral envelope upon excretion from host cells.

  Viruses that do not incorporate the envelope into their mature virions leave the host cell by other mechanisms. Some strategies that non-enveloped viruses use to release new virions from cells include cell lysis, either directly by the host immune response to infected cells (T cells or macrophages), or It can be caused by host cell protein synthesis or direct viral activity on cellular structure. An example of a virus that alters cell structure and induces cell lysis is an adenovirus. Adenoviruses express several proteins later in the infection that alter the structural integrity of cells by disrupting the filament network and protein synthesis. Some non-enveloped viruses can release their progeny virus via a non-destructive mechanism without any cytopathic effects. Poliovirus induces cell lysis rapidly (about 8 hours), but it is also released from cells within PS lipid vesicles that can infect new host cells. Poliovirus particles within PS vesicles are more efficient at infecting HeLa cells and primary macrophages than virus particles removed from PS vesicles, and blockade of vesicles by Annexin V is in a dose-dependent manner Inhibit vesicles from infected cells, suggesting that PS lipids are cofactors for poliovirus infection. In addition to poliovirus, coxsackievirus B3 and rhinovirus particles are also released into PS lipid vesicles (Chen et al., 2015), which are released by enteroviruses to selectively release mature particles without lysing cells. The common mechanism used is shown.

  With respect to SV40, SV40 may also be released from cells in PS-lipid vesicles of the type described above. For example, it has been reported that release of SV40 particles from cells may be seen before induction of a cytopathic effect (Clayson et al., 1989). SV40 virions are also observed in smooth cytoplasmic vesicles 48 hours after infection, and the release of SV40 particles is a sodium ion permeable pore that blocks intracellular protein transport by blocking cation transport across lipid membranes. , Was inhibited by monensin.

Also, many viruses need to induce activation of host cells to create an environment for efficient replication. Cell activation by either viral or non-viral activators results in an increase in intracellular calcium (Ca ²⁺ ) that activates PS translocation. Thus, potential mechanisms of action of PS-targeting antibodies such as bavituximab include interference with their ability to mediate protein or viral release required for cell activation, reversal of PS-mediated immunosuppression, and immune clearance Clearance of infected cells or virus by mechanisms.

  The in vivo virus model demonstrates increased survival of animals infected with the virus treated with PS targeting antibodies. Potential mechanisms by which PS-targeting antibodies such as bavituximab have been shown to exert such antiviral properties include: 1) binding to viral particles, 2) binding to infected cells, and 3) virus. Inhibition of replication and 4) enhancement of the immune response by blocking immunosuppressive cell receptors that bind to PS. Data in the HIV-1 model demonstrates that virions produced by virus-infected macrophages have elevated levels of PS, which serve as cofactors for macrophage HIV-1 infection. Blocking PS on HIV-1 with a PS targeting antibody may prevent cell-cell interactions and block viral target cell fusion. The results also show that bavituximab binds to pitindevirus particles and that treatment of guinea pigs infected with pitindevirus enhances the development of both anti-pitinde antibodies and cellular responses.

  Overall, the treatment of all viral infections, including enveloped and non-enveloped viruses, using PS-targeting antibodies, such as bavituximab, is described in US Pat. No. 7,611,704, which supplements this disclosure with respect to such treatment. Taught in U.S. Patent No. 7,906,115. In particular, Table H, Table J, and Table G of these patents describe the virus in animals and humans, along with common antiviral drugs (Table G) that can be used in combination therapy with PS targeting antibodies such as Bavituximab. FIG. 5 illustrates the treatment of infections and related diseases (Table H, Table J).

K. Treatment of Cancer The broad section of this application relates to treating tumors and cancer using PS-targeting antibodies such as Bavituximab. Treatment of benign tumors such as acoustic neuromas, acoustic tumors, neurofibromas, trachoma, purulent granulomas and BPH are included. The treatment of malignancies is preferred. As used herein, “tumor (s) and cancer (s)” are intended to indicate a malignant tumor, unless expressly stated otherwise.

  Treatment of blood-derived tumors such as leukemias and lymphomas, as well as various acute or chronic neoplastic diseases of the bone marrow are included. Preferably, the tumor to be treated is a solid tumor or an angiogenic tumor, including tumors in which angiogenesis is active and tumors having prothrombotic blood vessels. “Solid” and “angiogenic” tumors are tumors that have a vascular component, ie, require tumor blood vessels to provide oxygen and nutrients to tumor cells.

  Breast, ovarian, breast, lung, liver (hepatocellular, HCC), colon, colorectal, rectal, prostate, pancreatic, brain cancer, whether primary or metastatic (Gliomas and glioblastomas), cervical cancer, uterine cancer, endometrial cancer, head and neck cancer, parotid cancer, esophageal cancer, larynx cancer, thyroid cancer, gastrointestinal cancer, gastric cancer, kidney cancer (kidney) Cell carcinoma (RCC), biliary tract, bladder, testis, and melanoma, Merkel cell carcinoma, and hematological malignancies, as well as cell tumors (squamous and non-squamous cell carcinomas, small cell carcinomas and non-small cell carcinomas) Cancers), adenocarcinoma, and all cancers, exemplified by other cancers, including neuroblastoma. In certain embodiments, the invention applies particularly to non-small cell lung cancer (NSCLC) or breast, pancreatic, liver, kidney, rectal, or ovarian, or melanoma. In particular, the invention applies to NSCLC, such as non-squamous NSCLC.

  In addition to the published literature, the treatment of all cancers using PS-targeting antibodies such as bavituximab is taught in several US patents. For example, U.S. Patent Nos. 6,406,693, 7,422,738, 8,486,391, 7,247,303, and 7,572,448. , All of which supplement this disclosure with respect to such treatment. See also the discussion above regarding therapeutically effective anti-cancer doses (Section H2). Because the mechanism of action of PS-targeting antibodies such as bavituximab is substantially or completely the same in all solid tumors, the present invention It is understood that it is widely applicable to the treatment of solid tumors.

L. Combination Therapy Many sections of this application, the published literature, and several US patents also relate to treating cancer using PS-targeting antibodies such as bavituximab in combination therapy (eg, US Pat. U.S. Patent No. 8,486,391; U.S. Patent No. 7,572,448).

  Thus, the biomarkers and methods of treatment can be used in conjunction with any other method commonly used in the treatment of certain diseases or disorders presented by animals or patients, particularly cancer and viral infections and viral diseases. Unless the given therapeutic approach itself is known to be detrimental to the patient's condition and does not significantly counteract PS-targeted antibody therapy, it is contemplated to use it in conjunction with the present invention. Combination therapies for non-malignant diseases are also contemplated.

  For the treatment of cancer, the present invention may be used in conjunction with classical approaches such as surgery, chemotherapy, radiation therapy, cytokine therapy, anti-angiogenesis, and newer approaches such as immuno-oncology (IO) agents. be able to. Accordingly, the present invention relates to the use of a PS-targeting antibody, such as bavituximab, in conjunction with, prior to, or subsequent to surgery or radiation therapy, or a conventional chemotherapeutic or radiotherapeutic agent, cytokine, anti-angiogenic agent. Biomarkers and combination therapies that are administered to a patient before or after with an apoptosis-inducing agent, targeted therapies, IO agents and the like.

  For surgery, any surgical intervention may be performed in conjunction with the present invention. In the context of radiation therapy, any mechanism for inducing DNA damage locally in tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, and even electron emission is contemplated. Directed delivery of radioisotopes to tumor cells is also contemplated, and may be used in connection with targeting antibodies or other targeting means.

  The general use of substance combinations in the treatment of cancer is well known. When one or more agents are used in combination with a PS-targeting antibody, such as bavituximab, the result of the combination need not be an additive effect observed when each treatment is performed separately. At least an additive effect is generally desirable, but any increase in therapeutic effect or benefit (eg, reduction of side effects) over one of the monotherapy would be worthwhile. Also, it is not necessary that the combination treatment exhibit a synergistic effect, but this is possible and advantageous.

  As used herein, the “primary therapeutic agent” or “first anticancer agent” used in the present invention is a PS targeting antibody such as Bavituximab. As used herein, a "secondary or tertiary therapeutic agent" or "at least a second or third anticancer agent" is a second or third different therapeutic, anticancer, or antiviral agent, i. A therapeutic agent "other than" a therapeutic agent, an anticancer agent, or an antiviral agent. In the combination therapy of the invention, any second or third therapeutic agent can be used. Also, a second or tertiary therapeutic agent, a "second or third anti-cancer agent", or a "second or third anti-viral agent" may have an additive effect, It can be selected for the purpose of achieving an effect that exceeds the effect, and potentially a synergistic effect.

  To perform combination therapy, anti-tumor therapy, or anti-viral therapy, animals or patients can be treated with a PS-targeted antibody, such as bavituximab, in an animal or patient with their combined therapeutic, anti-cancer, or anti-viral effects. It only needs to be administered in combination with another, ie, a second or third, different therapeutic, anti-cancer or anti-viral agent, in a manner effective to effect its action. Thus, the agents exert their combined therapeutic effects in providing their presence in the disease site (eg, tumor, tumor environment, or tumor microenvironment) and / or in an animal or patient. In particular, they are preferably provided in an amount and for an effective period effective to exert their combined therapeutic effect on the animal or patient's immune system. To this end, the primary therapeutic agent and the second or third different therapeutic agent are substantially combined in a single composition or as two or three different compositions using different routes of administration. At the same time.

  Alternatively, a PS targeting antibody such as bavituximab may precede or follow a second or third different therapeutic, anti-cancer or anti-viral agent, for example, by intervals ranging from minutes to weeks or months. You may. In certain embodiments where the primary therapeutic agent and the second or third different therapeutic agent are separately applied to the animal or patient, the time between each delivery may be such that each agent can still exert an advantageous combined effect. Ensure that no dead periods exist. From standard practice, including clinical experience to date with bavituximab, one or two weeks is not a period of inactivity between administration of bavituximab and administration of a second or third different therapeutic agent. In fact, an interval of about one week may be preferred.

  In addition, one preferred combination tumor therapy involves administering to a animal or patient at substantially the same time, or preferably at intervals of several weeks, a PS targeting antibody such as bavituximab in combination with a second, different anticancer agent, Administering a third different anti-cancer agent at a subsequent time and continuing regular administration of the third different anti-cancer agent over a subsequent effective time period (eg, months).

  Secondary or tertiary therapeutic agents for separately timed combination therapies may be selected based on certain criteria, including those discussed herein and known in the art. However, although it may be preferable to select one or more second or third different therapeutic agents for subsequent administration, this does not preclude their use in substantially simultaneous administration, if necessary.

  With respect to cancer, a second or third different anti-cancer agent selected to be administered “prior to” the first therapeutic agent and designed to achieve an increased and potentially synergistic effect is within the tumor microenvironment. And an agent that induces the expression of PS. For example, it stimulates localized calcium production, activates membrane transporters that move PS to the outer surface of the plasma membrane, damages tumor endothelium, causes pre-apoptotic changes, and / or causes tumor endothelium or tumor Agents that induce apoptosis in cells will generally result in increased PS expression. Examples of such agents are docetaxel and paclitaxel. The PS can then be targeted using a PS targeting antibody such as bavituximab, thereby amplifying the overall therapeutic effect and also via host effectors (complement, ADCC, antibody-mediated phagocytosis, CDC) Increase the attacks made.

  Drugs that have selectivity for angiogenesis, remodeling, or activated endothelial cells that are present in tumor blood vessels but not in normal quiescent blood vessels are also exposed to PS in the tumor microenvironment. Can be used to selectively cause Examples of such agents are combretastatin and docetaxel. This will also result in increased antibody binding and enhanced initiation of host effector mechanisms.

  A second or third different anti-cancer agent that is selected for "subsequent" administration to the first therapeutic agent and designed to achieve an increased and potentially synergistic effect is the effect of the first therapeutic agent. Includes drugs that benefit from PS targeting antibodies, such as bavituximab, cause tumor necrosis. Thus, a second or third different anti-cancer agent that is effective for subsequent administration includes anti-angiogenic agents that inhibit metastasis, agents that target necrotic tumor cells (specific for intracellular antigens made available from malignant cells in vivo) Chemotherapeutic agents and anti-tumor cell immune complexes that attack any peripherally viable tumor cells (US Patent Nos. 5,019,368 and 5,882,626). . As described below, the currently most preferred second or third different anti-cancer agent for subsequent administration to a PS targeting antibody such as Bavituximab is an immune checkpoint inhibitor.

  Depending on the condition, it may be desirable to extend the treatment period significantly, for several days (2, 3, 4, 5, 6, or 7) or weeks (1, 2, 3, 4, 5, 6, 7, or 8) or several months (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration. This means that one treatment is intended to substantially destroy the tumor while another is preventing micrometastasis or tumor regrowth and / or stimulating or assisting the host response to the tumor (anti-angiogenic agents or immune checks). (Point inhibitors). However, to allow for effective wound healing, anti-angiogenic agents should be administered at a careful time after surgery. Thereafter, the anti-angiogenic agent may be administered for the life of the patient.

  It is also envisioned to utilize multiple doses of the primary therapeutic agent or any of the second or third different therapeutic agents. The first therapeutic agent and the second or third different therapeutic agent may be administered interchangeably every other day or every other week, or a series of one drug treatment is given, followed by a series of other treatments. (S) may be provided. In any case, to achieve a therapeutic effect using the combination therapy, regardless of the time of administration, it is necessary to deliver two or more drugs in a combined amount effective to achieve the therapeutic effect. is there.

L1. Chemotherapy Whether administered substantially simultaneously or sequentially, a PS targeting antibody such as bavituximab can be administered in combination with one or more chemotherapeutic agents or agents. Chemotherapeutic agents can kill proliferating tumor cells and enhance the necrotic areas created by the entire treatment. Therefore, the drug can enhance the action of the primary therapeutic agent of the present invention.

  Most cancer chemotherapeutics are selective for dividing oxygenating cells. They have advantages in combination therapy because chemotherapeutic drugs act on different targets than primary therapeutics, resulting in more complete antitumor effects. For example, chemotherapeutic agents are selectively active against rapidly dividing oxygenated tumor cells in the tumor periphery. Anti-angiogenic drugs that are selective for hyperoxygenated angiogenesis in the periphery of the tumor would also be effective in the combination.

  By inducing thrombus formation in tumor blood vessels, the first-line therapeutics of the invention can also enhance the effects of chemotherapeutic drugs by retaining or trapping the drug within the tumor. Thus, the chemotherapeutic agent is retained in the tumor while the remaining drug is removed from the body. Thus, tumor cells are exposed to higher concentrations of the drug for longer periods of time. This confinement of the drug within the tumor allows for a reduced dose of the drug, making the treatment safer and more effective.

  Additional drugs for use in combination in the present invention are required to achieve their anti-tumor effect, as they act on cells "sensitized" to the drug by the action of the primary therapeutic agent The dose of the second drug is reduced. For example, this can occur if the tumor component of the action of the second drug is exerted on tumor blood vessels and the antibody or agent of the invention sensitizes cells to the drug. This is also the case when the primary therapeutic agent of the present invention sensitizes tumor cells to a second drug, either directly or through stimulation of cytokine release.

  Other second or third anti-cancer agents suitable for combination therapy are those that enhance the activity of host effector cells, for example, by selectively inhibiting the activity of immunosuppressive components of the immune system. Such agents can cause the primary therapeutics of the present invention to stimulate aggression by effector cells as part of their mechanism to function more aggressively. Examples of such agents are docetaxel and immune checkpoint inhibitors.

  An understanding of the exact mechanism (s) of action of the primary therapeutic agent is not necessary for the practice of the treatments of the invention, but may be combined in the present invention using data and reasonable inferences regarding such mechanisms. A particular second or third anti-cancer agent can be selected for the treatment. In turn, the efficacy of the selected combination therapy supports the original data and the proposed mechanism of action, and also results in a preferred category of second or third anti-cancer agent for performing the combination therapy.

  Drugs that induce apoptosis may be used in combination therapy. For example, docetaxel induces apoptosis by binding to microtubules and disrupting cell mitosis, thus inducing PS exposure (Hottchkiss et al., 2002). Treatment of endothelial cells and tumor cells overlying tumor vessels with subclinical concentrations of docetaxel is known to induce cell surface PS expression.

  The anti-tumor effects of PS-targeting antibodies such as bavituximab include Fc domain-mediated increases in immune effector functions such as ADCC, CDC, stimulation of cytokine production, and combinations of such mechanisms. This is also related to docetaxel, which has been shown in other studies that treatment of breast cancer patients with docetaxel resulted in increased serum IFNγ, IL-2, IL-6, and GM-CSF cytokine levels, and that natural killer ( NK) cells and lymphokine-activated killer cell (LAK) cells, thereby enhancing the anti-tumor immune response in these patients.

  Thus, docetaxel induces both PS expression and binding of the administered antibody, and also enhances the activity of immune effectors that mediate antitumor effects. Based on the foregoing considerations, the combination of antibody and docetaxel is also a preferred embodiment when used in conjunction with or following treatment with an immune checkpoint inhibitor, as described below. .

  Thus, docetaxel and other chemotherapeutic agents that induce apoptosis are certain preferred agents for use in the combination therapies of the invention. Combination with chemotherapeutic agents that induce apoptosis, such as docetaxel, synergistically attacks tumor vasculature endothelial cells and tumor cell compartments, resulting in significantly enhanced therapeutic efficacy as well as reduced toxicity. These combinations are contemplated for use in treating breast cancer, particularly metronomic chemotherapy using docetaxel and a PS targeting antibody.

Exemplary chemotherapeutic agents for combination therapy are described in U.S. Patent No. 7,572,448 and U.S. Patent No. 9,421,256 (e.g., U.S. Patent No. 7,572,448). Table D and Table C of US Patent No. 9,421,256), such as, for example, pemetrexed, temozolomide, tamoxifen, erlotinib, sunitinib, sorafenib, paclitaxel, carboplatin, gemcitabine, and docetaxel. For example, other therapeutic antibodies such as trastuzumab, rituximab, and bevacizumab can also be used. Each of those chemotherapeutic agents, antibodies, and other drugs known in the art are exemplary and not limiting. Variations in dosage may occur depending on the condition being treated. The treating physician will be able to determine the appropriate dose for the individual subject. In certain preferred embodiments, docetaxel is used 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 ² .

M. Combining Immunotherapy (IO) The challenge for effective immunotherapy is to overcome multiple pathways that inhibit natural 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 deplete activated tumor-specific T cells and attenuate their tumoricidal activity. PD-1 is absent on na ー ブ ve T cells, B cells, macrophages, DCs, and monocytes, but is highly expressed on their activated counterparts. In particular, tumors and associated bone marrow cells utilize the PD-1 pathway to generate innate and adaptive immune tolerance through up-regulation of PD-L1 expression. Mechanistic studies have shown that blocking these immune checkpoints is most effective when there is a new or existing anti-tumor immune response. Unfortunately, due to exposure of PS and other immunosuppressive factors that govern the tumor microenvironment, existing tumor-specific immune activities are limited in cancer patients.

  In several cancer types, a sustained anti-tumor immune response with drugs that block PD-1 signaling has been observed, but only a subset of patients are responding and consequently unsatisfactory important medical The need remains. In particular, patients expressing low levels of PD-1 and PD-L1 (biomarkers of immunosuppression and lack of T cell activation) in the tumor microenvironment appear to be less responsive to checkpoint blockade therapy. Observations of immune activation indicate that PS-targeting antibodies, such as bavituximab, can increase the proportion of patients who can benefit from anti-PD-1 / PD-L1 and other checkpoint therapies.

  Presented herein is clinical data demonstrating for the first time that human patients treated with bavituximab and immunotherapy have significant survival advantages. In particular, the results of Example XIX show that patients treated with bavituximab (and docetaxel) followed by subsequent immunotherapy ("SACT-IO") compared patients treated with placebo (docetaxel alone) followed by immunotherapy. Demonstrate that they have a statistically significant and better overall survival. Since the mOS of Bavituximab patients undergoing subsequent IO has not yet been reached, the prolongation of survival was statistically significant (p = 0.006) and even more impressive (Example XIX, FIG. 26, Table 16). Thus, bavituximab actually enhances the activity of immunotherapeutic agents in human patients.

  Thus, as exemplified by the data in Example XIX, an important embodiment of the present invention is the use of PS targeting antibodies, such as bavituximab, in combination with immunotherapeutic or immune tumor (IO) agents to treat cancer patients. Treatment. Exemplary immunotherapeutic agents for combination therapy are listed in Table C of Provisional Application No. 62 / 406,727, filed October 11, 2016, and in Provisional Application No. 62 / 406,727, filed April 3, 2017. No. 62 / 480,994 and provisional application No. 62 / 507,580 filed May 17, 2017, each of which is listed in Table D, including those with NK cell therapy and CAR-T therapy. Combinations are presently preferred.

  Particular preferred examples of IO agents are described in Provisional Application No. 62 / 480,994 filed on April 3, 2017 and Provisional Application No. 62 / 507,580 filed on May 17, 2017, respectively. Such as those listed in Table E of Approved in Clinical Therapy or Human Clinical Trials, preferably Late Clinical Trials. Illustrated by the details in Table E of each of provisional application No. 62 / 480,994 filed on April 3, 2017 and provisional application No. 62 / 507,580 filed on May 17, 2017. As such, dosages for use and indications for treatment are well known to those skilled in the art. For example, melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), classical Hodgkin's lymphoma, squamous cell carcinoma of the head and neck (head and neck cancer), bladder cancer, small cell lung cancer, brain cancer ( Malignant glioma, anaplastic astrocytoma (AA), and glioblastoma multiforme (GBM), hepatocellular carcinoma (HCC), esophageal cancer, gastric cancer, mesothelioma, and myeloma Is nivolumab at 240 mg or 3 mg / kg every 2 weeks; melanoma, NSCLC, classical Hodgkin's lymphoma, head and neck cancer, gastric cancer, breast cancer, bladder cancer, all solid tumors, colorectal cancer, RCC, multiple myeloma, esophagus 200 mg or 2 mg / kg pembrolizumab every 3 weeks for treatment of cancer and HCC; for treatment of bladder cancer, NSCLC, RCC, colorectal cancer, prostate cancer, melanoma, breast cancer, ovarian cancer, and small cell lung cancer 3 1200 mg atezolizumab per interval; 2 for the treatment of metastatic Merkel cell carcinoma, NSCLC, ovarian cancer, gastric cancer, bladder cancer, RCC, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, and head and neck cancer Averumab 10 mg weekly; and 10 mg / kg Durvalumab (MEDI 4736) every 2 weeks for treatment of NSCLC, head and neck, bladder, and small cell lung cancer.

  Other suitable IO agents are 3 mg / kg or 10 mg / kg ipilimumab every 3 weeks for treatment of unresectable or metastatic melanoma; 15 mg / kg tremelimumab every 3 months for treatment of melanoma; NSCLC REGN2810 for the treatment of melanoma; PDR001 for the treatment of melanoma; racotumomab for the treatment of NSCLC; MEDI0562 and GSK3174998 for the treatment of advanced solid tumors; ulelumab for the treatment of melanoma; utmilumab for the treatment of DLBCL; For tumor treatment, each of BMS-986016, LAG525, JNJ-6610588, TSR-022, MBG453, MEDI1873 and INCAGN01876.

  As directly supported by the data in Example XIX, a particularly preferred IO agent for combination therapy with a PS targeting antibody such as Bavituximab is a "checkpoint inhibitor", also referred to herein as an "immune checkpoint antibody". is there. Suitable "immune checkpoint antibodies" include activating immune checkpoints such as CD28, OX40, and / or GITR, agonist (activating) antibodies that bind to a receptor or molecule, and PD-1, PD-L1 , CTLA-4, TIM-3, and / or LAG-3. Such blocking antibodies are conventionally referred to as "immune checkpoint inhibitors", which are also used herein. Some such antibodies have also been approved as clinical treatments or late-stage clinical trials as provisional application Ser. No. 62 / 480,994, filed Apr. 3, 2017 and May 17, 2017. No. 62 / 507,580, filed in Table E.

  The currently most preferred example of an immune checkpoint antibody (immune checkpoint inhibitor) is "a blocking antibody that binds to CTLA-4, PD-1, or PD-L1". Some such blocking antibodies that bind to CTLA-4, PD-1, or PD-L1, and methods involving functional assays for their selection, preparation, and use, are described in Table B. In addition, it is well known to those skilled in the art. These include blocking antibodies against CTLA-4, such as ipilimumab and tremelimumab; blocking antibodies against PD-1, such as nivolumab (Brahmer et al., 2015), REGN2810, and pembrolizumab (Garon et al., 2015); Included are blocking antibodies to PD-L1 such as Fehrenbacher et al., 2016); as well as combinations of any one or more of such antibodies, known as "IO doublets." Of these, tremelimumab, nivolumab, durvalumab, and atezolizumab are presently preferred. The main US patents for tremelimumab, nivolumab, durvalumab, and atezolizumab are US Pat. No. 6,682,736, US Pat. No. 8,008,449, US Pat. No. 8,779,108, and US Pat. No. 8,217,149.

  In addition to Table B, other suitable examples of anti-CTLA-4 antibodies are those described in U.S. Patent No. 6,207,156, which is specifically derived from the deposited hybrid cells. An anti-CTLA-4 antibody comprising a CDR (CDR3, CDR2, or CDR1) selected from the defined antibodies.

  In addition to Table B, other suitable examples of anti-PD-L1 antibodies are described in U.S. Patent No. 8,168,179, which specifically discloses human anti-PD-L1, including a combination of chemotherapy. U.S. Patent No. 9,402,899, which specifically relates to tumors with antibodies to PD-L1, including chimeric, humanized, and human antibodies. And US Pat. No. 9,439,962, which specifically relates to the treatment of cancer with anti-PD-L1 antibodies and chemotherapy. These anti-PD-L1 antibody compositions and methods include those being developed by Ono Pharmaceuticals and coworkers.

  Additional suitable antibodies to PD-L1 are those of U.S. Patent Nos. 7,943,743, 9,580,505, and 9,580,507, and kits thereof (U.S. Patent No. , 580, 507), as well as nucleic acids encoding those antibodies (US Patent No. 8,383,796). Such antibodies bind to PD-L1 and compete with the reference antibody for binding, are defined by the VH and VL genes, or have heavy and light chain CDR3s of defined sequences (U.S. Pat. No. 7,943). 743) or heavy chain CDR3 (US Patent No. 8,383,796) or conservative modifications thereof, or have 90% or 95% sequence identity to a reference antibody. These anti-PD-L1 antibodies also include those with defined quantitative (including binding affinity) and qualitative properties, immunoconjugates, and bispecific antibodies. Such antibodies, as well as those having defined quantitative (including binding affinity) and qualitative properties (both in single-chain format and antibodies in the isolated CDRs), in enhancing the immune response (Including antibodies) (US Patent No. 9,102,725). As in U.S. Patent No. 9,102,725, enhancement of the immune response can be used to treat cancer or an infection, such as a pathogenic infection caused by a virus, bacterium, fungus, or parasite. . These anti-PD-L1 antibody compositions and methods include the product of BMS936559.

Further suitable antibodies to PD-L1 are those in US Patent Application No. 2016/0009805, which discloses specific epitopes on PD-L1, including antibodies of defined CDR sequences and competing antibodies. Antibodies, nucleic acids, vectors, host cells, immune complexes; detection, diagnostic, prognostic, and biomarker methods; and therapeutic methods.



* * *

  The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed in the following examples represent techniques that have been found by the inventor to work well in the practice of the present invention, and may therefore be considered to constitute a preferred mode for its practice. Should be understood by those skilled in the art. However, one of ordinary skill in the art, in light of the present disclosure, may make many changes and still achieve similar or similar results in the particular embodiments disclosed without departing from the spirit and scope of the invention. You should understand.

Example I
Generation of 3G4 Antibody This example describes the immunization protocol, generation, and initial characterization of a mouse PS targeting antibody called 3G4.

  In order to present anionic phospholipids, mainly PS, as a stronger immunogen to the immune system, they were formulated in the context of cells, especially as PS positive cells. PS exposed to the membrane, surrounded by other membrane components, has a better conformation for producing antibodies. The intent is to immunize immunocompetent animals with autologous cells that express PS, where the animals do not produce antibodies to all self-surface antigens, but instead have PS exposed to the membrane as an external element. Recognize.

Mouse endothelioma cells bEnd. 3 (immortalized mouse (BALB / c strain) endothelial cells) were cultured in 10% DMEM with 9 ml / 500 ml HEPES buffer in a 10% CO ₂ incubator. Until the desired number of cells is obtained, bEnd. Three cells were grown in T175 TC flasks. Typically, each flask at about 70-80% confluency has about 3 × 10 ⁶ cells, and each mouse has 1 × 10 ^{6 to} 20 × 10 ⁶ cells, up to 1 × 10 ⁷ cells are received.

bEnd. Three cells were treated with 50 μM to 200 μM hydrogen peroxide for 1 or 2 hours at 37 ° C. to expose anionic phospholipids, especially PS, before immunization. The H ₂ O ₂ stock was [9.8M], 30% by volume. This was diluted 1: 1000, after which 0.4 ml was added to a T175 TC flask with 40 ml of medium to give a final concentration of H ₂ O ₂ of 100 μM. Cells were maintained at 37 ° C. for 1 hour. For harvesting, the cells were washed three times with warm phosphate buffered saline (PBS) +10 mM EDTA in order to remove any BSA or serum proteins in the medium. Cells were removed by gentle trypsinization, washed, and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated, and the cells were resuspended in DMEM to the appropriate volume without additives (each mouse received approximately 1 x 10 ⁷ cells in 200 μl) and kept on ice.

  Cells treated in this way were injected intraperitoneally into BALB / c mice using a 1 ml syringe and a 23 gauge needle (200 μl of cell suspension). First, mice were immunized 3-7 times at 3-4 week intervals. Immune sera was collected by bleeding mice 10 days after each boost, starting with a second boost. Serum titers of PS antibodies were tested by ELISA.

  These immunizations with autologous PS-positive cells do not result in the non-restrictive production of autoantibodies, but rather the production of antibodies reactive with PS, mainly in combination with other anionic phospholipids. Was limited to production. Mice with very high titers of antibodies reactive with anionic phospholipids such as PS were obtained. The mice did not show any signs of toxicity.

  In a further immunization, various mice were treated with hydrogen peroxide treated bEnd. Three immunizations with 3 cells were performed and sera were tested 54 days after the first immunization. IgG antibodies reactive with PS in serum were detected with anti-mouse IgG, Fc-specific secondary antibodies (IgM antibodies in serum were detected with anti-mouse IgG mu-specific secondary antibodies). Using this immunization protocol, several effective antisera with IgG and IgM antibodies reactive with PS were obtained, of which antisera with IgG antibodies were generally more effective.

  Typically, fusions were performed to produce monoclonal antibodies when the desired antiserum to PS had an IgG titer of over 200,000, but the PC titer was below 50,000. . Hybrid cells were obtained by fusing splenocytes of the immunized animals with myeloma partner P3X63AG8.653 cells (ATCC, Rockville, MD).

  One important aspect of this technology for preparing monoclonal antibodies useful for treating tumors is a selection strategy involving screening to select antibodies that bind to anionic phospholipids but not to neutral phospholipids. Met. Another important aspect was to select antibodies that bind to the PS coated plate as strongly in the absence of serum as in the presence of serum. This was performed to eliminate antibodies that recognize complexes of PS and serum proteins, such as complexes of PS and β2GPI.

  A strategy for isolating monoclonal antibodies reactive with PS involved screening hybrid cell supernatants on PS coated plates using anti-mouse IgG, Fc gamma-specific secondary antibodies. First, screening was performed on four phospholipids (PS, phosphatidylserine; PE, phosphatidylethanolamine; CL, cardiolipin; and PC, phosphatidylcholine), and bEnd3 cells. Clones that were reactive with PC, a neutral phospholipid, were discarded, and clones that were not reactive with bEnd3 cells were similarly discarded. Clones with high binding to PS were selected. First, wells with PS-only reactivity or strong selectivity for PS were subcloned, and second, wells that exhibited PS reactivity in combination with binding to other anionic phospholipids.

  The isotype of each selected hybrid cell was determined. The production of IgG class antibodies is particularly desirable because they typically have higher affinity, lower clearance rates in vivo, and a number of advantages over IgM, including simplicity of purification, modification, and handling. Was done. Wells containing IgM or a mixture of different Igs were discarded or recloned to focus on wells with a homogeneous IgG isotype. Subcloning of highly positive clones was repeated 3-4 times.

Initially called "F3-G4", was named again as 3G4, was selected advantageous mouse IgG antibody (IgG ₃ kappa). The 3G4 antibody was tested for binding to PS in an ELISA in the presence and absence of serum and was initially characterized as "serum independent", that is, an antibody that binds PS in the absence of serum.

  The 3G4 antibody was studied using the following "standard ELISA" used to test binding to PS or other phospholipids. Aliquot a stock solution of phospholipid antigen (PS antigen, 25 mg of P-6641 in a 2.5 ml bottle, 10 mg / ml (solvent: 95: 5 chloroform: MeOH)) and store at −30 ° C. in an airtight container. A preferred 96-well plate is Dynatech U bottom Immulon 1 (from Dynatech Labs, catalog number 011-010-3550).

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 color development solution was 10 ml of 0.2 M Na ₂ PO ₄ , 10 ml of 0.1 M citric acid, one 10 mg OPD tablet, and 10 μL of hydrogen peroxide. Stop solution was _H 2 SO ₄ in 0.18 M.

  The protocol involved coating a 96-well plate with PS as follows: The PS stock solution was diluted to 10 μg / ml in n-hexane and mixed well. 50 μl was added to each well and evaporated for 1 hour. 200 μl of 10% serum (as blocking buffer) was added to each well, covered and maintained at room temperature for 2 hours or at 4 ° C. overnight. Plates were washed three times with PBS. Primary antibody (diluted in blocking buffer) was added and incubated at 37 ° C for 2 hours. Plates were washed three times with PBS. 100 μl of a secondary antibody (typically goat anti-mouse IgG-HRP) was added per well and incubated at 37 ° C. for 1 hour. Plates were washed three times with PBS. The ELISA was developed by adding 100 μl of color development solution to each of the wells and allowed to develop for 10 minutes, after which 100 μl of stop solution was added to each plate and the optical density was read at 490 nm.

  That the 3G4 antibody has an improved relative affinity for PS as compared to conventional antibodies, and that the 3G4 antibody has PS, CL, PI (phosphatidylinositol), PA (phosphatidic acid), and PG (phosphatidylglycerol) ). Consistent with a model for targeting PS differentially expressed in tumors, the 3G4 antibody did not react with neutral phospholipids, PC, and SM.

  Using standard protein A procedures, the 3G4 antibody was purified from cultured hybrid supernatants to a homogeneous appearance. Briefly, a sample containing 3G4 antibody at physiological pH was applied to a Protein A column and allowed to pass slowly to allow IgG to bind to immobilized Protein A. The column was washed with wash buffer to remove unbound serum components. The 3G4 antibody was eluted from the column using acidic elution buffer (about pH 2.8), after which the fraction containing the eluted antibody was neutralized or dialyzed back to physiological pH. In testing this highly purified 3G4 antibody for binding to PS in an ELISA performed in the absence of serum, it is still believed that 3G4 binds directly to PS, ie, without serum or serum proteins. Was.

Example II
Preclinical anti-tumor effects of 3G4 antibodies In this example, data is provided to illustrate early preclinical experience demonstrating some of the anti-tumor effects of 3G4 antibodies in syngeneic and xenogeneic tumor models.

Protocol for Animal Tumor Studies The effect of 3G4 was first tested in syngeneic and xenogeneic tumor models. A general protocol for animal tumor treatment studies is as follows.

  Animals were obtained from Charles Rivers Laboratories. Mice were 4-5 week old female C. leptum. B-17 SCID or Fox Chase SCID mice. Mice were housed in an autoclave cage by aseptic handling and fed sterile food and water. All procedures were performed in a laminar flow hood. Mice were acclimated for one week, after which the ears were tagged and blood samples (approximately 75-100 μl) were taken from the tail vein to check for leaks by ELISA. Mice that failed the leaky ELISA test were not used in the test procedure. Two to three days after ear tagging and blood sample removal, tumor cells were injected orthotopically into the mammary fat pad (MFP) or subcutaneously into the right flank.

In the orthotopic model, 1 × 10 ⁷ cells in 0.1 ml of DMEM were injected into the MFP of a typically anesthetized mouse. Mice were anesthetized by intraperitoneal injection of 0.075 ml of mouse cocktail. The mouse cocktail is 5 ml ketamine (100 mg / ml), 2.5 ml xylazine (20 mg / ml), 1 ml acepromazine (10 mg / ml), 11 ml sterile water. Dosage was 0.1 ml per 20-30 grams body weight via the intraperitoneal route over 30 minutes.

  After anesthesia of the mouse (measured by no response to pinch / foot pinch), the mouse is placed on its left side and the area just behind the head and around the right forearm / back is 70% And wiped with ethanol. A 2-3 mm incision was made immediately behind the right forearm (lateral chest) and a whitish fat pad appeared when the flap was raised. Using a 1 ml syringe and a 27 gauge needle, 0.1 ml of cells was injected into the fat pad, resulting in vesicles 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 woke up from anesthesia and was able to move. Post-operative health was determined and if any signs of distress were observed, the animals were given acetaminophen (0.24 mg / ml) + codeine (0.024 mg / ml) in drinking water. One week later, the wound clips were removed. This method was used to ensure that cells were correctly located at selected sites rather than subcutaneous regions. Tumors were approximately 200 [mu] l in volume (length x width x weight) for 14-15 days and the engraftment rate was essentially 100%.

In the subcutaneous model, mice were typically injected with 1 × 10 ⁷ cells in 0.2 ml. The mice were not anesthetized, but were tightly grasped and restrained, exposing the right flank. Injection of 1 × 10 ⁷ cells in 200 μl just below the mouse skin using a 1 ml syringe with a 23 gauge needle showed blebs. It was not uncommon to observe a small leak from the injection site. To reduce this leakage, a torsional movement was used when withdrawing the needle from the hypodermic injection. Tumor volume was measured by length x width x height.

  For the perfusion protocol, mice were injected intravenously with 1000 U of heparin in 0.2 ml of saline. The mice were then sedated by intraperitoneal injection of the mice with 0.1 ml of the mouse cocktail. After the mice are fully sedated (measured by no reflexes when pinching the toes / paws), the thoracic cavity is opened to expose the heart and lungs. A 30 gauge needle attached to the tubing and perfusion pump was inserted into the left ventricle. The right ventricle was cut out so that blood could drip. Physiological saline was pumped at a rate of 1 ml per minute for 12 minutes. At the end of the perfusion, the needle and tube were removed. Tissues were removed for further studies, either immunohistochemistry or pathology.

  Differences in tumor growth rates were tested for statistical significance using a non-parametric test (Mann-Whitney rank sum test).

B. Tumor treatment results In a syngeneic model, fibrosarcoma tumor cells of Meth A mice were used with BALB / c mice. In a heterologous model, human MDA-MB-231 breast tumor cells or MDA-MB-435 cells were seeded on the breast fat pad of SCID mice. In another heterologous model, cells were injected, by growing tumors to a size of 500 mm ³ than before treatment, were established large human Hodgkin's lymphoma L540 xenograft. (Referred to as BBG3, Babesia bovis mouse IgG ₃ kappa antibodies to the antigen, secreted from the hybrid cells obtained as 23.8.34.24 ATCC; HB-10113) control antibody in contrast to the tumor bearing mice (group 1 Per 8 to 10 animals) were injected intraperitoneally with 100 μg of the 3G4 antibody purified to homogeneity in appearance. The treatment was repeated three times a week. Animals were monitored twice or three times a week for tumor measurements.

  Growth of both syngeneic and xenogeneic tumors was effectively inhibited by treatment with the 3G4 antibody (P <0.05). At the end of the study, in contrast to control mice, the average reduction in tumor growth in 3G4 treated mice was 65% for MDA-MB-435 (FIG. 1A), 75% for MDA-MB-231 (FIG. 1B), Meth A was 90% (FIG. 1C) and L540 was 50% (FIG. 1D). Control mice treated with the isotype-matched control antibody, BBG3, did not cause tumor growth delay. The treatment of syngeneic Meth A tumor cells has been particularly successful. Even in mice bearing large L540 tumors known to be resistant to necrosis, 3G4 antibody treatment inhibited tumor growth as compared to controls. No toxicity was observed in mice treated with the 3G4 antibody.

  Thus, in summary, the 3G4 antibody caused tumor vascular injury, localized thrombosis, tumor necrosis, and delayed tumor growth without evidence of toxicity.

Example III
Production of Chimeric 3G4 Antibody Bavituximab This example provides the entire sequence of the heavy and light chain variable regions of the 3G4 antibody, including the six complementarity-determining regions (CDRs) in total, and is referred to as the mouse-now called bavituximab. The generation of chimeric versions of 3G4 antibodies, including human chimeric antibodies (ch3G4), is described.

A. 3G4 Antibody Sequence The original sequence of the 3G4 antibody variable region was obtained by RACE from hybrid cells producing the 3G4 antibody and the sequence was verified. The nucleic acid and amino acid sequences of the heavy chain variable region (Vh) of the 3G4 antibody are shown in FIG. 18A of US Pat. No. 7,572,448. The sequence of the heavy chain variable region includes VH CDR1, VH CDR2, and VH CDR3 at positions predictable by Kabat (Kabat et al., 1991). The BstEII site in the nucleic acid sequence can be used as a convenient site, for example, to prepare a functional mouse variable region for use in transplantation into a human constant region.

  In fact, the 3G4-2BVH sequence has been grafted into the human gamma 1 constant region at the BstEII site using the Lonza pEE vector. The resulting product contains a mouse leader sequence whose VH is conjugated to the human CH1 sequence in the manner shown in FIG. 18A of US Pat. No. 7,572,448.

  The nucleic acid and amino acid sequences of the light chain variable region (Vκ) of the 3G4 antibody are shown in FIG. 18B of US Pat. No. 7,572,448. Light chain variable region sequences include VL CDR1, VL CDR2, and VL CDR3 at positions predictable by Kabat (Kabat et al., 1991). A BbsI site within a nucleic acid sequence can be used as a convenient site, for example, to prepare a functional mouse variable region for use in transplantation into a human constant region.

  In fact, the 3G4-2BVL sequence has been grafted into the human kappa constant region at the BbsI site using the Lonza pEE vector. The resulting product contains a mouse leader sequence, the VL of which is joined within the human CL1 sequence in the manner shown in FIG. 18B of US Pat. No. 7,572,448.

B. As described generation directly below the mouse chimeric antibody 2AG4, murine 3G4 antibody of human chimera (ch3G4) is a human IgG ₁ isotype (hIgG _1). The mouse IgG homolog of ch3G4 corresponds to the IgG _2a isotype (mIgG _2a ). The construct was prepared, was tested and shown to behave original murine IgG ₃ antibodies essentially the same way.

  Briefly, the 3G4 light chain coding sequence was amplified by RT-PCR from total RNA isolated from a 3G4 hybrid cell line. RT-PCR primers were designed for cloning into the pEE12.4 vector, a Lonza expression vector, such that the amplified fragment contained Xmal and EcoRI restriction enzyme sites at both ends of the amplification product. The 3G4 heavy chain variable region was amplified by RT-PCR from total RNA isolated from a 3G4 hybrid cell line. Primers were designed so that the amplified fragment contained HindIII and XmaI restriction enzyme sites at both ends of the amplification product for cloning into the pEE6.4 vector, which is a Lonza expression vector.

  The mouse IgG2a constant region was amplified from a plasmid vector by PCR. The PCR primers were designed to have BstII and EcoRI restriction sites at both ends of the amplification product for cloning into the pEE6.4 + 3G4VH vector. The BstEII site was designed to be in frame with the upstream 3G4 VH variable region sequence. The heavy and light chain constructs were combined into a single dual gene vector (12.4 3G4 IgG2a) by cutting both vectors with SalI and NotI. The heavy and light chain coding regions were verified by sequencing.

  12.4 The 3G4 IgG2a vector was transfected into NS0 cells by electroporation. After transfection, NS0 cells were diluted and plated in 96-well plates in medium lacking glutamine. Only cells transfected with the construct (containing the glutamine synthase gene for positive selection) are able to grow in the absence of glutamine. Using the standard ELISA of Example I, transfectants were identified, screened for antibody secretion, and those transfectants secreting the highest amounts of antibody were grown in large cultures. Purified antibodies were generated.

  The resulting 2aG4 antibody was purified to homogeneity in appearance and shown to have essentially the same affinity and binding profile as the 3G4 antibody.

C. Generation of Human Chimeric Antibody ch3G4 (Bavituximab) A chimeric construct containing a mouse variable region and a human constant region was generated (ch3G4) and shown to have essentially the same characteristics as the original mouse antibody.

The mouse 3G4 antibody was converted to a human-mouse chimeric antibody. Mouse _{V H} was cloned and grafted onto human gamma ₁ constant region of BstEII site of the Lonza 2BVH vector. Mouse _{V K} was cloned and grafted onto human K constant region of BbsI site of the Lonza 2BVL vector. The sequence was verified. All constructs were expressed in CHO (Chinese Hamster Ovary) cells and the antibodies were purified. This is an antibody now called bavituximab.

  Using the standard ELISA of Example I, the resulting ch3G4 bound to the phospholipid-coated ELISA plate at least to the same extent as mouse 3G4. The in vitro binding profile of chimeric 3G4 to phospholipids PS, PA, CL, PI, and PG was shown to be the same as 3G4. Binding was antigen-specific because no binding was observed with a control antibody of irrelevant specificity. In vivo, ch3G4 has also been shown to localize to tumor vascular endothelium and to exert antitumor and antiviral effects in extensive studies.

  However, when the chimeric 3G4 construct was expressed in CHO cells under serum-free conditions and the purified antibody was tested for binding to PS in an ELISA in the absence of serum, binding to PS was lost.

Example IV
3G4 Antibody and Bavituximab Target PS in a β2GPI-Dependent Manner This example demonstrates the apparent PS binding profile of the 3G4 antibody from the original hybrid cell with the chimeric antibody expressed in CHO cells Provide data to resolve conflicts. In doing so, this example demonstrates that the interaction between the 3G4 antibody and PS is dependent on plasma protein β2-glycoprotein I (β2GPI).

A. Materials and Methods Materials Dulbecco's Modified Eagle's Medium (DMEM) and Trypsin / EDTA were purchased 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 hybrid cell culture medium, Synthechol NS0 supplement, L-α-phosphatidylserine (PS), bovine serum albumin (BSA), and ovalbumin from chicken egg white (OVA) are available from Sigma Chemical Co. (St. Louis, MO). DEAE cellulose, heparin-Sepharose, and Hybond-P membranes were obtained from Amersham Biosciences (Buckinghamshire, UK). 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine [lysophosphatidylcholine (LPC)] was obtained from Avanti Polar Lipids (Alabaster, AL). 96-well Immulon-1B and Immulon-2HB microtiter plates were obtained from Thermo Lab Systems (Franklin, Mass.). Tris-HCl gradient SDS-PAGE gels and Opti-4CN substrate kits were obtained from Biorad (Hercules, CA). Eight-well glass chamber slides were obtained from BD Biosciences (Bedford, MA).

2. Antibodies The 3G4 mouse monoclonal antibody generated to bind to the anionic phospholipid PS is the antibody described in Example I. 3G4 was first produced in hybrid cell supernatants (Example I, Example II). 3G4 was also converted to the mouse IgG2a isotype (Examples III, B) and was also produced in the NS0 mouse myeloma cell line. NS0 cells were cultured in DMEM supplemented with 10% FBS or in serum-free hybrid cell culture medium with Synthechol NS0 supplement. A human chimeric version of 3G4 (ch3G4, Bavituximab) was generated (Examples III, C) and antibodies were produced from CHO cells under serum-free conditions.

  Mouse anti-human β2GPI (anti-β2GPI or α-β2GPI) mAbs were obtained from US Biological (Swampscott, MA). A hybrid cell that secretes C44, a colchicine-specific mouse IgG2a mAb, was obtained from the American Type Culture Collection (Rockville, MD) and was used as a control for 3G4 and anti-β2GPI. Rituximab (a human IgG1 chimeric mAb) was used as a control for ch3G4. All secondary antibodies were obtained from Jackson Immunoresearch Labs (West Grove, PA).

3. Preparation of Antibody Fragments 3G4 F (ab ') ₂ was generated by incubation with the protease pepsin. 3G4 Fab and control Fab 7H11 (anti-adenovirus) were generated by incubation with the protease papain. All antibody cleavage products were purified by FPLC and verified by SDS-PAGE.

4. Purification of β2GPI from Human Plasma Human β2GPI (hβ2GPI) for use in this 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 the pooled plasma to a final concentration of 1.57% by volume. The precipitate was discarded and the supernatant was adjusted to pH 7.5 with saturated Na ₂ CO 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. Thereafter, the fraction passing through the DEAE column was applied to a heparin-Sepharose affinity column equilibrated with 50 mM Tris (pH 8.0), and the bound protein was eluted using 1.0 M NaCl. Finally, the β2GPI preparation was dialyzed against PBS and further purified by protein A / G to remove contaminating IgG. The final preparation contained a homogenous band at 50 kDa as indicated by non-reducing SDS / PAGE and Coomassie® staining.

5. Construction and Expression of β2GPI and β2GPI Domain To produce pure recombinant full-length and deleted forms of β2GPI, the yeast shuttle expression vector pPIC6αA (Invitrogen) and the host strain Mut ⁺ X-33 (Invitrogen) were used. The expression vector contains the 5 'promoter sequence and 3' transcription termination sequence of the alcohol (methanol) oxidase gene (AOX1). This vector also has a yeast α mating factor signal sequence downstream of the AOX1 promoter to which an external cDNA can be fused to secrete the recombinant heterologous protein into the medium. P. Expression in P. pastoris provides glycosylation and disulfide bond formation similar to those in mammalian cells.

  The following five expression constructs, without their cognate signal peptide, using human β2GPI cDNA as described in Luster et al., 2006 and US Pat. No. 8,956,616 to generate expression constructs, β2GPI cDNA entire coding region (domains IV); domain I deletions (domains II-V); domain I and II deletions (domains III-V); domain I, II, and III deletions (domains IV- V); and only domain V (domains I, II, III, and IV deletions). A common 3 'primer was used for PCR of all fragments.

  The PCR amplified fragment was inserted in frame between the EcoR1 and XbaI restriction sites of pPICαA, immediately downstream of the α mating factor signal sequence. A stop codon was introduced at the end of each fragment to prevent the recombinant protein from fusing to the c-myc epitope or C-terminal His tag. Plasmid constructs were isolated from E. coli in the presence of 100 μg / ml blasticidin. coli and verified by restriction analysis and nucleotide sequencing. The recombinant proteins expressed by the above five constructs encoded proteins of about 36, 29, 24, 16, and 9 kDa, respectively, before glycosylation.

  For transformation and screening of expression clones, the recombinant plasmid construct was linearized with the restriction enzyme SacI, purified and 10 μg was used to transform host strain X-33 by the spheroplast method (Invitrogen). Transformants of each of these constructs were selected for 4 days on YPD (yeast extract peptone dextrose medium) plates containing 400 μg / ml blasticidin. Several clones of each of these constructs were re-streaked on YPD plates with 400 μg / ml blasticidin to determine true integrants. Thereafter, ten clones of each construct were streaked on minimal dextrose (MD) and minimal methanol (MM) plates. Thereafter, five clones of each construct that grew equally well on both MD and MM plates were grown in liquid MD and MM media for 24, 48, 72, 96, and 120 hours. The supernatant and pellet of each clone at each time point were analyzed by Western blot using an anti-human β2GPI polyclonal antibody. Clones that showed the highest protein expression in the supernatant were further used for large-scale preparations.

  For large-scale purification of the recombinant protein, the recombinant protein was produced using culture conditions recommended by Invitrogen. The starting culture of each clone was cultured overnight at 30 ° C. with vigorous shaking in 5 ml of buffered minimal glycerol complex medium (BMGY). Cells were harvested and used to inoculate 25 ml of BMGY and grown for 2 days. The cells from the 25 ml culture were then used to inoculate 1 L of buffered minimal methanol complex (BMMY) medium (1.0% methanol). Cultures were continued for 4 days with vigorous shaking at 30 ° C., and 100% methanol was added every 24 hours (1.0% final concentration) to maintain protein expression. The medium was clarified by centrifugation (4000 × g, 15 minutes), the supernatant was dialyzed in 50 mM Tris buffer at 4 ° C. for 2 days, and then applied to a DEAE-Sephacel column equilibrated with 50 mM Tris buffer. Applied. The flow through solution was collected and applied to a heparin-Sepharose column. β2GPI was eluted from a heparin-Sepharose column with 1 M NaCl, dialyzed against 50 mM Tris buffer, concentrated using an Amicon concentrator, and analyzed by Western blot. The N-terminus of each protein was sequenced to confirm cleavage of the α-factor leader sequence. Protein yields varied from 10 mg / L (full length β2GPI) to 25 mg / L (β2GPI domain V).

6. Preparation of “Nicked” hβ2GPI Nicked hβ2GPI was prepared from intact hβ2GPI purified from human plasma as described above in this example. hβ2GPI was incubated with plasmin-coated beads at 37 ° C. for 17 hours. The beads were removed by centrifugation and the supernatant containing the cleaved protein was collected. Western blot of the purified product showed that the nicked β2GPI preparation contained no plasmin and no plasmin autoproteolytic products (no reactivity with anti-plasmin or anti-angiostatin antibodies). N-terminal sequence analysis revealed two N-termini, corresponding to the N-terminus of β2GPI, and one new sequence generated at the Lys317 / Thr318 cleavage site.

7. PS ELISA
The standard ELISA of Example I was adapted with the following changes. PS-coated Immunlon IB microtiter plates were blocked overnight in 1 wt% OVA. The next day, serial two-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 serum from bovine, human, rat, or mouse. Plates were incubated at 37 ° C. for 1 hour to detect 3G4 binding. All ELISA studies were performed at least three times.

8. Anti-hβ2GPI ELISA
The assay was performed as described above with the following changes. hβ2GPI, nicked hβ2GPI, or recombinant hβ2GPI peptide was coated overnight at a concentration of 10 μg / ml onto 96-well Immunlon 2HB microtiter plates. The plates were then blocked for 1 hour at room temperature in 1% OVA. 3G4, ch3G4, or anti-β2GPI were diluted in 1% OVA to an initial concentration of 13.33 nM to prepare a two-fold serial dilution. Plates were incubated at 37 ° C. for 1 hour to detect antibody binding. All ELISA studies were performed at least three times.

9. Western Blot Protein samples were heated to 95 ° C. in non-reduced SDS sample buffer for 5 minutes. The samples were then loaded on a 4-15% gradient SDS-PAGE gel of Tris-HCl and separated using a Mini Protean II instrument (Biorad). Separated proteins were transferred to a PVDF membrane and blocked overnight in 3 wt% BSA. Membranes were probed with anti-β2GPI, 3G4, or control mouse IgG diluted to 1 μg / ml in 3% BSA, thoroughly washed, and incubated with peroxidase-labeled goat anti-mouse IgG. The membrane was developed using the Opti-4CN substrate kit.

10. Induction and Detection of PS Exposure on Endothelial Cells Adult bovine aortic endothelial (ABAE) cells were maintained in DMEM supplemented with 10% FBS and 2 mM L-glutamine. ABAE cells are removed from subconfluent cultures by brief exposure to 0.25% trypsin / 0.02% EDTA and seeded at 8 × 10 ⁴ cells per well in 8-well chamber slides did. After overnight culture, cells were gently washed with PBS and treated with 200 μM lysophosphatidylcholine (LPC) to induce PS exposure. LPC treatment was performed in either 10% FBS or 10% normal mouse serum (MS) for 30 minutes at 37 ° C. in the presence of 3G4, ch3G4, or control IgG. When LPC treatment was performed with 10% MS, hβ2GPI was added as a cofactor because 3G4 / ch3G4 could not bind to PS in MS (see results below in this example).

  PS exposure was determined by immunofluorescence staining. Cells were washed extensively in PBS, fixed in 4 wt% paraformaldehyde, and incubated with a biotin-conjugated anti-mouse secondary antibody. The cells were then incubated with FITC-conjugated streptavidin (Jackson Immunoresearch) to detect antibody binding. The cells were then permeabilized with 0.1% Triton-X100 in PBS and treated with Texas Red conjugated phalloidin (Molecular Probes, Eugene, OR) and 4 ', 6-diamidino-2-phenylindole (DAPI, Molecular Probes). Counterstained. Images were captured using a Coolsnap digital camera (Photometrics, Tucson, AZ) attached to a Nikon microscope and processed with MetaVue software (Universal Imaging Corporation, Downtown, PA).

11. Quantification of Antibody Binding to ABAE Cells The area of antibody binding was determined using MetaVue image analysis software that can quantify the number of illuminated pixels in the image. Antibody binding was quantified using images of FITC fluorescence. FITC images were normalized for the number of cells present in the field of view using the corresponding DAPI fluorescence image. A small FITC / DAPI ratio indicates a small antibody binding area, while a large FITC / DAPI ratio indicates a large binding area. The FITC / DAPI ratio was used to determine the increase or decrease in antibody binding area relative to basal amount of antibody binding under selected conditions. Five images at 200 × magnification were used for each analysis. Data are analyzed as the average relative FITC / DAPI ratio with the standard deviation.

B. Results Overall, the following data demonstrate that the interaction between the 3G4 (and bavituximab) antibody and PS is dependent on the plasma protein β2GPI. 3G4 has been shown to bind to β2GPI in domain II, which generally recognizes β2GPI domain I and is not associated with pathogenic antibodies isolated from patients with anti-phospholipid antibody syndrome (APS). This data indicates that a divalent 3G4 / β2GPI complex was required for enhanced PS binding, including binding to PS positive cells, because the 3G4 Fab ′ fragment does not have this activity.

1.3G4 requires serum factors for binding to PS-coated microtiter plates 3G4 antibody purified from serum-containing medium (SCM) or serum-free medium (SFM) performs serial dilutions in 10% FBS Then, it binds to the PS-coated microtiter plate (FIG. 2A, solid line). In contrast, when performing serial dilutions in 1% OVA (lacking bovine serum protein), 3G4 purified from SFM no longer binds to PS (FIG. 2A, dashed line, ■). This finding indicates that factors present in bovine serum mediate the interaction between 3G4 and PS.

  Interestingly, when performing serial dilutions in 1% OVA, 3G4 purified from SCM still binds to PS (FIG. 2A, dashed line, ▲). Clearly, serum proteins from the serum-containing medium mediate the interaction between 3G4 and PS. This indicates that even when purifying 3G4 grown in SCM, the low levels of serum proteins that may be present in the purified antibody are still sufficient to support PS binding.

  In light of these findings, the following studies were performed using 3G4 purified from SFM.

2. Binding of 3G4 to PS in sera from different species To determine if sera from other mammalian species can mediate the interaction between the 3G4 antibody and PS, 10% mouse, rat Serial dilutions of 3G4 were performed in human, human, or other serum. The 3G4 antibody bound PS in the presence of rat and human serum almost as well as in the presence of bovine serum (FIG. 2B). However, 3G4 did not bind to PS in the presence of mouse serum (FIG. 2B). In a related study, 3G4 bound to PS in the presence of hamster, ferret, guinea pig, rabbit, and monkey serum. Thus, the serum protein epitope recognized by 3G4 is conserved among all mammalian species tested except mice.

3.3G4 Binds Serum Glycoprotein β2GPI In the early 1990's, many so-called anti-phospholipid (aPL) antibodies did not recognize phospholipids directly, but instead bound to serum proteins, which turned It has been shown to have affinity for lipids (Galli et al., 1990; McNeil et al., 1990). Therefore, a panel of human serum proteins known to interact with anionic phospholipids was screened for reactivity with the 3G4 antibody.

  For example, human β2GPI (hβ2GPI) was coated on microtiter plates and incubated with mouse anti-human β2GPI antibodies (anti-β2GPI), 3G4 antibodies, or control mouse IgG2a with irrelevant specificity (control mIgG). As expected, anti-β2GPI bound to hβ2GPI, whereas control mIgG did not (FIG. 3). The 3G4 antibody also bound strongly to hβ2GPI coated plates (FIG. 3).

  To determine if β2GPI was the only serum protein recognized by the 3G4 antibody, purified hβ2GPI and 10% human serum were run on an SDS-PAGE gel and transferred to a membrane support for immunoblotting. 3G4 detected a 50 kDa purified hβ2GPI and a single band of similar size in human serum. Importantly, the 3G4 immunoblot was substantially identical to the blot generated using the anti-β2GPI antibody. The control mIgG antibody did not detect any protein.

  Other human serum proteins known to interact with anionic phospholipids were tested to confirm lack of reactivity with the 3G4 antibody in an ELISA. Equal amounts of specific proteins were coated on microtiter plates, blocked in 1% OVA, and incubated with serial dilutions of 3G4 antibody. Plates were thoroughly washed and antibody binding was detected with a peroxidase-labeled secondary detection antibody. All studies included positive and negative control antibodies that performed as expected. The results of the immunoblot and ELISA studies were positive for β2GPI, negative for Annexin V, Factor XII, kininogen (low or high molecular weight), oxidized LDL, protein C, protein S, prothrombin, and tissue plasminogen activator ( tPA). Taken together, these data indicate that the 3G4 antibody binds to the serum protein β2GPI.

4.3G4 binds to β2GPI at domain II β2GPI has five domains, the fifth of which is involved in binding to anionic phospholipids such as PS. To determine which domains of β2GPI were recognized by the 3G4 antibody, recombinant human β2GPI constructs with different domain structures were generated and tested with recombinant full-length hβ2GPI. These domain constructs were created by successive truncations from the N-terminus and, in turn, lack each of the N-terminal domains as follows. Recombinant full-length hβ2GPI contains domains IV, hβ2GPI lacking domain I contains domains II-V, and hβ2GPI lacking domains I and II contains domains III-V. Hβ2GPI lacking domains I, II, and III contain domains IV-V, and hβ2GPI lacking domains I, II, III, and IV contain only domain V.

  Equal amounts of full-length hβ2GPI and each of the above hβ2GPI domain constructs were coated on microtiter plates and incubated with serial dilutions of the 3G4 antibody. This study showed that only hβ2GPI constructs containing domain II of β2GPI (domains IV and II-V) were detected by 3G4 (FIG. 4). When domain I was deleted, 3G4 bound equally well to domains II-V (FIG. 4). Thus, the 3G4 antibody binds to β2GPI at domain II.

  Given the information known about pathogenic antibodies isolated from APS patients, the discovery that the 3G4 antibody binds to β2GPI at domain II is significant. Pathogenic anti-β2GPI antibodies isolated from APS patients generally recognize domain I of β2GPI (de Laat et al., 2005). Anti-β2GPI antibodies from APS patients that recognize domain II are often not pathogenic. This may explain the lack of toxicity associated with 3G4 after toxicological studies (such as those described herein) performed in various animal models and in extensive clinical experience. high.

5. Co-binding 3G4 and β2GPI to cells with exposed PS In order to validate the above findings under more physiological conditions, a live cell binding assay was developed. This assay detects and measures antibody binding to the cell membrane surface after treatment with the membrane disruptor lysophosphatidylcholine (LPC) to induce PS disruption.

  In this assay, ABAE cells were incubated with the 3G4 antibody or control mIgG in DMEM + 10% FBS for 30 minutes in the presence or absence of 200 μM LPC. The cells were then washed, fixed, and stained with a fluorescent marker to visualize the binding of the antibody to the cell surface. Pixel area of 3G4 or mIgG binding was quantified using MetaVue software. All values are for 3G4 binding to cells not treated with LPC and were set to 1.

  When the 3G4 antibody was added to the ABAE cell culture medium under normal conditions, no binding to cells was observed. However, when ABAE cells are incubated with 3G4 in the presence of LPC, numerous pinpoints of 3G4 antibody binding are readily detectable. LPC is known to induce transient membrane distortion (Kogure et al., 2003), which is likely to cause loss of membrane asymmetry and PS exposure.

Quantitation showed that the area of 3G4 antibody binding increased over 500-fold upon LPC treatment, while binding of control mIgG remained undetectable. Similar results have been obtained previously, where it was shown that 3G4 and the PS binding molecule Annexin V bind to endothelial cells following induction of PS exposure with H ₂ O ₂ (Ran et al., 2005; USA Patent No. 8,486,391). The LPC-treated ABAE cells were not stained by the membrane-impermeable dyes propidium iodide or DAPI, indicating that 3G4 binds to PS on the cell membrane rather than the inner lobe of the plasma membrane.

  To confirm that β2GPI is required for the 3G4 antibody to bind to cells with exposed PS, a live cell binding assay was performed in media containing 10% mouse serum but not 10% FBS. Performed to prevent interference from bovine β2GPI. As demonstrated above, the 3G4 antibody does not bind to PS in the presence of mouse serum. In addition, the 3G4 antibody did not detect any protein in 10% mouse serum by immunoblot, indicating that 3G4 did not recognize mouse β2GPI.

  For this study, a human chimeric 3G4 antibody (ch3G4) was used to prevent background caused by the detection of mouse IgG present in mouse serum. No antibody binding was detected when ABAE cells were incubated with ch3G4 in the presence of 10% mouse serum and LPC (FIG. 5). In contrast, the addition of purified hβ2GPI to the binding reaction supported extensive binding of ch3G4 (FIG. 5), demonstrating that ch3G4 antibody binding was dependent on hβ2GPI. In all situations, ch3G4 binding was dependent on LPC treatment and no binding was detected when using control human IgG of irrelevant specificity.

  Interestingly, when ABAE cells were incubated with hβ2GPI in the presence of 10% mouse serum and LPC, washed extensively, and then incubated with ch3G4 antibody, very little ch3G4 binding was detected when hβ2GPI binding was detected. Was detected (FIG. 5). This finding indicates that hβ2GPI does not bind to cells with exposed PS in the absence of the ch3G4 antibody, reporting that β2GPI has a low affinity for the PS membrane surface under physiological conditions. Consistent (Willems et al., 1996; Bevers et al., 2004; Bevers et al., 2005). Taken together, these data indicate that in order to bind to ABAE cells with exposed PS, the ch3G4 antibody and hβ2GPI must be present simultaneously, indicating that the ch3G4 antibody has an affinity for β2GPI for PS. Suggest enhancement.

6. Confirm that the lipid binding domain of β2GPI is required for co-coupling of 3G4 The lipid binding domain of β2GPI is required for co-coupling of β2GPI and 3G4 and ch3G4 antibodies to cells with exposed PS To this end, a live cell binding assay was performed using “nicked” hβ2GPI. Nicked hβ2GPI is unable to bind to PS due to plasmin-mediated cleavage in the lipid-binding region of domain V (Hunt et al., 1993; Hunt & Krilis, 1994).

  When ABAE cells are incubated with ch3G4 antibody and hβ2GPI or nicked hβ2GPI in the absence of LPC, no ch3G4 binding is detected (FIG. 6A). In the presence of LPC, hβ2GPI can mediate binding of ch3G4 to ABAE cells with exposed PS, whereas nicked hβ2GPI cannot mediate binding (FIG. 6A). The lack of binding in the live cell assay is due to the inability of the ch3G4 antibody to bind to nicked hβ2GPI because ch3G4 binds to nicked hβ2GPI when equivalent amounts of protein are coated on the microtiter plate. Not (FIG. 6B). These findings demonstrate that the ch3G4 / hβ2GPI complex detects PS exposed on ABAE cells through the lipid binding region of hβ2GPI domain V.

7. Antibody bivalent is required for β2GPI co-binding The data presented above suggest that the 3G4 antibody detects PS by enhancing the binding activity of β2GPI to anionic phospholipids. To determine whether binding of 3G4 / β2GPI to cells with exposed PS requires divalentity, 3G4 F (ab ′) ₂ and 3G4 Fab ′ monomers were generated and intact Used for live cell binding assay with 3G4 antibody. As expected, intact 3G4 antibody bound to LPC-treated ABAE cells but not to untreated cells. Equal concentrations of 3G4 F (ab ′) ₂ also bound to LPC-treated ABAE cells (FIG. 7A), but negligible 3G4 Fab ′ binding (FIG. 7A). A clear decrease in binding of 3G4 F (ab ') ₂ was detected as compared to 3G4, indicating the binding of the polyclonal secondary antibody to the Fc epitope missing on 3G4F (ab') _2. Is likely to be due to the loss of Even at a concentration of 2 μM, 1,000 times higher than that required to bind β2GPI coated on microtiter plates, no binding of 3G4 Fab ′ was detectable on ABAE cells.

  Furthermore, 3G4 Fab 'inhibited binding of ch3G4 / β2GPI to LPC-treated ABAE cells in a concentration-dependent manner (FIG. 7B), whereas control Fab' of irrelevant specificity did not. The ability of 3G4 Fab 'to inhibit ch3G4 binding confirms that 3G4 Fab' is able to bind to β2GPI and that the monomeric 3G4 Fab '/ β2GPI complex is unable to bind to cells with exposed PS. I do. These data indicate that a bivalent 3G4 / β2GPI complex is required to bind to exposed PS on the cell surface.

  In summary, as shown in FIG. 4, the 3G4 antibody binds to β2GPI at domain II, and as shown in FIGS. 6A and 6B, the lipid binding region of β2GPI domain V binds to PS exposed on cells. 3G4 (and ch3G4) and β2GPI are required for co-coupling. In addition, antibody bivalency is required for such co-coupling of 3G4 (and ch3G4) and β2GPI to exposed PS, as demonstrated in FIGS. 7A and 7B. Thus, we present a model of antibody and β2GPI co-binding to exposed PS on the outer surface of the membrane, such as occurs in activated endothelial cells, tumor vascular endothelial cells and tumor cells, and virus infected cells (FIG. 8). ).

Example V
Preclinical Modeling of the Interaction Between Bavituximab and β2GPI This example provides preclinical data on the interaction between the antibody β2GPI of the bavituximab family and PS. Overall, the data indicate that relatively low levels of β2GPI, significantly below typical amounts in the human population, are sufficient for effective binding of bavituximab to PS.

A. Low β2GPI supports anti-tumor effect in mice In the initial development of mouse 3G4 antibodies, the antibodies were purified from supernatants of cultured hybrid cells to homogenous appearance using standard Protein A procedures (implemented Example I). Initial studies in mice have shown that this purified antibody exerts an antitumor effect in several models (Example II).

  After determining that the 3G4 antibody required β2GPI for PS binding (Example IV) and that all species of β2GPI except mice support PS binding (FIG. 2B), the antitumor of Example II was determined. It was inferred that the effect was an underestimation of the potency of the 3G4 antibody. That is, the anti-tumor effect of the 3G4 antibody in FIGS. 1A, 1B, 1C, and 1D is supported only by low levels of bovine β2GPI that may have been co-purified with the 3G4 antibody through a protein A column. Basically, most of the protein delivered was pure 3G4 antibody, but low levels of bovine β2GPI would have been co-administered to the mice (10% fetal bovine used for hybrid cell culture). From serum (FBS)). Thus, these initial data suggest that 3G4 antibodies do not require high levels of β2GPI for inhibiting tumor growth in vivo.

  The 3G4 antibody was purified to homogeneity in appearance using a protein A column that purifies the antibody based on affinity for IgG. There is an additional reason that β2GPI from the culture medium was separated from the 3G4 antibody during purification. First, due to the low affinity of β2GPI for the 3G4 antibody (and less than the affinity between protein A and 3G4), β2GPI may have separated from 3G4 during loading and washing. Second, the low pH elution step (to separate the 3G4 antibody from Protein A) may have removed β2GPI complexed to 3G4. It is also possible that during collection of the eluted protein at the major antibody peak, no smaller β2GPI protein was collected. Nevertheless, as shown in FIGS. 9A and 9B, 10-20% of the apparently pure 3G4 antibody delivered to mice was actually in the form of a 3G4-β2GPI complex. Even if you take a hypothetical position, such levels of bovine β2GPI are still very low, absolutely and in comparison to antibodies.

  More specifically, 3G4 antibody was produced from hybrid cells in 10% FBS. One liter volume will typically produce 10 mg / L of 3G4 antibody. Assuming that FBS contains 200 μg / ml bovine β2GPI (similar to the level of human β2GPI in human serum), 10% FBS contains 20 μg / ml bovine β2GPI. At harvest, one liter of hybrid cell supernatant will contain 10 mg of 3G4 antibody and up to 20 mg of bovine β2GPI (20 μg / ml × 1000 = 20 mg). Upon purification on protein A, the resulting material will contain 10 mg of protein. Assuming a 3G4 purity of 80-90%, the remaining 10-20% is a 3G4: β2GPI “complex” where one antibody binds to two β2GPIs (3G4-2 × β2GPI).

  Mice received 100 μg of such protein. At 90% purity, 90 μg of the administered 100 μg protein is pure 3G4, with 10 μg of the 3G4-2 × β2GPI complex. Assuming that the molecular weight (MW) of the antibody is 145 kD and the MW of β2GPI is 50 kD (Examples IV, A4, B3; McNeil et al., 1990; Luster et al., 2006), two β2GPIs for each antibody in the complex The ratio is about 3: 2 (exactly 59.2% antibody and 40.8% antibody). Thus, of the 10 μg complex, approximately 6 μg is 3G4 and 4 μg is β2GPI (exactly 5.92 μg of 3G4, 4.08 μg is β2GPI).

  The volume of mouse blood is 2 ml. Pure 3G4 antibody is present at 90 μg per mouse, or 45 μg / ml. At a ratio of about 3: 2, 10 μg of conjugate per mouse contains about 6 μg of 3G4 and about 4 μg of β2GPI (3 μg / ml of 3G4 and 2 μg / ml of β2GPI). At 2 μg / ml β2GPI capable of antibody binding, this was about 1% of the average β2GPI level in human serum and still supported antitumor activity (FIGS. 1A, 1B, 1C, and 1D).

  2 μg / ml β2GPI in mice corresponds to 0.04 μM. For 3G4, 3 μg / ml 3G4 from the administered 3G4-2 × β2GPI complex is added to 45 μg / ml pure 3G4, and 48 μg / ml 3G4 antibody is present. This corresponds to 0.33 μM antibody. With a starting point of 90% purity, for 0.04 μM β2GPI and 0.33 μM 3G4 antibody, this is a molar ratio of β2GPI to antibody of 0.12 (FIG. 9A). Even at a starting point of 80% purity, the same calculations indicate that the in vivo concentration of β2GPI is 4 μg / ml (0.08 μM), and the in vivo antibody concentration is 46 μg / ml (0.32 μM). This is still a molar ratio of β2GPI to antibody of only 0.25 (FIG. 9B). Subsequent studies, including sensitive Western blots of the antibody preparation, which could subsequently identify the β2GPI band on the gel, confirmed that the 3G4 antibody was always at least 80-90% pure. Thus, these calculations of a molar ratio of β2GPI to antibody of 0.12-0.25 indicate that initial mouse data using the 3G4 antibody indicate that high levels of β2GPI are not required for anti-tumor activity. Add a quantitative perspective to the description.

B. Low β2GPI Supports Cell Binding In a first study to analyze the level of β2GPI required for binding of the 3G4-β2GPI complex to PS on cells, treating ABAE cells with LPC and exposing PS exposure Was induced (Example IV) and then incubated with 40 nM of purified human β2GPI and various concentrations of the ch3G4 antibody (bavituximab). Relative ch3G4 binding increased in a concentration dependent manner from 320 pM to the apparent peak of 80 nM ch3G4, which is a 2: 1 antibody to β2GPI ratio (FIG. 10). As shown in Table 1 (MW of antibody, 145 kD; MW of human β2GPI, 50 kD), the apparent peak binding in this study corresponds to a molar ratio of β2GPI to antibody of 0.5.

  The bell-shaped relationship between the concentration of ch3G4 and binding to cells with exposed PS (FIG. 10) further supports the formation of the bivalent ch3G42 × β2GPI complex on the membrane surface, This suggests that the ch3G4-β2GPI complex is formed at very high antibody concentrations. At such concentrations, competition between the monovalent (unbound) complex and the divalent (bound) complex caused a decrease in the amount of ch3G42 × β2GPI complex bound to the cells (“hook effect”). Also known as).

  The maximum relative binding in this study occurs at an antibody concentration of 80 nM, which is a β2GPI to antibody ratio of only 0.5 (FIG. 10, Table 1). This is a lower ratio than expected for the bivalent interaction shown in FIG. 8, but is generally consistent with the ratio reported above for treatment of mice with 3G4 antibody purified from hybrid cells. However, in this study, the exact density of PS on cells is not known. More importantly, no saturation binding (plateau) was observed at the ch3G4 concentrations tested, and further testing at ch3G4 concentrations of 20-80 nM and 80-320 nM would be beneficial. Nevertheless, without such an intermediate test, it can be concluded that optimal molar antibody binding occurs at β2GPI to antibody molar ratios between 0.125, 0.5, and 2. These in vitro numbers are in good agreement with those reported above for in vivo therapy using hybrid cell purified 3G4 antibodies, where the molar ratio of β2GPI to antibody was 0.12-0.25 depending on antibody purity. . Therefore, this first in vitro study also shows that low levels of β2GPI effectively support binding of bavituximab to cells with exposed PS.

Low β2GPI supports PS binding In subsequent studies, binding of the 2aG4 antibody to PS (Example III) was tested in ELISA in the presence of various concentrations of human β2GPI.

Solutions containing a fixed amount of 2aG4 antibody and increasing amounts of human β2GPI were prepared. Briefly, 62.5 ng / ml (0.4 nM) of 2aG4 was added to ovalbumin 0.0032, 0.016, 0.08, 0.4, 2, 10, or 50 nM human β2GPI. Different 2aG4-β2GPI mixtures were added to PS microtiter plates and the molecules allowed to bind at 37 ° C. for 1-2 hours. Unbound molecules were removed by washing with PBS. The secondary antibody was HRP-conjugated anti-human IgG in binding buffer. The plate was left at 37 ° C. for 1 hour and then unbound antibody was removed by washing 5 times with PBS. TMB substrate was added to each well in a volume of 100 μl and left at room temperature for 15 minutes to generate a colorimetric reaction. The reaction was stopped by adding 100 μl of 2M H ₂ SO ₄ . Within 30 minutes of adding the stop solution, the absorbance (optical density, OD) was read by a plate spectrometer at a wavelength of 450 nm and analyzed using SoftMax Pro software (the average OD of the control without β2GPI was Subtraction from the average OD of the test sample).

This study showed that binding of the 2aG4 antibody to PS began to plateau at a molar ratio of β2GPI to antibody of about 1, where both molecules were present at about 0.4 nM (FIG. 11). More precisely, as shown in Table 2 (MW of antibody, 145 kD; MW of human β2GPI, 50 kD), the molar ratio of β2GPI to antibody of 0.93 indicates the antibody binding to PS-coated plates. It is effective to support.

  Extending the observations of the first in vitro study above, this study showed that antibody binding to PS already reached saturation at low β2GPI to antibody molar ratio and then reached a plateau (50 nM). A slight loss of PS binding was observed with β2GPI (FIG. 11), which is likely related to the typical saturating effect commonly detected in ELISA assay formats). In this study, the effective molar ratio of β2GPI to antibody was approximately 1 (0.93). Increasing the ratio of β2GPI to antibody above 5 did not result in improved binding (FIG. 11, Table 2).

  A series of related studies were performed to test the binding of bavituximab to PS in an ELISA in the presence of various concentrations of human β2GPI in ovalbumin. Both a bivituximab titration and a β2GPI titration were performed. These studies also showed that low levels of β2GPI, including concentrations up to 0.5 μg / ml, were effective in supporting various antibody concentrations in binding to PS-coated plates. .

D. Antibody Binding and Activity in Diluted Human Serum Another series of studies was performed to test the binding and function of bavituximab to PS in various dilutions of human serum. These included binding to PS in ELISA, FACS analysis using PS positive cells, and functional assays in the form of NFAT alternative ADCC bioassay.

1. ELISA
An ELISA was performed to test the binding of bavituximab to PS in various percentages of six different individual human serum samples. Different human sera (BioReclamation IVT, from North America) were diluted in PBS to prepare various ranges of% human serum up to 0.1%. 2 μg / ml Bavituximab-HRP (Example XVI, A3) was added to each% human serum solution. Different Bavituximab-HRP mixtures were added to PS microtiter plates and allowed to bind at 37 ° C. for 1-2 hours. Plates were washed with PBS. TMB substrate was added to each well in a volume of 100 μl and left at room temperature for 15 minutes to generate a colorimetric reaction. The reaction was stopped by adding 100 μl of 2M H ₂ SO ₄ . Within 30 minutes of adding the stop solution, the absorbance was read on a plate spectrometer at a wavelength of 450 nm and analyzed using SoftMax Pro software.

2. FACS
Binding of bavituximab to PS-positive cells (HT1080 cells treated with etoposide) was tested using different percentages of FBS as a source of bovine β2GPI, and by fluorescence-labeled cell sorting (FACS), also known as flow cytometry. It was measured.

  Different percentages of FBS in PBS solution were made and 10 μg / ml of bavituximab was added to each solution. HT1080 cells were treated with 50 μM etoposide for 18 hours to induce PS exposure to the cell surface. The cells were then incubated with different percentages of Bavituximab in FBS, followed by secondary antibodies to Bavituximab to visualize the antibodies bound to the cells via flow cytometry. Negative controls included cells not treated with etoposide that did not expose PS on the surface, and PBS solutions lacking FBS.

3. NFAT
Activated T cell nuclear factor (NFAT) is a generic name applied to a family of transcription factors that have been shown to be important in the immune response. The NFAT signaling pathway and NFAT response element (NFAT-RE) have been used in the development of assays and commercial kits for monitoring the NFAT signaling pathway in cultured cells.

  An NFAT bioassay for use with the bavituximab antibody family has been developed (Larson et al., 2013). Jurkat cells (Promega), which have been engineered to express the FcγRIIIa-V158 receptor on the cell surface, also have a luciferase gene-containing genetic element under the control of a minimal TATA promoter containing multiple NFAT-REs. Had been transfected. These are NFAT effector cells co-cultured with PS positive target cells. PS targeting antibodies such as bavituximab bind to PS on the surface of target cells. Subsequently, the Fc region of the PS targeting antibody binds to the FcγRIIIa-V158 receptor on NFAT effector cells, causing signal transduction through the NFAT pathway. NFAT binds to NFAT-RE and activates quantifiable luciferase expression. Thus, this NFAT assay is an alternative ADCC bioassay for bavituximab and other PS targeting antibodies.

4. Results Exemplary results of the above ELISA assay are presented in FIG. 12, which also shows that diluted human sera with low levels of β2GPI effectively support antibody binding. Similar to the PS ELISA above using purified β2GPI (FIG. 11), there is some loss of PS binding at 50% and 100% human serum, especially when using undiluted serum. It relates to typical saturation effects commonly detected in assay formats. No such effect was observed in the FACS assay, indicating that the binding of bavituximab to cells with exposed PS is essentially the same in 50%, 75%, and 100% fetal calf serum. Was done.

From FIG. 12, it can be seen that binding of bavituximab to PS in the ELISA began to plateau at about 1% human serum. Normal human serum contains on average 200 μg / ml β2GPI (Steinkasserer et al., 1991; Mehdi et al., 1999; Miyakis et al., 2004), so 1% human serum is about 2 μg / ml or 0.04 μM Β2GPI. FIG. 12 shows that binding of bavituximab to PS is already saturated at this concentration of β2GPI. As shown in Table 3 (MW of antibody, 145 kD; MW of β2GPI, 50 kD), 1% of human serum in the ELISA corresponds to a molar ratio of β2GPI to antibody of 2.86. This is generally consistent with the rationale that each molecule of bavituximab needs to bind to two molecules of β2GPI in order to form a stable complex with PS on the cell surface (FIG. 8).

  As shown in FIG. 12, even with 0.5% human serum, binding of bavituximab to PS is close to a plateau, especially for serum sample 13, which shows a molar ratio of β2GPI to antibody of 1.43. (Table 3). The results of the NFAT surrogate ADCC bioassay also showed that molar ratios of β2GPI to antibody within this general range were effective in supporting bavituximab function. For example, while this study was not designed to specify an optimal ratio of bavituximab activity, a molar ratio of (bovine) β2GPI to antibody of 1.9 effectively supported bavituximab activity in the NFAT assay. It has been shown.

  In summary, this example demonstrates that a molar ratio of β2GPI to antibody as low as 0.12 to 2.86 supports antibody binding to PS and PS positive cells, promotes activity in functional assays, Indicates that effective treatment is possible. In view of all the above data and taking a prophylactic approach, to maximize binding and function of bavituximab, the molar ratio of β2GPI to antibody should be about 2.86 (Table 3), which is It was estimated that it need not be higher than about 3.

Example VI
Pharmacokinetics of Bavituximab in Clinical Studies This example relates to the pharmacokinetics of bavituximab when administered to human subjects with diseases where PS is a marker, particularly cancer and viral infections. Clinical experience has been shown to be consistent with preclinical modeling, as described above.

A. Initial Phase I Study A phase I multicenter, open label, dose escalation study was performed to evaluate the safety of bavituximab when administered intravenously (bavituximab monotherapy) to 26 patients with refractory advanced solid tumors, Tolerability and pharmacokinetics (PK) were evaluated. Patients were enrolled in four consecutive dose escalation cohorts (0.1, 0.3, 1, or 3 mg / kg of bavituximab weekly) on 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, 0, 7, 14, and On day 21 the patient was administered bavituximab.

Based on experience in the preclinical model (Example V) and other patient populations, an upper dose of 3 mg / kg per week was chosen. In a broad animal model study following the study of Example II, maximal efficacy was achieved with an antibody dose of 0.5 mg / kg three times a week, with a half-life of 48 hours and 2 μg / ml throughout the course of treatment. A simulated mean blood concentration of 5.5 resulted in a C _max of 5.5 μg / ml. Above such doses, it was estimated that PS binding by bavituximab would saturate based on the observation of concentrations at which the binding of bavituximab to PS-positive cells saturates in vitro (Example V).

  Prior to the study, samples were collected from patients in the 0.1 and 0.3 mg / kg dose cohorts on days 0, 1, 2, 4, 7, 10, 14, and every 7 days from day 21 to 70. . Samples from patients in the 1 and 3 mg / kg dose cohorts before study, every 0, 1, 2, 4, 7, 14, 21, 22, 23, 25, and every 7 days from day 28 to 56 Was collected. Bavituximab blood levels were determined by a valid ELISA.

Table 4 shows this phase I study including maximum concentration (C _max ), clearance (CL), half-life (t _1/2 ), and area under the plasma concentration-time curve from zero to infinity (AUC _inf ). A summary of the mean (coefficient of variation, CV) PK parameters of bavituximab after a single dose (day 0) and multiple doses (day 21) in is presented.

As shown in Table 4, after a single dose, the mean half-life of bavituximab was determined to be in the range of 37.2-43.9 hours. On day 0, the median time after administration when the maximum serum concentration (T _max ) was reached, the mean maximum serum concentration (C _max ) was 2.11-56.4 μg / ml (dose dependent). Range (values ranged from 2.04 to 3.73 hours). For bavituximab administered at 3 mg / kg, the maximum serum concentration was 56.4 μg / ml. For the entire study, bavituximab half-life ranged from 37 to 47 hours. In this study, the maximum tolerated dose was not reached.

  Bavituximab exhibited linear single dose (day 0) and multiple dose (day 21 or 42) PK characteristics (FIG. 13). Bavituximab did not show any apparent accumulation or time-dependent PK differences after multiple dose administration. In summary, this study showed that bavituximab was well tolerated at doses ranging up to 3 mg / kg per week and that pharmacokinetics supported a weekly dosing regimen. In particular, at the 1 mg / kg dose, the bavituximab concentration remained above the therapeutic threshold of 2 μg / ml predicted based on preclinical models for 6 days, and at the 3 mg / kg dose, the bavituximab concentration increased for 7 days. It was determined to remain above this 2 μg / ml (FIG. 13). Therefore, a dose of 3 mg / kg per week was selected for future use in oncology.

B. Further Pharmacokinetic Studies In addition to the Phase I studies described above, here, the PK of bavituximab administered as a single dose, once a week or as an infusion twice a week (60-90 minutes) was determined. Over 120 patients, across several other clinical studies in patients with cancer or viral infections. Bavituximab was confirmed to exhibit linear single and multiple dose PK characteristics at doses ranging from 0.1 to 6 mg / kg, with evidence of apparent accumulation of bavituximab or time-dependent PK differences. There was no. The median T _max was shown to occur within the first few hours after the end of the infusion. Serum bavituximab levels decrease in an apparently monoexponential or biexponential linear fashion. When observed, the more rapid distribution phase is essentially complete within 6 hours, with a final elimination half-life of about 1-2 days (21.9-46.8 hours).

1. PK in viral infections
The PK characteristics of bavituximab, with or without HIV, are generally similar in patients with cancer and chronic viral infections, as tested in patients chronically infected with HCV.

A phase I, open-label, single-center, dose-escalation study evaluated a single intravenous infusion of bavituximab in patients chronically infected with HCV (Examples VII, A). As shown in Table 5, the observed bavituximab concentrations were found to be very consistent with the predicted values of the PK modeling data.

  In a corresponding Phase Ib multicenter, open-label, non-randomized, escalating, repeated-dose study in chronic HCV patients, analysis of PK data showed a linear single-dose PK characteristic at day 0 at all dose levels. And a linear multiple dose profile at day 10 and there was no evidence of accumulation of bavituximab or time-dependent PK differences after 2 weeks of dosing.

  In a phase Ib study in patients co-infected with chronic HCV and HIV (Examples VII, C), bavituximab was administered on day 0 after weekly administration at doses ranging from 0.3 to 6 mg / kg. The eyes exhibited linear single dose PK characteristics and the day 49 exhibited linear multiple dose PK characteristics. Bavituximab did not show time-dependent PK differences or accumulation after multiple administrations once a week for 8 weeks.

2. PK in combination therapy
Importantly, there was no clinically relevant pharmacokinetic interaction for either drug when administered in combination with bavituximab and other drugs, especially chemotherapeutics. This includes the case where bavituximab and docetaxel are administered in combination.

In this regard, a phase Ib multicenter, open-label, non-randomized study was first conducted in patients with refractory advanced solid tumors in patients with gemcitabine, paclitaxel + carboplatin, or 3 mg / kg of bavituximab in combination with docetaxel. The safety, tolerability, and PK of weekly intravenous administration were evaluated. After a single dose (day 0) or multiple doses of bavituximab (day 21), it was determined that there were no significant differences in any measurable parameter between the three treatment groups. Evaluation of C _max and AUC indicated that there was no accumulation of bavituximab after multiple weekly dose administrations over 8 weeks.

  In a phase II randomized, double-blind, placebo-controlled study evaluating bavituximab plus docetaxel in previously treated locally advanced or metastatic non-squamous NSCLC patients (Example XIII), a subset of the entire study population ( (6 patients per group) also participated in a PK sub-study to investigate the drug-drug interaction between bavituximab and docetaxel. At designated time points in cycles 1 and 2, additional blood collections were performed on these patients. No clinically relevant pharmacokinetic drug-drug interactions were observed for bavituximab with docetaxel. In addition, docetaxel exhibited similar pharmacokinetic characteristics with and without administration of bavituximab. Therefore, no clinically relevant pharmacokinetic drug-drug interactions were observed for bavituximab with docetaxel in these patients.

Example VII
Treatment of Viral Infections in Patients Using Bavituximab In this example, data to illustrate some of the clinical experience in treating viral infections in patients using vituximab, including bavituximab in combination with ribavirin Is presented. Data are also presented to show that at selected clinical doses, administration of bavituximab does not appreciably reduce β2GPI levels in human subjects.

A. Phase I Study in HCV Patients Bavituximab was first evaluated in a Phase I open label escalating dose escalation study and a Phase Ib open label escalating escalating dose study in patients chronically infected with hepatitis C virus (HCV). . These studies involved 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, a continuous cohort of 6 patients), 0.3, 1, 3, and 6 mg / Kg dose was administered in Phase Ib (24 cohorts, 4 cohorts of 6 patients).

  In phase I and Ib studies in HCV patients, all dose levels of bavituximab were well tolerated. In Phase I, a transient decrease in viral load suggestive of antiviral activity was observed at all dose levels. In phase Ib, the viral load decreased slightly after treatment with doses of 0.3, 1, and 6 mg / kg of bavituximab, and these reductions were often transient, but at least 1% in each cohort. In human patients there was a sustained decrease in viral load. In particular, at a dose of 3 mg / kg bavituximab, a consistent reduction in HCV was demonstrated through study treatment and follow-up.

B. Bavituximab does not deplete β2GPI The Phase Ib study described above also measured levels of β2GPI in patients to determine whether administration of bavituximab altered β2GPI levels in these human subjects. The results are depicted in FIG. In patients receiving 1 mg / kg of bavituximab, β2GPI levels were substantially unchanged. In patients receiving 3 mg / kg bavituximab, a transient reduction (20-25%) in serum levels of β2GPI was observed. However, such a reduction did not change statistically significantly from the pre-dose level (FIG. 14). In fact, at a dose of 3 mg / kg of bavituximab, β2GPI levels remained within the normal range and returned to pre-treatment levels within 24 hours. In contrast, β2GPI was significantly reduced (p <0.02) in patients receiving 6 mg / kg bavituximab (FIG. 14). At the 6 mg / kg dose, β2GPI levels were reduced by 40% to near the lower end of the normal range, as compared to pre-treatment levels. Nevertheless, even in human subjects treated with 6 mg / kg bavituximab, β2GPI returned to baseline levels within 3 days.

  Thus, these data confirmed the selection of a 3 mg / kg dose of bavituximab for use in humans. This dose is an effective concentration at which bavituximab and β2GPI are effective to allow the binding of the bavituximab-β2GPI complex to the exposed PS on cells at the disease site without depleting plasma β2GPI levels. At the maximum dose. However, this data also shows that any reduction in β2GPI during bavituximab treatment is only transient, and that β2GPI levels are restored within 3 days.

C. Phase I Study in HCV-HIV Patients A separate Phase Ib multicenter, open-label, randomized, dose-escalating, repeated-dose study was performed to identify chronic HCV (most of HCV genotype 1) and human immunodeficiency virus (HIV). ) Was evaluated in patients co-infected. The primary objective was to determine safety, tolerability, PK profile, virus kinetics, MTD, and / or MED. The study involved a scheduled visit of 16 times over approximately 16 weeks. Babituximab was administered at the following doses: 0.3 mg / kg (6 patients), 1 mg / kg (6 patients), 3 mg / kg (9 patients), and 6 mg / kg (6 patients). Administered to a continuous cohort of patients. Patients received bavituximab intravenously every week for 8 weeks. All patients in the cohort proceeded with dose escalation after completing the first four weeks of dosing without a thrombotic event classified as a serious adverse event (SAE).

The median baseline HCV viral load was 6.76 log ₁₀ and the median HIV baseline was 3.99 log ₁₀ . At specific times during the study, plasma viral loads of HCV and HIV were measured. When treated with bavituximab at all dose levels, some patients in each treatment group displayed transient antiviral activity (maximal 0.5 log ₁₀ or more HCV and / or HIV viral load from baseline). Reduction).

D. Phase II Study in HCV Patients A phase II multicenter randomized active control study was performed to evaluate bavituximab in combination with ribavirin for the initial treatment of chronic HCV (genotype 1) infection. The primary endpoint was the percentage of patients who had an early virological response (EVR) at study week 12, which was defined as a 2-log ₁₀ international unit (IU) or greater reduction in HCV RNA levels. . Safety was included as a secondary endpoint.

  Patients received a maximum of 28 days of screening / wash-out period, followed by randomization (at a ratio of 1: 1: 1), all of which were given twice daily oral ribavirin 1000 mg (<75 kg body weight) or 1200 mg (> 75 kg body weight) With 0.3 or 3 mg / kg of weekly infusion of bavituximab or 12 weeks of subcutaneous injection of pegylated interferon alpha-2a (pegylated interferon, also referred to as PEG-IFNα-2a). Patients who exhibited EVR after 12 weeks received off-study treatment with pegylated interferon plus ribavirin for up to 48 weeks.

  A total of 66 patients (38 men, 28 women) with an average age of 39.1 years were enrolled in this study. Each of the 22 patients received 0.3 mg / kg of bavituximab, 3 mg / kg of bavituximab, and pegylated interferon. The median doses of 0.3 and 3 mg / kg of bavituximab received were 12 doses each, with an average treatment period of 78 and 75 days, respectively.

  In this study, some patients treated with bavituximab plus ribavirin showed a gradual viral reduction over 12 weeks. Interestingly, EVR was seen in twice as many patients in patients treated with lower doses of bavituximab (0.3 mg / kg), as opposed to higher doses of 3 mg / kg bavituximab. (18% vs. 9%). At all doses, EVR rates were higher in patients receiving pegylated interferon than in patients receiving bavituximab, but bavituximab showed a more favorable safety profile, indicating that pegylated Almost twice as many patients in the interferon group reported AEs.

Example VIII
Treatment of Breast Cancer Patients with Bavituximab and Paclitaxel Turning to clinical cancer treatment, this example provides data from the treatment of patients with HER2-negative metastatic breast cancer using bavituximab in combination with taxane, paclitaxel .

In a single-center investigator-initiated study, 14 patients with HER2-negative metastatic breast cancer had 1 patient with paclitaxel (80 mg / m ² ) administered on days 1, 8, and 15 in a 4-week cycle. They received 3 mg / kg of bavituximab once a week. Bone pain, fatigue, headache, and neutropenia were the most common adverse events (AEs). The addressable infusion-related response was the most common AE associated with bavituximab. Bavituximab showed no evidence of increased thrombogenicity. Treatment resulted in an overall response rate (ORR) of 85%, two patients showing a complete response, and a median progression-free survival (PFS) of 7.3 months (95% CI: 2.8, 10. 8).

  In summary, this study showed that bavituximab in combination with paclitaxel was well tolerated for the treatment of patients with metastatic breast cancer, with promising results observed with respect to clinical response rate (RR) and PFS.

Example IX
Treatment of Breast Cancer Patients with Bavituximab and Paclitaxel-Carboplatin This example evaluates the safety and efficacy of bavituximab plus paclitaxel and carboplatin in patients with locally advanced or metastatic breast cancer that is not restricted to hormonal or HER2 status. We report the results of a phase II, open-label, single-arm study.

  This Phase II study utilized Simon's two-stage design. Fifteen patients were enrolled in Stage A and the study was expanded to a further 31 patients in Stage B, for a total of 46 patients. The primary objective was to determine the complete response (CR) + partial response (PR), or overall response rate (ORR), defined as CR + PR. Secondary objectives included time to tumor progression, duration of response (DOR or DR), overall survival (OS), and safety.

Up to 6 cycles, on days 1, 8, and 15 of a 28-day cycle, in combination with carboplatin (AUC = 2 dose) and paclitaxel 100 mg / m ² once a week until disease progression with bavituximab (3 mg / day) kg). Sixteen of the 46 patients (34.8%) were naive.

  The most common adverse event (TEAE) that occurred under Grade 4 treatment was neutropenia (12 patients, 26.1%), which was treated with the chemotherapy used in this study Expected incidence in patients. The most common grade 3 TEAEs are leukopenia (11 patients, 23.9%), neutropenia (9 patients, 19.6%), and anemia (5 patients, 10. 9%). These are also the expected incidence in patients treated with the chemotherapy used in this study.

  Objective response according to the Solid Tumor Response Criteria (RECIST) occurred in 34 of 46 patients (73.9%), 5 of 46 patients (10.9%) had CR, 29 One patient (63.0%) had a PR. The mean duration of response (DOR) is 3.7 months (95% confidence interval [CI]: 3.1, 5.8), and the median PFS is 6.9 months (95% CI: 5.6; 7.7). At the end of the study, the median OS was determined to be 23.2 months (95 CI: 553 days until "not determined"). These results are very promising for the ongoing development of Bavituximab, especially in combination therapy.

Example X
Example 3 Treatment of Breast Cancer Patients with Bavituximab and Docetaxel This example demonstrates another phase II study to evaluate the safety and efficacy of bavituximab in combination with docetaxel in patients with locally advanced or metastatic breast cancer. We report the results of a blinded, single-arm study.

  This study was also a Phase II multicenter study utilizing Simon's two-stage design. Fifteen patients were enrolled in Stage A and the study was expanded to a further 31 patients in Stage B, for a total of 46 patients. The main objective was to determine ORR (CR + PR). Secondary objectives included time to tumor progression, DOR, OS, and safety.

Administer bavituximab (3 mg / kg) once a week until progression in combination with docetaxel (35 mg / m ² ) given on days 1, 8, and 15 of a 4-week planned cycle for up to 6 cycles Was. All patients received one prior chemotherapy regimen. Of the most common TEAEs reported, only fatigue, headache, back pain, and hypertension were grade 3 or higher.

  In this study, 28 (60.9%) of 46 patients were determined to have an objective response, and 5 (10.9%) of 46 patients had a CR and 46 Twenty-three (50.0%) of the patients had PR. The median DOR was 6.1 months (95% CI: 5.7, 7.5) and the median PFS was 7.4 months (95% CI: 6.1, 9.1). . At the time of the final analysis, the median OS was approximately 20.7 months (95% CI: 16.1 months to "not determined"). These data provide strong support for further development of bavituximab, including combination therapy with docetaxel.

Example XI
Treatment of Liver Cancer Patients with Bavituximab and Sorafenib This example presents data from the treatment of a patient with advanced hepatocellular carcinoma (HCC) using bavituximab in combination with sorafenib.

  A phase II single center study of bavituximab and sorafenib in advanced hepatocellular carcinoma (HCC) was performed. Patients received 3 mg / kg of bavituximab intravenously (IV) once a week and 400 mg of sorafenib orally twice daily (PO BID) until radiological progression. Secondary endpoints included overall survival (OS), disease-specific survival, 4-month progression-free survival, safety, and response rate. The study recruited 38 patients.

  In the relevant bridging data from six patients in this study, similar to that shown for relevant PS-targeted antibodies in multiple preclinical cancer models, half of the evaluated patients had anti-virus after one cycle of bavituximab treatment. It was determined to have an increase in tumor immune cells. In addition, increased immune responses were associated with patients who continued study treatment for longer periods, suggesting a clinically significant anti-tumor immune response. Three of the six patients evaluated had increased infiltration of activated anti-tumor T cells (CD8) into the tumor microenvironment, which correlated with an increased time to disease progression. In addition, these responding patients first show lower levels of PD-1-positive cells (which are well-established markers of T cell activation and disease outcome) prior to treatment initiation and can subsequently be measured after bavituximab treatment Showed a significant rise.

  Clinically, no grade 4 or 5 adverse events were recorded. The most common all-grade events were diarrhea (32%), fatigue (26%), and anorexia (24%). The median OS (mOS) was 6.2 months. Two patients achieved a partial response with a 4-month PFS of 61%.

  These results demonstrated that bavituximab and sorafenib were well tolerated in patients with advanced HCC without the signs of autoimmune adverse events seen with other checkpoint immunotherapy. Clinical outcomes of time to progression, disease control rate, and 4-month progression-free survival indicate that many prior treatments have very poor prognosis due to their unfavorable disease biology, including high rates of macrovascular invasion Particularly promising in this patient cohort with a history.

Example XII
Treatment of Pancreatic Cancer Patients with Bavituximab and Gemcitabine This example presents data for the treatment of patients with previously untreated stage IV pancreatic cancer using gemcitabine in combination with bavituximab.

  This study (PPHM1002) is a phase II, randomized, open-label study to assess gevicitabine when administered with or without bavituximab in patients with previously untreated stage IV pancreatic cancer Research. The main objective was to compare patients' OS between treatment groups. Secondary objectives included comparing PFS, ORR, DR, and safety.

Enrolled patients were randomized in a 1: 1 ratio to receive study treatment with gemcitabine alone or in combination with 3 mg / kg of bavituximab once a week. Gemcitabine (1000 mg / m ² ) was given on days 1, 8, and 15 of each 28-day cycle (4 weeks) until disease progression or unacceptable toxicity. A total of 70 patients were enrolled in this study. In general, the patient population has a very broad disease burden, which may reduce the response in both treatment groups.

  The most common TEAEs in the bavituximab plus gemcitabine arm were nausea (44.1%), anemia (35.3%), and fatigue, constipation, and anorexia (each occurring in 32.4% of patients). Was. Three (9.1%) patients randomized to gemcitabine alone had grade 5 (fatal) events (sudden death [1], liver abscess [1], and cardiac arrest [1]) It had. No grade 5 (fatal) events occurred in the gemcitabine + bavituximab group.

  Although most efficacy endpoints were comparable between treatment groups, there was a numerically higher response rate and survival probability at 1 year in the bavituximab and gemcitabine groups. At the end of the study, the median overall survival (95% CI) was 5.2 (4.0-6.3) months for the gemcitabine alone treatment group and 5.6 (4.7-4.7) for the bavituximab plus gemcitabine treatment group. 7.0) months. These results for the addition of bavituximab are particularly promising in this patient population with a very broad disease burden.

  After a functional β2GPI analysis of Example XVII and a functional β2GPI analysis of Example XVII showing that functional β2GPI levels correlate with therapeutic results, the archival samples from this Phase II study were also tested for functional β2GPI did. As reported in Example XVIII, the results of these analyzes reinforce the discovery that functional β2GPI levels are a biomarker for successful treatment of bavituximab.

Example XIII
Phase II Study of Bavituximab and Docetaxel in NSCLC Patients Based on phase I and single-arm phase II studies, this example demonstrates the use of previously treated stage IIIb / IV non-squamous non-small cell lung cancer (NSCLC). For a phase II trial of testing bavituximab plus docetaxel in patients with.

  This study (PPHM0902) is a phase II randomized, double-blind, placebo-controlled study evaluating bavituximab plus docetaxel in patients with previously treated locally advanced or metastatic non-squamous NSCLC. The main purpose of this study was to compare the ORR (CR + PR) between treatment groups. Secondary objectives included comparing PFS, DR, OS, security, and PK.

Patients were randomized in a 1: 1: 1 ratio to receive docetaxel + placebo, docetaxel + 1 mg / kg bavituximab, or docetaxel + 3 mg / kg bavituximab. Docetaxel 75 mg / m ² was given on the first day of each 21-day cycle for up to six cycles, and placebo or the assigned dose of bavituximab was administered once a week. Until progression or toxicity, patients continued to receive their assigned blinded treatment (placebo, 1 mg / kg bavituximab, or 3 mg / kg bavituximab) once a week.

  A subset of the entire study population (6 patients per group) participated in a PK sub-study to investigate drug-drug interactions between bavituximab and docetaxel. At specific points in cycles 1 and 2, additional blood collections were performed on these patients.

  A total of 121 patients (76 men and 45 women) with an average age of 60.0 years were enrolled in this study. The Independent Data Monitoring Committee (IDMC) has determined that open labeling of study treatment is recommended because the ORR primary endpoint has been reached, and unblinded study treatment following this meeting. In addition, no safety concerns or issues were identified by the IDMC.

  After open labeling of the study, packaging and labeling label errors were found involving the placebo and 1 mg / kg groups. A study summary was submitted to the Food and Drug Administration (FDA) to pool data from patients receiving placebo or 1 mg / kg bavituximab to form a composite control group for exploratory analysis and comparison to the 3 mg / kg bavituximab group did.

  Overall, no significant differences in the incidence of AEs between treatment groups by toxicity grade were observed. No significant difference in SAE was observed between treatment groups. Three patients in the combined control group (3.8%) and two patients in the 3 mg / kg bavituximab group with docetaxel (5.0%) had grade 5 (fatal) events. Combined control patients with fatal events included 1 patient with sepsis, 1 patient with a cerebral vascular attack, and 1 patient experiencing both pneumonia and pseudosepsis. In the 3 mg / kg bavituximab plus docetaxel group, one patient had fatal sepsis unrelated to bavituximab and one patient also had a senile event unrelated to bavituximab.

A summary of efficacy endpoints is presented in Table 6, in which the analysis is based on Intent to Treat (ITT) population and central decision data. All endpoints (ORR, PFS, and OS) demonstrated a tendency for 3 mg / kg bavituximab to predominate as compared to the combined control group (placebo or 1 mg / kg bavituximab). Compared to the combined group, the ORR of 3 mg / kg bavituximab was approximately 50% higher. The median PFS was similar between the combined group and the 3 mg / kg bavituximab group, but the median OS of patients receiving 3 mg / kg bavituximab was about 60% longer. In particular, patients in the combined group had only 7.3 months of mOS, whereas patients treated with 3 mg / kg of bavituximab plus docetaxel had 11.7 months of mOS (HR = 0.66).

  Following the Phase III study of Example XIV, and the analysis of functional β2GPI in Example XVII, which showed that functional β2GPI levels correlated with therapeutic outcome, the stored samples from this Phase II study were also functional. Was tested for sex β2GPI. The results of these analyzes, described in Example XVIII, further confirm that functional β2GPI levels are a biomarker for successful treatment of bavituximab.

Example XIV
Phase III Study of Bavituximab and Docetaxel in NSCLC Patients As reported in the previous examples, the overall results of the Phase I and Phase II studies demonstrated a clinically significant therapeutic effect of Bavituximab. Based on such results, especially the double blind phase II study described above, a phase III study was performed and this example describes the phase III study and the data obtained.

The phase III trial (PPHM1202) was a randomized, double-blind, placebo-controlled, multicenter trial of bavituximab plus docetaxel in previously treated stage IIIb / IV non-squamous non-small cell lung cancer (NSCLC) patients. This global, double-blind, phase III study was initiated in 2012. The selection criteria were for patients with stage IIIb / IV non-squamous NSCLC that had progressed platinum-doublet chemotherapy (in the case of known EGFR or ALK mutations, they would have progressed with appropriate targeted therapy). Yes (ECOG PS0-1 and previous immunotherapy allowed). In this study, 597 such patients were recruited in a 1: 1 ratio and administered docetaxel (75 mg / m ² ) for up to 6 cycles of 21 days at 3 mg / kg weekly until progression or toxicity. They were given in combination with bavituximab (babituximab + docetaxel) or placebo (docetaxel alone). The primary endpoint was overall survival (OS), and secondary endpoints were objective response rate (independent central judgment, ICR), progression-free survival (ICR), safety, PK, quality of life (OS). LCSS), and exploratory biomarkers including immune correlation. The baseline characteristics of the selected patients are shown in Table 7, where the "placebo" column refers to patients treated with docetaxel alone and the "babituximab" column refers to patients treated with babituximab + docetaxel.

A. Safety Median OS (mOS) was evaluated because 70% of targeted OS events were reached (see below). Throughout the study, it was determined that safety profiles were generally similar between groups. A summary of treatment and safety has recently been published (Palmero et al., 2017). As reported therein, the safety profile of the combination of bavituximab and docetaxel is similar to the combination of placebo + docetaxel. Grade ≥3 adverse events occurred in 68% of patients in the bavituximab plus docetaxel group and in 60% of patients in the docetaxel alone group. Treatment-related AEs reported in more than 15% of patients have recently been reported (Palmero et al., 2017). In addition, treatment-related grade 3/4 febrile neutropenia was slightly higher with bavituximab plus docetaxel (8%) than with docetaxel alone (5%). The number of patients treated with docetaxel alone (placebo, n = 300) in the baseline characteristics of Table 7 was based on the ITT population, ie, including all randomized patients, while published in Palmero et al., 2017 Note that the number of patients treated with docetaxel alone (placebo, n = 299) is based on a safety population, ie, includes all randomized patients who received treatment.

B. Efficacy Since reaching 70% of targeted OS events, the mOS in 297 patients in the bavituximab plus docetaxel group was 10.7 months (95% confidence interval [CI], 8.6-11.5). MOS in 300 patients in the docetaxel alone group was 10.8 months (95% CI, 9.2 to 12.6) (mortality hazard ratio (HR), 1.10 (0.89, .37)). Progression-free survival (PFS) was also similar in the two groups when reaching 70% of targeted OS events, with a median PFS of 4.1 months in the bavituximab + docetaxel group and 3 months in the docetaxel alone group. .9 months. Subsequent immunotherapy received about 15% of the patients in the study and was evenly distributed between the bavituximab plus docetaxel group and the docetaxel alone group (see Example XIX).

  A follow-up period of 12 months has elapsed since the last patient was randomized and reached approximately 85% of targeted OS events, with a median OS of 10.2 for 297 patients in the bavituximab + docetaxel arm. At 5 months (95% confidence interval [CI], 8.4 to 11.9) and at 10.9 months (95% CI, 9.2 to 12.1) in 300 patients in the docetaxel alone group (HR, 1.06; P = 0.533). PFS at this stage was 4.2 months (95% CI, 3.9-4.6) in the bavituximab plus docetaxel group and 4.1 months (95% CI, 3.2-2.5) in the docetaxel alone group. 4.8) (HR, 1.02; P = 0.876). The ORR at this stage was 15% in the bavituximab + docetaxel group, whereas it was 11% in the docetaxel alone group (odds ratio 0.7, P = 0.15).

The efficacy analysis (ITT) at this stage is listed in Table 8, where the P-value is based on the two-sided stratified Cochran-Mantel-Haenszel exact method. Stratification factors include stage (IIIB vs. IV), geographic region (North America, Europe, other regions), previous maintenance and / or targeted therapies (yes vs. no).

  These results of the median OS were unexpectedly different from the phase II data described above in Example XIII and the hypothetical mOS used for the power of the study, the latter of which was 9.1 months with bavituximab + docetaxel And docetaxel alone was 7.0 months mOS (assuming 9.1 vs. 7.0 months mOS, providing 80% power and a one-sided significance level of 2.5%) 473 OS events; HR 0.77).

  A retrospective VeriStrat® proteomics study demonstrated a good VS sign in 80% of the bavituximab plus docetaxel group and 84% of the docetaxel alone group (Example XV). Although this Phase III study in patients with previously treated non-squamous NSCLC did not meet the main purpose of superior OS in the bavituximab plus docetaxel group, this result was notable, especially in the docetaxel alone group. It may have been affected by a higher percentage of VS good signatures than expected.

Example XV
Initial Biomarker Analysis of Phase III Trials of Bavituximab In connection with the Phase III trials described above, one or more biomarkers, or a pattern of biomarkers (of bavituximab Biomarker analysis was performed to identify the "sign". This example describes the first proteomic code analysis for a sample collection technique applied to later studies.

A. Sample Collection A Phase III study was designed to obtain informed consent for the collection of patient blood samples. Patient blood samples were obtained using appropriate phlebotomy techniques. The tourniquet was placed 7-10 cm above the venipuncture site, but the application of the tourniquet for preliminary vein selection did not exceed 1 minute. The patient was closed without grasping his fist and the venipuncture site was washed with a 70% isopropyl alcohol pad and air-dried using a circular motion from center to periphery.

  Using a 21 gauge needle, the patient's blood was collected in 5.0 ml gold-covered serum separator tubes (SST). The tourniquet was removed as soon as blood began to flow and the tubing was completely filled. Immediately after collection, the tubes were inverted five times and allowed to solidify for at least 30 minutes. The tubes were centrifuged at 1,000-1,300 g for 15 minutes within 30-60 minutes after collection to separate the serum. Using a pipette, approximately 1.25 ml of serum was transferred to two 3.6 ml cryovial tubes and the samples were frozen.

  The frozen vial was placed in a sample bag and tightly sealed. Dry ice was layered on the bottom of the dry ice transport box, and the sample bag was placed in this box. Dry ice was added until the box was full, the lid was locked in place, and the samples were transported to the central laboratory for storage at -70 ° C.

  The central lab prepared vials for partial aliquots by thawing the samples. Using a pipette, at least 250 μl of the serum was transferred to four 2 ml naturally capped cryovial tubes and refrozen at −70 ° C. The same transport instructions were repeated and the partially aliquoted samples were transported to the laboratory frozen on dry ice for test biomarkers.

B. VeriStrat® Analysis Understanding the multidimensional features of cancer is important for patient selection and treatment planning. The VeriStrat® test is a blood-based, commercial, predictive and prognostic proteomics test for patients with advanced NSCLC. In addition to being prognostic, VeriStrat predicts the differential therapeutic benefit when choosing from monotherapy options. VeriStrat was performed retrospectively on patient samples from Phase III trials.

  Using mass spectrometry to classify patients as poor VeriStrat (VS) correlating with more invasive disease (VS-P) or better VS correlating with better prognosis (VS-G), Patient pre-treatment serum samples were tested for protein expression. OS was analyzed by the VeriStrat subgroup using Kaplan-Meier statistics.

  VeriStrat classification was available for 569 of 597 randomized patients. In the bavituximab + docetaxel group, 80% had good VS and 20% had poor VS. In the docetaxel alone group, 84% had good VS and 16% had poor VS. Thus, in the Phase III study, the good / bad signs of VeriStrat were roughly balanced between treatment groups.

  The median overall survival (mOS) was 11.5 months (95% confidence interval [CI], 10.6-12.9) for all good VS, and 5.7 months (95% for poor VS). CI, 4.2-7.2) (p <0.0001). HR OS (VS good vs. VS bad) 0.49 (95% CI 0.37-0.64); p <0.001. These VeriStrat results are consistent with the PROSE test (Gregorc et al., 2014) and the overall prognosis of PFS and OS.

  Among patients with good VS, the mOS of the bavituximab + docetaxel group was 11.2 months (95% CI, 10.2 to 12.8), and the docetaxel alone group was 11.8 months (95% CI, 10.2 months). 4-13.5) (p = 0.38). Among the patients with poor VS, the mOS in the bavituximab + docetaxel group was 5.8 months (95% CI, 5.0 to 11.3), and the docetaxel alone group was 4.7 months (95% CI, 3.0%). 4 to 7.2) (p = 0.27). Given the limited treatment options for this group of patients, the ability of bavituximab to improve OS in patients with poor VS is important.

  In conclusion, the VeriStrat results of the Phase III trial are an overall prognosis for PFS and OS, but do not predict a response to bavituximab treatment. Unexpected OS results in the docetaxel group may have been affected by a relatively high overall proportion of VeriStrat good patients. Notably, the percentage (> 80%) of VeriStrat good patients in this phase III study was higher than previously reported (about 67%), indicating that patients had an overall good prognosis. Thus, the performance of the docetaxel group that is better than expected is partially explained.

  Apart from the aforementioned Veristrat analysis, a separate proteomics approach was also explored, especially for Bavituximab. Although extensive mass spectrometry and correlation analysis were performed to investigate studies that could identify a subgroup of patients that would benefit from the addition of bavituximab, enrichment analysis of such gene sets has The lack of identification of any markers relevant to clinical benefit has emphasized the need for further research and potential new approaches.

Example XVI
Assay for Functional β2GPI This example relates to the development of a specifically designed β2GPI assay for the detection of functional (active) β2GPI in a fluid sample. This test method is uniquely adapted to detect and quantify functional β2GPI (meaning β2GPI capable of binding to both PS and Bavituximab). Thus, this example provides a previously unavailable means required for further meaningful biomarker analysis in connection with Bavituximab treatment.

A. Materials and Methods Materials and Devices The following specific materials and devices were used in the assays to produce the results presented in sections B1 and B2 of this example. Materials: 96-well medium-bound flat bottom plate (Greiner BioOne, cat # 655001), 96-well non-bound round bottom plate (Costar, cat # 3605), hexane (Sigma, cat # 32293), PS antigen (Sigma, catalog #) P6641), ovalbumin (Sigma, catalog No. A5503), chromogenic substrate tetramethylbenzidine (TMB) (KPL, catalog No. 50-76-00), 2M in _H 2 _SO 4 (Fisher, catalog number SA818-4), Plate cover (Fisher 015-027-11), adhesive plate sealer (VWR232701), reagent reservoir (VistaLab cat # 3054-1000). 1.5 ml microcentrifuge tubes, 50 ml conical tubes, and 15 ml conical tubes were also utilized.

  Equipment: Vortex (Scientific Industries), timer (VWR62344-64), 10-1000 [mu] l pipettor (Rainin), 100-300 [mu] l multi-channel pipettor (Rainin), 450 and 650 nm plate reader (EN1835). A balance, stir bar, and 37 ° C. incubator were also utilized. SoftMax® Pro software was used for this assay.

2. Buffers and Techniques The wash buffer is 1 × phosphate buffered saline (PBS) and the blocking buffer is 2% ovalbumin in 1 × PBS.

  When working in large volumes (eg, 500 μl or more) throughout the assay, subtractive pipetting was utilized. All amounts of diluent were pipetted first. An equal amount of diluent was removed before adding additional reagents. All potentially dangerous vapors were handled in the draft.

3. Bavituximab-HRP
Bavituximab antibody was conjugated to horseradish peroxidase (HRP) to prepare a bavituximab-HRP detector for use in the assay. Using EZ-Link® Plus Activated Peroxidase (Thermo Scientific, Cat. No. 31487) followed by a procedure (provided by the manufacturer) to conjugate the activated peroxidase to the antibody at pH 7.2 Was executed. 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 and reconstituted. Immediately after reconstitution, 10 μl of a 5 M sodium cyanoborohydride solution was added to the reaction and incubated for 1 hour at room temperature. After completion of the incubation, 20 μl of stop buffer was added and incubated for 15 minutes at room temperature. Conjugated bavituximab-HRP (1 mg / ml) was stored at 4 ° C for up to 4 weeks.

4. Coating ELISA plates were coated with PS antigen as follows. 5 μg / ml PS antigen was prepared and diluted in 6 ml hexane in a draft with the blower off. Using a 12 channel pipette, 50 μl of PS solution was added to each well. The draft blower was turned on again and the hexane was allowed to evaporate for 30-45 minutes, typically 30 minutes.

5. Blocking PS coated ELISA plates were blocked as follows. 100 ml of blocking buffer (2% ovalbumin in 1 × PBS) was prepared per plate. Using a 12 channel pipette, 200 μl of blocking buffer was added to each well. Blocked ELISA plates were incubated at 37 ° C. for 120 minutes (± 10 minutes, which did not change assay performance).

6. Sample preparation Assay standards, positive controls, and sample preparation were performed as described below.

The β2GPI standard for the positive control was obtained from Haematologic Technologies, Inc. in a buffer of 0.2 M glycine, 0.15 M NaCl, pH 7.4. (HTI, catalog number B2G1-0001-C, 1.0 mg / ml). The vials of β2GPI were thawed and standards and positive control preparations were performed as follows.
Prepare 1 ml of 10 μg / ml β2GPI substock A in blocking buffer,
Prepare 1 ml of 1,000 ng / ml β2GPI substock B in blocking buffer by subtractive pipetting 100 μl from substock A,
Prepare 1 ml 250 ng / ml β2GPI standard in blocking buffer by subtractive pipetting 250 μl from substock B,
200 ng / ml, 75 ng / ml, 30 ng / ml, and 5 ng / ml control samples were prepared from the 1000 ng / ml substock according to Table 9 using subtractive pipetting.

  Unknown samples were prepared for testing as follows. Unknown samples are prepared in blocking buffer at a final dilution of 1: 4000 and 1: 8000, unknown samples at a 1: 100 dilution are prepared first, and a 1:40 dilution to a 1: 100 dilution. To achieve a dilution of 1: 4000, and a dilution of 1:80 was prepared from a dilution of 1: 100 to achieve a dilution of 1: 8000.

Preparation of unbound plates was performed as follows. 75 μl of blocking buffer is added to columns 1 to 3 of rows BH, 150 μl of a 250 ng / ml standard is added to columns 1 to 3, row A, and using a multichannel pipette, columns 1 to 3 Was serially diluted from row A to row G, 75 μl of positive control and sample were added to designated wells, and 75 μl of blocking buffer was added to all empty wells. The plate settings are shown in Table 10.

7. Detection Before termination of blocking, 6 ml of 300 ng / ml Bavituximab-HRP was prepared in blocking buffer. The assay plate was washed with 1 × PBS by pipetting 250 μl into each well, which was repeated twice more. A plate washer may be used, in which case the plate is washed once with 1 × PBS. The plate was as dry as possible.

  50 μl of 300 ng / ml Bavituximab-HRP was added to all wells of the assay plate. From each corresponding well of the unbound plate, 50 μl was added. Using assay and unbound plates in this way means that detectably labeled bavituximab is first added to the PS-coated assay plate before adding the sample containing β2GPI from the unbound plate. I do. This order avoids cross-contamination during pipetting. Samples containing bavituximab-HRP and β2GPI were incubated together on the plate and the incubation was performed at 37 ° C. for 90 minutes.

8. Color development TMB peroxidase substrate and TMB peroxidase solution B were removed from the refrigerator at least 1 hour before use. The assay plate was washed with 1 × PBS by pipetting 250 μl into each well, which was repeated twice more. A plate washer may be used, in which case the plate is washed once with 1 × PBS. The plate was as dry as possible.

A 12 ml TMB mixture was prepared by mixing 6 ml TMB peroxidase substrate with 6 ml TMB solution B. 100 μl of TMB solution was added to each well of the assay plate and allowed to develop for 5-6 minutes. Color development was stopped by adding 100 μl of 2M H ₂ SO ₄ to each well of the assay plate. The assay plate was read and the optical density (OD) was determined at 450 nm within 30 minutes after stopping the reaction. A microplate reader was used in conjunction with the SoftMaxPro plate data and analysis template to provide a printout of the assay data.

9. Preparation of Nicked β2GPI Samples of β2GPI purified from human plasma and recombinant human β2GPI were both treated with plasmin (enzymatic hydrolysis) to prepare samples containing the majority of nicked β2GPI. Nicked β2GPI was not purified to homogeneity in early studies, but it was determined that nicked β2GPI was present beyond nickless, or “intact,” β2GPI.

10. Assay for total β2GPI An assay was designed to detect total β2GPI based on the manufacturer's specifications for the antibodies used. This is an assay using an antibody commercially available from US Biological, in which the plate is coated with a capture antibody against β2GPI and all bound β2GPI is used using an anti-β2GPI-HRP conjugate as the detection antibody. Is detected. Antibody catalog numbers are capture antibody, US Biological No. A2299-81A, affinity purified anti-β2GPI and detection antibody, US Biological No. A2299-81B, peroxidase conjugated anti-β2GPI.

  A 1: 100 dilution of the capture antibody was prepared at pH 9.6 in carbonate buffer (50 mM sodium bicarbonate). 100 μl was added to each well of the ELISA plate and incubated at room temperature. Plates were washed with 1 × PBS buffer containing Tween-20, then blocked with 200 μl per well of assay diluent containing 1% BSA and incubated at 37 ° C. A two-fold dilution standard curve was prepared starting from 500 ng / ml in assay diluent using purified β2GPI. Samples were diluted in assay diluent to achieve concentrations within the linear region of the standard curve. After blocking incubation, plates were washed, followed by addition of 100 μl per well of standard curve and samples in duplicate or triplicate. After addition of the standard curve and the samples, the plates were incubated at 37 ° C. The detection antibody was diluted 1: 400 with assay diluent. After incubation of the sample and the standard curve, the plate was washed, followed by the addition of 100 μl detection antibody per well. Plates were incubated at 37 ° C. After incubation of the secondary antibody, the plates were washed and then developed with TMB. Plates were read on a plate reader at 450 nm and sample concentration was determined from a standard curve.

B. Result 1. Differentiation between functional β2GPI and nicked β2GPI β2GPI purified from human plasma (“human”) or recombinantly expressed β2GPI (“recombinant”) is treated with plasmin and plasmin-cleaved (nick) that does not bind to PS. In) A β2GPI test sample containing the majority of β2GPI was prepared. These samples were purified in this assay (Table 11B) and using an assay designed to detect total β2GPI using commercially available capture and detection antibodies (Table 11A), without plasmin (intact). Tested with β2GPI, and each 50:50 mixture. The results are shown below.

  Both the so-called “total β2GPI assay” using commercially available antibodies (Table 11A) and the “functional β2GPI assay” of the present invention (Table 11B) read similar concentrations of β2GPI (141 ng / ml and 136 ng / ml). It can first be understood that Using the whole β2GPI assay, there is no essential difference in detecting plasmin-treated recombinant β2GPI, with only a modest reduction in detection as the amount of plasmin-treated β2GPI from human plasma increases (141 to 104 ng / ml). In contrast, using a functional β2GPI assay, an increase in the amount of recombinant or plasma-derived plasmin-treated β2GPI results in a significant decrease in binding (136 to 33 ng / ml).

  Thus, consistent with the design of the assay, these results indicate that the assay effectively detects functional β2GPI (ie, β2GPI that binds to both PS and bavituximab), as opposed to nicked β2GPI. Show that you can do it. This distinguishes the functional β2GPI assay of the present invention from commercially available assay kits (and assays using commercially available anti-β2GPI antibodies) that also detect nicked β2GPI along with β2GPI that binds to PS (not to bind to PS). Is done.

2. Quantification of Functional β2GPI The assay can successfully determine the amount of functional β2GPI (which is both β2GPI binding to PS and Bavituximab) in a fluid sample. This assay is currently routinely performed to prepare a reproducible β2GPI standard curve. In this regard, a four-parameter logistic fit is used, which is the statistical equation used for nonlinear regression analysis. The four parameter fitting equation is

Where:
A is the Y value corresponding to the asymptote for the lower values of the X-axis (ie, the flat part of the curve);
B is a coefficient that describes how quickly the curve transitions from the asymptote at the center of the curve, and is commonly called the slope coefficient,
C is the X value corresponding to the midpoint between A and D, commonly called EC50,
D is the Y value corresponding to the asymptote for higher values on the X-axis.

  A representative example of a standard curve of functional β2GPI is shown in FIG. From such a standard curve, the concentration of functional β2GPI in a human blood sample, such as a plasma or serum sample, can be determined. Standard curves are prepared in ng / ml (nanogram / ml), mainly for accuracy, but also for the economics of sample preparation. Since the average level of β2GPI in the normal human population is about 200 μg / ml (micrograms / ml) (Mehdi et al., 1999; Miyakis et al., 2004), the standard curve is that the test sample is diluted before analysis in the assay. Prepared under the expectation that The diluted plasma or serum test sample can be subjected to the assay, and then the patient's β2GPI concentration can be calculated by adjusting the dilution factor.

  This assay is currently used to determine the level of functional β2GPI in patients in the above Phase III study, and the results are presented in Example XVII below, and in Examples XVIII and XX. .

3. Alternative equivalent assay components and steps In addition to the specific materials, devices, and assay steps described in Sections A1-A8 of this example, from the concept of an assay to detect and quantify functional β2GPI Variations in components and method steps may be made and executed without departing from the invention. The following results show that the related drugs can replace the drugs described in sections A1-A8, and that essentially the same results are achieved.

  Certain preferred ELISA plates are those optimized for lipid adsorption and can be used to replace the ELISA plates of section A1 above. An ELISA plate optimized for lipid adsorption is known, which has a surface chemistry that provides better lipid (PS) binding. One such ELISA plate is the ThermoFisher PolySorp® plate used in the new assay format.

  The hexane-based PS coating method of section A4 above preferably replaces the isopropanol-based PS coating method that can provide the user with certain safety benefits (by avoiding the use of hexane). You may. In the new assay format, when using isopropanol as the coating buffer, the ELISA plate is coated with PS antigen using 10 μg / ml PS antigen diluted in isopropanol and the incubation time is 90 minutes.

  To generate a valid β2GPI calibration curve, any known method of obtaining β2GPI can be used. For example, it is purchased from a commercial sales company such as HTI (section A6 above). For clear and reproducible calibration control, alternative β2GPI preparations can also be developed. One such preferred method is to express β2GPI in CHO cells and purify the expressed β2GPI.

  Preferred purification of β2GPI from CHO cells is a clarification of the harvest, a chromatin extraction step that removes contaminants and allows the clarified harvest to pass through a 0.2 μm filter; Use of a tangent flow filtration (TFF) system to buffer exchange the harvested sample and reduce its conductivity without increasing volume; in an anion flow-through mode to remove additional contaminants Capto Adhere step; strong cation step using Nuvia ™ S to remove aggregates and other contaminants, concentrate eluate, and facilitate any buffer exchange; and optionally, purified β2GPI Involves the use of a TFF system for buffer exchange and concentration. β2GPI has thus been expressed and purified and used in new assay formats.

  In addition to section A3 above, certain preferred bavituximab-HRP detectors include two commonly used non-proprietary crosslinkers, SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate). Or a conjugate cross-linked using either SATA (N-succinimidyl S-acetylthioacetate). Other preferred bavituximab-HRP detection agents provide conjugates in which the number of HRP exceeds the number of bavituximab antibodies, especially HRP: bavituximab ratios of 2: 1 or 3: 1 and have essentially free (unconjugated) antibodies. It does not. Such a conjugate is purified by an S-300 sizing column to remove unreacted reaction components. Bavituximab-HRP detectors having each of these components and properties are described in Columbia Biosciences, 4985 Winchester Blvd. , Frederick, Maryland, 21703 and used at 600 ng / ml in a new assay format.

Although one or more of the above alternative components and assay steps are preferred, especially for technical reasons, even the combination of all such alternatives is described in this example (i.e., sections A1-A8). A) provides a functional β2GPI that provides essentially the same results as the assay first described in). The results of such comparisons are shown in Table 12 below, which shows the functional β2GPI measured in two different assay formats using four random human samples (donors) from San Diego Blood Bank. Present the level.

Example XVII
Β2GPI Biomarker Analysis in the Bavituximab Phase III Study Utilizing the functional β2GPI assay described above, this example reports the level of functional β2GPI before treatment in patients in the Phase III study of Example XIV. By correlating the level of functional β2GPI with therapeutic outcome, this example also demonstrates the function as a biomarker for the success of bavituximab treatment, such as in NSCLC patients treated with bavituximab and docetaxel and other combination therapies. It also relates to sex β2GPI.

A. Functional β2GPI Levels in Patients 597 patients were recruited for the above Phase III study. The collection of a patient's blood sample in a phase III study is described in Examples XV, A. At the time of this analysis, there were 592 patient samples for which functional β2GPI could be evaluated. Aliquots of blood samples from these 592 patients were tested for functional β2GPI using the assay described in the immediately preceding example.

A summary of functional β2GPI levels and statistics before treatment at μg / ml is shown in Table 13, where the “Bavituximab” column refers to patients treated with Bavituximab + docetaxel and the “Placebo” column refers to patients treated with docetaxel alone Refers to patients who have had

  The levels of functional β2GPI before treatment ranged from 0.5 to 402 μg / ml, and the distribution of functional β2GPI for all patients is shown in FIGS. 17A and 17B. In patients treated with bavituximab plus docetaxel, functional β2GPI ranged from 22 to 365 μg / ml, as shown in FIG. 17C. The distribution of functional β2GPI in patients treated with docetaxel alone is shown in FIG. 17D, which covers the entire range of this study (0.5-402 μg / ml).

  For each treatment group (202 and 195 μg / ml), and for the entire study (198 μg / ml), the level of functional β2GPI before treatment is consistent with the 200 μg / ml average reported in the literature (20 mg / ml). dl according to Mehdi et al., 1999 and 200 mg / l according to Miyakis et al., 2004).

  The percentage of patients with pre-treatment functional β2GPI levels of 200 μg / ml or higher was determined to be 56% for patients treated with bavituximab plus docetaxel and 49% for patients treated with docetaxel alone.

B. Single cut-off β2GPI biomarker analysis A subgroup analysis performed to assess functional β2GPI as a predictor of response in patients receiving bavituximab plus docetaxel therapy demonstrated a strong trend toward increased survival .

  First, the patient's β2GPI data was evaluated using a single cut-off method. In searching for such an optimal cutoff, step 1 is to search for OS separation or significant OS separation of the high β2GPI group versus the low β2GPI group for patients in the bavituximab + docetaxel group. To explore OS segregation or significant OS segregation for the patients with high β2GPI in the bavituximab + docetaxel group versus the docetaxel alone group (placebo).

  The initial analysis of functional β2GPI as a potential biomarker by applying the single cut-off method to 578 evaluable patients was surprisingly surprising in patients with high β2GPI, in patients with high β2GPI, bavituximab + docetaxel The mOS was 11.9 months (95% CI, 9.0-14.7) in the 167 patients in the group and 9.4 months (95% CI, 7 in the 141 patients in the docetaxel alone group. 0.7 to 11.7) (dead HR, 0.77; P = 0.1). In these initial analyses, “high β2GPI” is defined as a pre-treatment level of functional β2GPI of 200 μg / ml or greater (≧ 200 μg / ml). Because these analyzes are based on a single cut-off, patients without “high β2GPI” have less than 200 μg / ml (<200 μg / ml) functional β2GPI.

  Subsequently, the single cut-off analysis was extended to 592 evaluable patients. Although not statistically significant, these analyzes also demonstrated a surprising trend to prolong survival in the bavituximab + docetaxel group when patients had pre-treatment levels of functional β2GPI greater than or equal to 200 μg / ml. These results are represented in FIGS. 18A and 18B by Kaplan-Meier survival curves for functional β2GPI above 200 μg / ml. Of the 592 evaluable patients, FIG. 18A shows that for patients treated with bavituximab, those with functional β2GPI greater than or equal to 200 μg / ml (167 patients) had mOS of 11.4 months. In contrast, 127 patients with “low β2GPI” less than 200 μg / ml show mOS of only 9.8 months (HR HR, 0.76; P = 0.54, CI (0.58 , 1.01)). As shown in FIG. 18B, the mOS in the 167 patients in the bavituximab + docetaxel group was 11 in patients with pre-treatment levels of functional β2GPI of 200 μg / ml or greater, representing about 52% of randomized patients. MOS at 10.2 months (95% CI, 8.5-11.9) in 146 patients in the docetaxel alone group at .4 months (95% CI, 8.4-16.6). (Dead HR, 0.82; P = 0.134, CI (0.63, 1.06)).

C. Two cut-off β2GPI biomarker analysis After the single cut-off analysis described above, the data in 592 evaluable patients was further analyzed using the two cut-off method (Klein & Moeschberger, 2003). In the two cut-off method, step 1 is to search for a significant separation of "in range" vs. "out of range" OS for patients treated with bavituximab (+ docetaxel) and step 2 is "in range" Is to explore significant OS segregation of the patients with bavituximab versus placebo.

These subgroup analyzes using the two-cut-off method in 592 evaluable patients showed several statistically significant ranges, each showing the survival benefit of bavituximab starting with functional β2GPI of 200 μg / ml or more. Of functional β2GPI, thus confirming the initial surprising finding that pre-treatment levels of functional β2GPI of 200 μg / ml or more are beneficial for treatment with bavituximab. In particular, the two cut-off method showed that pre-treatment levels of functional β2GPI in the range of 210-270, 210-280, 210-290, 200-280, and 200-290 μg / ml, respectively, were treated with docetaxel alone For patients, it was shown to be a statistically significant predictor of the overall survival benefit of patients treated with bavituximab plus docetaxel. These results for the functional β2GPI ranges of 210-270, 210-280, 210-290, 200-280, and 200-290 μg / ml are shown in Tables 14A and 14B.

  Summarizing the results in Table 14A, each of the functional β2GPI ranges from 210 to 270, 210 to 280, 210 to 290, 200 to 280, and 200 to 290 μg / ml has a hazard ratio and survival improvement of less than 1. Has a statistically significant P value. Table 14B shows that patients with functional β2GPI outside of these stated ranges, of course, have a statistically significant P value representing a hazard ratio greater than 1 and worse survival (or increased mortality). Opposite in point. For example, patients with pre-treatment β2GPI levels of 200-290 μg / ml, which account for about 49% of randomized patients, had 11.4 months of mOS when treated with bavituximab (+ docetaxel). And 10.1 months for the control group of patients with the same range of β2GPI levels. This increase of 11.4 months versus 10.1 months reflects a statistically significant improvement in mOS (HR 0.76, P = 0.049).

  There is no indication in the literature that pre-treatment levels of functional β2GPI above 200 μg / ml show a tendency to prolong survival during treatment with bavituximab, 210-270, 210-280, 210-290, 200-280 There is no suggestion that pre-treatment levels of functional β2GPI, and between 200 and 290 μg / ml, predict benefit in overall survival of patients treated with bavituximab. In fact, no significant previous clinical experience with bavituximab suggests such a result. Furthermore, such findings were highly inconsistent with data from extensive preclinical modeling, which indicated that varying levels of serum β2GPI did not significantly affect the treatment results of bavituximab. In particular, preclinical experience has shown that rather low levels of serum β2GPI, such as on the order of about 10-20 to 50-60 μg / ml, are sufficient to support binding and activity of bavituximab (implementation). Example V).

In particular, using different assays, Example V shows 0.12-0.25 (FIGS. 9A and 9B, along with FIGS. 1A, 1B, 1C and 1D), 0.125, 0.5 Β2GPI to antibody molar ratios of ２2 (FIG. 10), 0.93 (FIG. 11), and 1.43-2.86 (FIG. 12) are effective in supporting binding of bavituximab to PS. It indicates that. Considering several different binding and functional test systems, including preclinical data (Table 3) showing that bavituximab is effective at a molar ratio of β2GPI to antibody of about 2.86, the molar ratio of β2GPI to antibody is There is no need to exceed 3. Using a 3 mg / kg dose of bavituximab in this Phase III study, such a ratio is achieved with β2GPI levels below 60 μg / ml (FIG. 19). For reference, the amount of β2GPI, the amount of antibody, and the corresponding β2GPI-antibody ratio in the phase III study are shown in Table 15, where N = patients with functional β2GPI levels within each defined increment. Number (from 592 evaluable patients).

  When comparing Table 15 and Figure 17A with the data used for modeling (Table 1, Table 2, and Table 3), the majority of patients in the Phase III trial have bavituximab at its maximum blood concentration (56.4 μg). Example VI; Gerber et al., 2011) is even more than sufficient to saturate the bavituximab binding (> 2.86), ie, 60 μg / ml or 1.2 μM (Table 15, It can be seen that, starting from FIG. 19), it had functional β2GPI levels consistent with the β2GPI to antibody molar ratio. In fact, 4 out of 592 evaluable patients (0.68%) had pre-treatment levels of functional β2GPI less than 60 μg / ml. In addition, if the level of functional β2GPI is increased (this is true for the majority of patients in the study), the molar ratio of β2GPI to bavituximab is much higher than 2 or 3, exceeding 10 at 200 μg / ml. No previous preclinical modeling or clinical experience has indicated that such β2GPI levels or ratios are beneficial for bavituximab therapy. Rather, as shown in FIG. 19, the preclinical data shows that low levels of serum β2GPI (5 μg / ml β2GPI has a β2GPI: antibody molar ratio of 0.257) starting at about 10 μg / ml or less, and about 60 μg / Ml was simply shown to be sufficient to support binding and activity of bavituximab (Example V).

  Unexpectedly, these detailed analyzes of pre-treatment levels of functional β2GPI, as potential biomarkers for the performance of bavituximab, are very promising. Therefore, measuring the pre-treatment concentration of functional β2GPI in a patient is more likely or most likely to predict response to bavituximab therapy, ie, benefit from treatment with bavituximab Provide strategies for selecting patients. This was first observed, especially in NSCLC, when using bavituximab with docetaxel. However, the mechanism of binding of bavituximab in the complex with functional β2GPI and PS, and the overall mechanism of immune activation of bavituximab, is common to all bavituximab therapies, and therefore has a function of more than 200 μg / ml. Selection of patients based on pre-treatment levels of sex β2GPI can be included in all future trials and therapies using bavituximab with good hope that this will improve treatment outcome. Indeed, further evidence supporting this is provided in Examples XVIII and XX.

Example XVIII
Β2GPI Biomarker Analysis in Additional Bavituximab Clinical Trials Following the identification of functional β2GPI as a biomarker of successful bavituximab treatment in Example XVII, this example demonstrates the use of a functional β2GPI assay in an earlier Expand to test sample. The results below show that the same level of functional β2GPI also correlates with successful treatment results with Bavituximab, thus confirming functional β2GPI as a biomarker for Bavituximab.

A. Example XIII Phase II Test A sample of the NSCLC phase II test of Example XIII (PPHM0902) was tested using the functional β2GPI assay of Example XVI. There is a sample of 119 patients with evaluable pre-treatment levels of functional β2GPI, of which 40 patients are in the bavituximab 3 mg / kg group and 79 patients are in the combined control group (placebo or 1 mg / kg bavituximab). ).

  Pre-treatment levels of functional β2GPI ranged from 0.5 to 266 μg / ml for all patients (FIG. 20A). Functional β2GPI ranged from 0.5 to 266 μg / ml in patients treated with 3 mg / kg of bavituximab + docetaxel (FIG. 20B). The distribution of functional β2GPI in the patients of the composite control group was 0.5-257.4 μg / ml (FIG. 20C). For each treatment group (169.4 μg / ml for 3 mg / kg of bavituximab and 171.8 μg / ml for the combined control group), and for the entire study (171.0 μg / ml), pre-treatment levels of functional β2GPI were published in the literature. Consistent with reported averages.

  Using a “high β2GPI” cut-off, defined as the pre-treatment level of functional β2GPI of 200 μg / ml or greater (≧ 200 μg / ml), the bavituximab 3 mg / kg group had a total of β2GPI greater than 200 μg / ml It was determined that there was a tendency to increase with increasing survival time (FIGS. 21A and 21B), but not in the other group (FIG. 21C). For example, for patients treated with 3 mg / kg of bavituximab, those with functional β2GPI greater than or equal to 200 μg / ml had mOS of 16.8 months, whereas “low β2GPI” less than 200 μg / ml had less mOS. It was 9.4 months (FIG. 21A). Also, in patients with functional β2GPI greater than or equal to 200 μg / ml, the 16.8-month mOS of patients treated with 3 mg / kg of bavituximab exceeded that of only 8.7 months in patients in the composite control group. (FIG. 21B). The distinct separation of the curves in each of FIGS. 21A and 21B is compared to the overlaid curves of FIG. 21C (comparison of β2GPI of 200 μg / ml or greater in the composite control group with β2GPI of less than 200 μg / ml).

B. Example XII Phase II Study A sample of the Phase II pancreatic cancer study of Example XII (PPHM1002) was tested using the functional β2GPI assay of Example XVI. There were 31 patient samples with evaluable pre-treatment levels of functional β2GPI. Pre-treatment levels of functional β2GPI ranged from 82.5 to 343.2 μg / ml for all patients (FIG. 22). For these 31 patients, the mean level of functional β2GPI before treatment (219.2 μg / ml) was consistent with the mean reported in the literature.

  The sample size is small and the disease is very invasive, but using a “high β2GPI” cutoff of functional β2GPI of 200 μg / ml or more (≧ 200 μg / ml), 200 μg / ml for bavituximab It was determined that the above β2GPI tended to increase with increasing overall survival. Patients treated with bavituximab with functional β2GPI greater than or equal to 200 μg / ml had a mOS of 7.4 months, whereas 5.3 months with “low β2GPI” less than 200 μg / ml (FIG. 23).

C. Phase II Study of Bavituximab and Paclitaxel / Carboplatin in NSCLC A randomized, open-label, phase II study of paclitaxel / carboplatin, with or without Bavituximab (PPHM1001), was performed in a previously untreated locally advanced or metastatic non-squamous NSCLC Performed in patients with Samples of this test were tested using the functional β2GPI assay of Example XVI. There were 84 patient samples with evaluable pre-treatment levels of functional β2GPI, of which 44 patients were in the bavituximab group and 40 patients were in the paclitaxel / carboplatin group.

  Pre-treatment levels of functional β2GPI ranged from 0.5 to 326 μg / ml for all patients (FIG. 24A). Within patients treated with bavituximab, functional β2GPI ranged from 0.5 to 326 μg / ml (FIG. 24B). Functional β2GPI in patients in the paclitaxel / carboplatin group ranged from 88.8 to 292.7 μg / ml (FIG. 24C). For each treatment group (187.9 μg / ml for bavituximab, 186.4 μg / ml for paclitaxel / carboplatin group), and for the entire study (187.2 μg / ml), the pre-treatment levels of functional β2GPI are again in the literature Consistent with the average reported in

  Using the same “high β2GPI” cutoff that is the pre-treatment level of functional β2GPI of 200 μg / ml or greater (≧ 200 μg / ml), β2GPI of 200 μg / ml or greater is again in the bavituximab group. It was determined that it tended to increase with increasing overall survival, but not in the control (paclitaxel / carboplatin) group. For example, in patients treated with bavituximab, patients with functional β2GPI greater than or equal to 200 μg / ml had a mOS of 17.0 months, whereas “low β2GPI” less than 200 μg / ml at 14.2 months. (FIG. 25A). Also, in patients with functional β2GPI greater than or equal to 200 μg / ml, the 17.0 month mOS of patients treated with bavituximab exceeded the mOS of only 13.2 months of patients in the control group (FIG. 25B). Compare the separation of the curves in FIGS. 25A and 25B, and in particular in FIG. 25A, with FIG. 25C, where there is a tendency for patients of the control group to live longer when β2GPI is less than 200 μg / ml.

  In conclusion, the data for Example XVII and Example XVIII from four separate clinical trials confirm that functional β2GPI levels correlate with treatment outcome, thus confirming functional β2GPI levels as a biomarker of successful treatment with bavituximab Is consistently shown.

Example XIX
Survival of Bavituximab in Combination with Subsequent Immunotherapy Early analysis of the Phase III trial of Example XIV did not show superior OS in the bavituximab + docetaxel group compared to the docetaxel alone group, but did not An ongoing study was undertaken to identify other potential indicators of therapeutic benefit. This example demonstrates that patients treated with bavituximab and docetaxel followed by subsequent immunotherapy (SACT-IO) had a statistically significant better contrast to patients treated with docetaxel alone followed by immunotherapy. Indicates having mOS.

  After treatment with either bavituximab and docetaxel, or docetaxel alone, about 15% (93/597) of patients have a subsequent immune tumor (IO) therapy (IACT) using an immune checkpoint inhibitor (ICI). (IO) in the form of subsequent anti-cancer therapy (SACT). These 93 patients were equally balanced between the treatment groups, 46 patients received pretreatment with bavituximab and docetaxel, and 47 patients received pretreatment with docetaxel alone.

Surprisingly, it was determined that there was a dramatic increase in mOS in patients receiving prior treatment with bavituximab, as opposed to placebo, when treated with subsequent IO (FIG. 26). In particular, for patients receiving subsequent IO, mOS was still not reached in the bavituximab and docetaxel groups (95% CI, 15.2-not applicable), while 12.6 months (95%) in the docetaxel alone group CI, 10.4 to 17.8); HR = 0.46 and p = 0.006 (FIG. 26, Table 16). In patients who did not receive subsequent IO, mOS was 9.2 months in the bavituximab and docetaxel group and 10.2 months in the docetaxel alone group; HR = 1.16 and p = 0.172.

In the subsequent IO group, a specific immunotherapeutic of "first subsequent IO" was also identified. In 46 patients treated with bavituximab (and docetaxel) and subsequent IO, the immunotherapeutic agents are shown in Table 17, all of which are in the form of blocking antibodies that bind CTLA-4, PD-1, or PD-L1. Checkpoint inhibitor antibodies (immune checkpoint inhibitors). In particular, 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. . In summary, 42 of the 46 patients with bavituximab subsequently received nivolumab, two received durvalumab monotherapy, and two received tremelimumab plus durvalumab (Table 17).

  Four patients received more than one IO drug, ie, their “first subsequent IO” therapy itself was “IO concomitant”, ie, the first and second checkpoint inhibitor antibodies. Please note that Thus, in the "ITT" (treatment intent) analysis, 46 patients treated with bavituximab received the first subsequent IO, while in Table 17, there are 48 subsequent IO agents. This is because two patients received the "IO doublet." Overall, four patients received two or more subsequent IOs, each of which received MEDI 4736 (Durvalumab) and tremelimumab doublet. Of these four subjects, two were in the bavituximab group and two were in the placebo group.

  Of the 93 patients undergoing subsequent IO, patients previously treated with docetaxel alone (placebo) also received tremelimumab, nivolumab, or durvalumab (MEDI 4736). In addition, two patients in the placebo group received pembrolizumab (formerly MK-3475), and one patient in the placebo group received REGN2810, both of which are blocking antibodies that bind to PD-1. Overall, the first subsequent IO in 47 patients in the placebo group were tremelimumab (3), nivolumab (40), durvalumab (3), pembrolizumab (2), and REGN2810 (1). This was a total of 49 drugs in 47 patients, two of whom received the IO doublet of Durvalumab (MEDI 4736) -tremelimumab. That is, 40 of 47 patients in the control group subsequently received nivolumab, one received durvalumab monotherapy, one received tremelimumab monotherapy, two received tremelimumab + durvalumab, and two received One received pembrolizumab and one received REGN2810.

  FIG. 26 shows the survival benefit of initial treatment with bavituximab before subsequent IO with respect to time from randomization. The survival benefit of the initial bavituximab treatment before the subsequent IO is even more pronounced as measured as the time since the first subsequent IO treatment. In this context, for patients receiving subsequent IO, mOS is still not reached in the bavituximab and docetaxel groups (95% CI, 10.2 to n / a), while only 6.2 months in the docetaxel alone group (95% CI, 3.9-8.7); HR = 0.42 and p = 0.002.

  In conclusion, the data of this example show for the first time that bavituximab enhances the activity of immunotherapeutic agents in human patients. Thus, these results strongly support ongoing and future treatment of cancer patients with bavituximab in combination with immunotherapeutic agents, particularly immune checkpoint inhibitors.

Example XX
Β2GPI Biomarker Analysis of Bavituximab and Subsequent Immunotherapy As shown in Example XIX, patients treated with bavituximab (+ docetaxel) and subsequent IO have significantly better mOS than patients treated with docetaxel alone and subsequent IO . This example further confirms the use of functional β2GPI as a bavituximab biomarker, indicating that the same level of functional β2GPI also correlates with successful treatment with bavituximab in combination with immunotherapy. .

  Using the assay of Example XVI, it is shown that functional β2GPI levels of 200 μg / ml or more correlate with successful bavituximab treatment, including phase III studies (Example XVII). Based on the same “high β2GPI” cut-off as is the pre-treatment level of functional β2GPI ≧ 200 μg / ml (≧ 200 μg / ml), β2GPI ≧ 200 μg / ml was treated with bavituximab and subsequent IO Again correlated with an increase in overall survival, but not in control patients receiving subsequent IO.

  In particular, for patients with functional β2GPI greater than or equal to 200 μg / ml, mOS was still not reached in patients treated with bavituximab and subsequent IO, while mOS was 12.3 months in patients treated with docetaxel and subsequent IO. (10.2 to 17.6) (FIG. 27, p = 0.002). As predicted by the data in Example XVII, β2GPI greater than 200 μg / ml in patients treated with bavituximab (10.5 months) compared to controls (9.2 months) in patients without subsequent IO Although still tending to increase with increasing overall survival, the separation of the curves is not as pronounced as observed for subsequent IO patients (FIG. 27). In contrast to bavituximab treatment, for both patients with and without subsequent IO, patients in the control group tend to have longer survival when β2GPI is less than 200 μg / ml. A detailed analysis of the data for the group with β2GPI less than 200 μg / ml shows that patients treated with bavituximab (n = 12) and placebo (n = 19) and subsequent IO in the group with β2GPI less than 200 μg / ml Hindered by being small.

Thus, these clinical data indicate that 200 μg / ml or more of functional β2GPI could be used in combination with immunotherapy, especially in combination with immune checkpoint inhibitors such as tremelimumab, nivolumab, pembrolizumab, durvalumab, and atezolizumab. Shows that it is a biomarker of successful treatment with bavituximab.
* * *

  All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, without departing from the concept, spirit, and scope of the present invention, the compositions and methods, and the method steps or methods described herein. It will be apparent to those skilled in the art that changes can be made in the order of the steps. More specifically, it will be apparent that certain agents, both chemically and physiologically relevant, may replace the agents described herein, while achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims (40)

  Bavituximab for use in a method of treating cancer in a patient, wherein the functional β2-glycoprotein 1 (β2GPI) is present in a blood sample of said patient at a concentration of 200 μg / ml or more, wherein said functional β2GPI Binds to both phosphatidylserine (PS) and bavituximab, wherein bavituximab is the first anti-cancer agent, wherein the method further comprises administering at least a second anti-cancer agent. The method comprises:
(A) identifying the concentration of the functional β2GPI in the patient's blood sample;
(B) administering bavituximab and at least a second anti-cancer agent to said patient when the blood concentration of functional β2GPI is at least 200 μg / ml, wherein said at least one anticancer agent is administered to said patient.   If the patient has ovarian cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, breast cancer, esophageal cancer, malignant glioma, glioblastoma, prostate cancer, melanoma, head and neck cancer, renal cell cancer, bladder cancer, pancreatic cancer Or bavituximab for use according to claim 1 or 2 having lung cancer.   3. Bavituximab for use according to claim 1 or 2, wherein the patient has pancreatic cancer or non-small cell lung cancer (NSCLC).   5. Bavituximab for use according to any of the preceding claims, wherein said at least a second anti-cancer agent is a chemotherapeutic agent or an immune checkpoint antibody.   6. Bavituximab for use according to claim 5, wherein the chemotherapeutic agent is sorafenib, paclitaxel, carboplatin, gemcitabine, or docetaxel.   6. Bavituximab for use according to claim 5, wherein the immune checkpoint antibody is a blocking antibody that binds CTLA-4, PD-1, or PD-L1.   6. The Bavituximab for use according to claim 5, wherein the immune checkpoint antibody is tremelimumab, nivolumab, pembrolizumab, durvalumab, or atezolizumab.   9. Babituximab for use according to any one of claims 1 to 8, wherein the method further comprises administering a third anti-cancer agent.   Bavituximab for use according to any one of claims 1 to 9, wherein the blood concentration of functional β2GPI is from 200 μg / ml to 290 μg / ml. The functional β2GPI is:
(A) coating the ELISA plate with phosphatidylserine (PS) to prepare a PS coated ELISA plate;
(B) adding the bavituximab and the blood sample to the PS coated ELISA plate under conditions effective to bind β2GPI in the blood sample to both the bavituximab and the PS coated ELISA plate;
And (c) detecting the binding of bavituximab and β2GPI to the PS-coated ELISA plate, thereby measuring the functional β2GPI in the blood sample. Babituximab for use according to any one of claims 10 to 13.   12. Bavituximab for use according to any one of claims 1 to 11, wherein the blood sample is a plasma sample.   12. Bavituximab for use according to any one of claims 1 to 11, wherein the blood sample is a serum sample.   A method of diagnosing a cancer patient treatable with a first anti-cancer agent and at least a second anti-cancer agent, wherein said first anti-cancer agent is bavituximab, said method comprising: Determining the concentration of glycoprotein 1 (β2GPI), wherein if the blood concentration of the functional β2GPI is greater than or equal to 200 μg / ml, the patient is determined to be treatable with bavituximab and the at least a second anticancer agent. Wherein the functional β2GPI binds to both phosphatidylserine (PS) and bavituximab.   15. The method of claim 14, wherein said patient is treatable with said first and at least second and third anti-cancer agents.   A method for treating cancer in a human patient, comprising administering a first and at least a second anti-cancer agent to the patient, wherein the first anti-cancer agent is bavituximab, and wherein the patient comprises 200 μg / ml. A method comprising a pretreatment blood concentration of functional β2-glycoprotein 1 (β2GPI) as described above, wherein said functional β2GPI binds to both phosphatidylserine (PS) and bavituximab. A method for treating cancer in a human patient, comprising:
(A) measuring the concentration of functional β2-glycoprotein 1 (β2GPI) in a pre-treatment blood sample obtained from the patient, wherein the functional β2GPI comprises both phosphatidylserine (PS) and bavituximab Measuring, binding to
(B) administering a first and at least a second anticancer agent to the patient having a pretreatment blood concentration of functional β2GPI of 200 μg / ml or more, wherein the first anticancer agent is bavituximab; Administering. A method for treating cancer in a human patient, comprising:
(A) obtaining a pre-treatment blood sample from the patient;
(B) measuring the concentration of functional β2-glycoprotein 1 (β2GPI) in the pre-treatment blood sample, wherein the functional β2GPI binds to both phosphatidylserine (PS) and bavituximab. To do
(C) administering a first and at least a second anti-cancer agent to the patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or more, wherein the first anti-cancer agent is bavituximab; Administering. A method for identifying a human cancer patient treatable with a first and at least a second anticancer agent, wherein the first anticancer agent is bavituximab, wherein treating the patient comprises:
(A) measuring the concentration of functional β2-glycoprotein 1 (β2GPI) in a pre-treatment blood sample obtained from the patient, wherein the functional β2GPI comprises both phosphatidylserine (PS) and bavituximab Measuring, binding to
(B) if the concentration of functional β2GPI in the pre-treatment blood sample is greater than or equal to 200 μg / ml, identifying the patient as being treatable with bavituximab and the at least a second anticancer agent;
(C) administering bavituximab and the at least a second anti-cancer agent to the patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or greater. A method for identifying a human cancer patient treatable with a first and at least a second anticancer agent, wherein the first anticancer agent is bavituximab, wherein treating the patient comprises:
(A) obtaining a pre-treatment blood sample from the patient;
(B) measuring the concentration of functional β2-glycoprotein 1 (β2GPI) in the pre-treatment blood sample, wherein the functional β2GPI binds to both phosphatidylserine (PS) and bavituximab. To do
(C) if the concentration of functional β2GPI in the pre-treatment blood sample is greater than or equal to 200 μg / ml, identifying the patient as being treatable with bavituximab and the at least a second anticancer agent;
(D) administering bavituximab and the at least a second anticancer agent to the patient having a pre-treatment blood concentration of functional β2GPI of 200 μg / ml or greater.   The method of any one of claims 16 to 0, wherein said bavituximab is administered to said patient in an amount of 3 mg / kg.   22. The method according to any one of claims 16 to 21, further comprising administering at least a second and a third anti-cancer agent to the patient.   If the patient has ovarian cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, breast cancer, esophageal cancer, malignant glioma, glioblastoma, prostate cancer, melanoma, head and neck cancer, renal cell cancer, bladder cancer, pancreatic cancer 23. The method of any one of claims 14 to 22, wherein the patient has lung cancer.   24. The method of claim 23, wherein the patient has pancreatic cancer or non-small cell lung cancer (NSCLC).   25. The method of claim 24, wherein the non-small cell lung cancer (NSCLC) is a non-squamous non-small cell lung cancer.   26. The method of any one of claims 14 to 25, wherein said at least a second anti-cancer agent is a chemotherapeutic agent or an immune checkpoint antibody.   27. The method of claim 26, wherein the chemotherapeutic agent is sorafenib, paclitaxel, carboplatin, gemcitabine, or docetaxel.   27. The method of claim 26, wherein the immune checkpoint antibody is a blocking antibody that binds to CTLA-4, PD-1, or PD-L1.   29. The method of claim 28, wherein the immune checkpoint antibody is tremelimumab, nivolumab, pembrolizumab, durvalumab, or atezolizumab.   16. The method of claim 14 or 15, wherein the patient is determined to be treatable with bavituximab and at least a second anticancer agent if the blood level of functional [beta] 2GPI is between 200 [mu] g / ml and 290 [mu] g / ml.   30. The method of any one of claims 16-29, wherein the patient has a pre-treatment blood concentration of functional [beta] 2GPI of between 200 [mu] g / ml and 290 [mu] g / ml.   32. The method according to any one of claims 14, 15, or 17 to 31, wherein the blood sample is a plasma sample.   32. The method according to any one of claims 14, 15, or 17 to 31, wherein the blood sample is a serum sample. The functional β2GPI is:
(A) coating the ELISA plate with phosphatidylserine (PS) to prepare a PS coated ELISA plate;
(B) adding the bavituximab and the blood sample to the PS coated ELISA plate under conditions effective to bind β2GPI in the blood sample to both the bavituximab and the PS coated ELISA plate;
15. detecting the binding of bavituximab and β2GPI to the PS-coated ELISA plate, thereby measuring the functional β2GPI in the blood sample. 35. The method according to any one of claims 17 to 33. A method for measuring functional β2-glycoprotein 1 (β2GPI), wherein the functional β2GPI binds to both phosphatidylserine (PS) and bavituximab, and the method comprises:
(A) coating the ELISA plate with phosphatidylserine (PS) to prepare a PS coated ELISA plate;
(B) binding the biological sample suspected of containing bavituximab and β2GPI to the PS coating under conditions effective to bind β2GPI in the sample to both the bavituximab and the PS coated ELISA plate. Adding to the ELISA plate,
(C) detecting the binding of bavituximab and β2GPI to the PS-coated ELISA plate, thereby measuring the functional β2GPI in the sample.   The method of claim 0, wherein the biological sample is a blood sample.   37. The method of claim 34 or 36, wherein the blood sample is a plasma sample.   37. The method of claim 34 or 36, wherein the blood sample is a serum sample.   The bavituximab is conjugated to a detectable substance that produces a detectable signal, and the binding of bavituximab and β2GPI to the PS-coated ELISA plate is detected and measured by detecting and measuring the detectable signal. A method according to any one of claims 34 to 38.   40. The method of any one of claims 34-39, wherein the bavituximab is added to the PS coated ELISA plate prior to the addition of the sample containing β2GPI.