Pharmaceutical combinations for treatment of cancer

Abstract

Provided herein is a pharmaceutical combination comprising an SFRP2 antagonist and a PD-1 antibody antagonist. The invention also provides a method for treating cancer comprising administering to a patient in need thereof a therapeutically effective amount of an SFRP2 antagonist and a PD-1 antagonist.

Description

[0001] This application is a divisional application of the invention patent application with application date of September 27, 2019, application number 2019800766083, and invention name “Drug Combination for Treating Cancer”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 737,155, filed on September 27, 2018, the entire disclosure of which is hereby incorporated herein by reference. Technical Field

[0004] The present invention relates to a therapy for treating cancer comprising administering a SFRP2 antagonist as a monotherapy or in combination with a PD-1 antagonist simultaneously or sequentially to a patient in need thereof.

[0005] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes, and to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein should not be construed as an admission that this is prior art to the present invention. Background Art

[0006] Wnt ligands are secreted glycoproteins that activate downstream effectors by binding to cell surface G protein-coupled transmembrane receptors called Frizzled receptors. Activation of Wnt signaling is involved in normal embryonic development, but dysregulation of this pathway has been associated with tumor progression in various cancers (1, 2). Secreted Frizzled-related proteins (SFRPs) were previously thought to be inhibitors of the canonical Wnt-β-catenin pathway (1), suggesting that SFRP2 may be a tumor suppressor. However, several additional studies have shown that SFRP2 can act as a β-catenin agonist rather than an antagonist (3-7), suggesting that it plays a role in tumor promotion.

[0007] A large body of evidence now strongly supports the contribution of SFRP2 to tumor growth in breast cancer (5, 8-11), angiosarcoma (9, 10), osteosarcoma (12), rhabdomyosarcoma (13), alveolar soft tissue sarcoma (14), glioblastoma (15), multiple myeloma (16), renal cell carcinoma (2), prostate cancer (17), lung cancer (18), and melanoma (19). Furthermore, in vivo SFRP2 molecular imaging showed that SFRP2 expression increased proportionally with tumor size (20), and the present inventors showed that murine SFRP2 monoclonal antibodies inhibited angiosarcoma and breast cancer growth in vivo (21). Furthermore, Techavichit et al. showed that SFRP2 is highly overexpressed in metastatic osteosarcoma, and overexpression in low metastatic osteosarcoma cells increased metastasis in vivo, whereas knockdown of SFRP2 in highly metastatic osteosarcoma reduced cell migration and invasion in vitro (12). In addition to the direct effects of SFRP2 on tumor cells, SFRP2 is also involved in tumor angiogenesis (9, 10, 19, 22-24). Therefore, SFRP2 plays a dual role in directly activating tumor growth and plays a secondary role in activating angiogenesis.

[0008] In endothelial cells, SFRP2 activates non-canonical Wnt / Ca 2 pathway, rather than the canonical β-catenin pathway, to stimulate angiogenesis (22, 24). 2+ Pathway is mediated by activated G protein and phospholipase. This leads to the short-term increase of cytoplasmic free calcium and the activation of phosphatase calcineurin, which dephosphorylates the nuclear factor (NFAT) of activated T cells and then translocates from the cytoplasm to the nucleus. More and more data support the key role of NFAT in mediating tumor growth (including cell growth, survival, invasion and angiogenesis) (25). NFAT protein also plays an important role in the development and function of the immune system (including the activation of T cells). Specifically, nuclear NFAT cooperates with other transcription factors to regulate a series of genes involved in the immune system function (26), including IL2 and cyclooxygenase 2 (27).

[0009] Combination therapy

[0010] The administration of two drugs to treat a given condition (such as cancer) creates many potential problems. The in vivo interactions between two drugs are complex. The effects of any single drug are related to its absorption, distribution, and elimination. When two drugs are introduced into the body, each drug may affect the absorption, distribution, and elimination of the other drug, and thus change the effect of the other drug. For example, a drug can inhibit, activate, or induce the production of enzymes involved in the metabolic pathway that eliminates another drug (44). In one example, it has been experimentally demonstrated that the combined administration of glatiramer acetate (GA) and interferon (IFN) can eliminate the clinical effectiveness of either therapy (49). In another experiment, it was reported that the addition of prednisone to a combination therapy with IFN-β antagonized its upregulatory effects (48). Therefore, when two drugs are administered to treat the same condition, it is impossible to predict whether each drug will supplement the therapeutic activity of the other drug in the subject, have no effect on it, or interfere with it.

[0011] Not only may the interaction between two drugs affect the intended therapeutic activity of each drug, but the interaction may also increase the levels of toxic metabolites (44). The interaction may also increase or decrease the side effects of each drug. Therefore, when two drugs are administered to treat a disease, it is impossible to predict how the side effect profile of each drug will change. In one example, the combination of natalizumab and interferon beta-1a was observed to increase the risk of unexpected side effects (47, 45, 46).

[0012] Furthermore, it is difficult to predict exactly when the effects of an interaction between two drugs will become apparent. For example, a metabolic interaction between drugs may become apparent upon initial administration of the second drug, after both drugs reach steady-state concentrations, or upon discontinuation of one of the drugs (44).

[0013] Therefore, the state of the art at the time of filing is that the effect of the addition of two drugs or combination therapy (particularly a SFRP2 antagonist together with a PD-1 antagonist) cannot be predicted with any reasonable certainty until the results of combination studies are available. Summary of the invention

[0014] The present invention relates to a drug combination comprising a therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonists and a therapeutically effective amount of PD-1 antagonists. The present invention also relates to a method for treating cancer, comprising administering a therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonists and a therapeutically effective amount of PD-1 antagonists to a patient in need thereof simultaneously or sequentially. The present invention also relates to a method for treating certain cancers, comprising administering a therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonists to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings described below are for illustration purposes only and are not intended to limit the scope of the present invention.

[0016] Figure 1 : Gp100 reactive mouse spleen T cells were cultured for 3 days alone or in the presence of Hs578T (upper row) or RF420 cells (lower row) and treated for 3 days. The intensity of each condition was measured by FACS analysis. This experiment used anti-CD3 and anti-CD28 antibodies (TCR stimulation) as positive controls. The percentage of suppression was calculated based on the division index method. The division index is calculated by multiplying the proliferation index by the percentage of dividing cells, thus representing the division status of the entire population. The experiment was repeated three times. Representative overlaps are presented on the left, and the cumulative data of all repeats are presented in the bar graph (*p<0.01).

[0017] Figure 2 : A) FZD5 protein is present in T cells. BC) T cells were treated with SFRP2 (30 nM) for 1 h, and (B) nuclear and (C) cytoplasmic fractions were isolated. Samples were probed with antibodies against the indicated protein markers. D) T cells were treated with antigen gp100 (0.87 μM) or hSFRP2 mAb (10 μM) alone or in combination for 60 min, and the nuclear fraction was isolated. The protein levels of NFATc3 in SFRP2-treated cells were compared with untreated cells. AC) Actin: loading control for cytoplasmic fraction; histone H3 and TATA: loading control for nuclear fraction. BD) Densitometry was performed using ImageJ, and density was calculated by multiplying the average intensity by the surface of each band. The loading control was used to eliminate inter-sample variability. The final results were obtained by normalizing each value to the untreated control (BD) or antigen-treated sample (D).

[0018] Figure 3.A) Splenic T cells were treated with IL2, IL2+TCR antigen, IL2+TCR antigen+TGFb. Or IL2+TCR antigen+TGFb and hSFRP2 mAb. Protein lysates were extracted and probed for SFRP2 by western blot. This shows that SFRP2 increased in the case of TCR and TGF b and decreased in the case of hSFRP2mAb. B) NAD+ concentration Mouse spleen cells were treated with IL-2 (6,000u / well), with or without TCR / TGFβ (5ng / ml), and with or without hSFRP2 mAb (10nM) for 3 days (n=3 per group). Compared with untreated controls, hSFRP2 mAb increased NAD+ concentration (*p=0.02). C) CD38+ cell number (Z axis). Cells were treated as above, but for 36 hours. In the presence of TCR / TGFβ addition, CD38+ cells increased, which was significantly inhibited by hSFRP2 mAb (n=3, **p<0.001).

[0019] Figure 4 SFRP2 mAb inhibits PD-1 in T cells. Splenic T cells were treated with IL2 alone, or IL2 with TCR antigen and TGFB, or IL2 with TCR antigen and TGFB and hSFRP2 mAb. Cells were analyzed by FACS. TCR and TGFB increased PD-1 histogram, which was reversed by hsFRP2 mAb.

[0020] Figure 5 .A) Osteosarcoma RF420 cells were injected intravenously into C57BL6 mice. Treatment with IgG1 control or hSFRP2 mAb (4 mg / kg, every 3 days) started 10 days after injection of tumor cells. After 3 weeks, the animals were euthanized, their lungs were removed and surface nodules were counted (*: p≤0.0001; n=12). B) Representative lung with tumor metastasis. C) T cells isolated from the spleen of C57BL / 6 mice injected with RF420 cells and treated with IgG1 control or hSFRP2 mAb. Cells were stained with CD38 and fluorescent dyes, and the mean fluorescence intensity (MFI) was analyzed by FACS. The bar graph shows the fluorescence measurements obtained from T cells isolated from 4 different spleens for each treatment (n=4). In both splenocytes and TILs, CD38 was statistically different when using hSFRP2 mAb (*p≤0.001).

[0021] Figure 6RF420 mouse osteosarcoma cells were injected into the tail vein of C57BL / 6 mice. Starting on day 7, mice were treated for 21 days with IgG1 control, hSFRP2 mAb, mouse PD-1 mAb, or a combination of both antibodies. Mice were euthanized and lungs were harvested. The number of surface metastases and micrometastases in each group was counted by H&E. There was no reduction in the number of metastases in the case of PD-1 mAb treatment. In the case of hSFRP2 as a monotherapy, the number of metastases was significantly reduced (p<0.001), and the reduction was further promoted in the case of combination (p<0.001).

[0022] Figure 7 :In vitro activity of humanized SFRP2 mAb. (A) Concentration-response curve EC 50 : half maximal effective concentration; Kd: equilibrium dissociation constant; Hill: Hill coefficient. (B) Bar graph; (CH) Bar graph showing the effects of increasing concentrations of hSFRP2 mAb (0 to 10 μM) on apoptosis (C, F; n=8) and necrosis (D, G; n=8), proliferation (E, H; n=12) in Hs578T breast cancer cells (CE) and SVR angiosarcoma cells (FH). *: p≤0.05; **: p≤0.001. Proliferation was measured, while apoptosis and necrosis were measured using Annexin V and propidium iodide. The results for apoptosis and necrosis are a compilation of 2 independent experiments, each containing 4 wells, n = 8. The results presented for proliferation are a compilation of 3 experiments, each containing 4 replicates (n = 12).

[0023] Figure 8 : Effect of humanized SFRP2 mAb on tumor growth in angiosarcoma and breast cancer. A) AUC: area under the curve; T 1 / 2: half-life; CL: clearance; Vd: volume of distribution; Cmax: maximum serum concentration. Each data point represents the mean ± SEM of measurements of at least 3 independent samples (n=3 per time point). A) Days are counted from the baseline date, which is 30 days from tumor inoculation.

[0024] Fig. 9 : Humanized SFRP2 mAb treatment promotes tumor apoptosis. The upper bar graph shows an increase in the number of apoptotic cells in tumors treated with hSFRP2 mAb (white bars) compared to IgG1 control-treated tumors (black bars). *: P≤0.05. Lower images: Paraffin-embedded SVR (upper panel) and Hs578T (lower panel) tumors were sectioned and processed for TUNEL staining. For each tumor, a total of 5 fields were photographed, the number of apoptotic cells (brown) in each field was counted, and the average was taken for each tumor. A total of 10 tumors per treatment (n=10) were used for analysis.

[0025] Fig.10 : Humanized SFRP2 mAb reduces metastatic osteosarcoma growth. A) Number of lung surface nodules after treatment. B) Splenocytes and TILs were harvested from mice treated with IgG1 control and hSFRP2 mAb and subjected to flow cytometry.

[0026] Fig.11 : Combination of humanized SFRP2 mAb and nivolumab inhibits metastatic osteosarcoma growth. A) Number of lung surface nodules after various treatments. B) Graph shows fluorescence measurements obtained from T cells isolated from 4 different spleens for each treatment (n=4), ***p≤0.001. Mean fluorescence intensity (MFI).

[0027] Fig.12 : SFRP2 competition ELISA using variant antibodies.

[0028] Fig.13 : SDS Page. 1 μg of purified lead hSFRP2 mAb on 4-12% NuPAGE-SDS gel.

[0029] Fig.14 : Healthy donor T cell proliferation response to test antibodies. DETAILED DESCRIPTION

[0030] It should be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. In addition, although any methods, devices and materials similar or equivalent to the methods, devices and materials described herein can be used in the practice or testing of the present invention, certain methods, devices and materials are now described.

[0031] The present invention provides a method for treating cancer, comprising administering to a subject in need thereof a certain amount of an SFRP2 antagonist and a certain amount of a PD-1 antagonist, wherein the amount is effective for the subject when taken together. The present invention also provides a drug combination comprising a certain amount of an SFRP2 antagonist (such as an SFRP2 mAb) and a certain amount of a PD-1 antagonist (such as an anti-PD-1 antibody). In one embodiment, a novel humanized SFRP2 monoclonal antibody (hSFRP2mAb) is provided, which reduces CD38 in splenocytes and tumor infiltrating lymphocytes (TILs) in vivo and has an excellent concomitant effect with a PD-1 antibody in inhibiting tumor growth in vivo. In another embodiment, the humanized SFRP2 monoclonal antibody reduces PD-1 in lymphocytes in vitro. Therefore, the hSFRP2 mAb of the present invention affects cell function by inhibiting the non-classical WNT pathway in a variety of cell types.

[0032] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist, a CD38 antagonist and / or a PD-1 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0033] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist, a CD38 antagonist, and a PD-1 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0034] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist and / or a CD38 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0035] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist and a CD38 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0036] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist or a CD38 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0037] In another embodiment, the present invention provides a method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0038] In one embodiment, the SFRP2 antagonist is: (a) an antibody or an antigen-binding fragment of an antibody that specifically binds to a SFRP2 receptor and inhibits its activation; or (b) a soluble form of a SFRP2 receptor that specifically binds to a SFRP2 ligand and inhibits the binding of the SFRP2 ligand to the SFRP2 receptor.

[0039] In one embodiment, the PD-1 antagonist is: (a) an antibody or an antigen-binding fragment of an antibody that specifically binds to a PD-1 receptor and inhibits its activation; or (b) a soluble form of a PD-1 receptor that specifically binds to a PD-1 ligand and inhibits the binding of the PD-1 ligand to the PD-1 receptor.

[0040] Sarcomas are a heterogeneous group of malignancies that include >50 different subtypes, each with unique clinical and pathological characteristics. Generally, there is a 50% mortality rate, and most cures are achieved with complete surgical resection with or without radiation therapy. The results of chemotherapeutic agents for unresectable or metastatic disease have been disappointing, with minimal long-term benefit and a 5-year survival rate of only 15% for patients with metastatic disease (34). The response rate to doxorubicin is 20% to 25%. PD-1 inhibitors are currently being studied for use in sarcomas. In a retrospective study of 28 patients with metastatic soft tissue sarcomas treated with nivolumab, 50% of patients had partial responses or stable disease (35). Although targeted agents have some activity in sarcomas, improved therapeutic agents and novel therapeutic combinations are necessary to improve responses and outcomes. In this study, the inventors report the development of a humanized SFRP2 mAb that is non-immunogenic and binds to SFRP2 with high affinity. Not only did the hSFPR2 mAb suppress tumor growth as a single agent in three tumor cell lines (angiosarcoma, osteosarcoma, and breast carcinoma sarcoma), but it also did so far better than PD-1 inhibitors alone in osteosarcoma.

[0041] In phase III trials in patients with melanoma, non-small cell lung cancer, and renal cancer, blockade of the PD-1 receptor or its ligand PD-L1 improved overall survival. Early studies suggest that PD-1 pathway blockade may benefit subsets of patients with many other cancer types. However, the majority of patients do not respond to PD-1 pathway blockade, and understanding of how to improve response rates is urgently needed (36).

[0042] Although the inventors and others have previously demonstrated the effects of SFRP2 on angiogenesis and tumor cells (9, 10, 19, 20, 22, 24), the inventors' current studies reveal a new mechanism: SFRP2 stimulates NFAT not only in endothelial cells and tumor cells, but also in T cells. Given that NFAT is required for PD-1 induction following TCR stimulation of CD4 and CD8 T-cells, and because the calcineurin / NFAT pathway inhibitor cyclosporine A is able to block PD-1 (37, 38), the inventors hypothesized that blocking SFRP2 would reduce the depletion of effector T-cells and result in better tumor control. The inventors' data showed that while the depletion markers CD5 and CD103 expression were unchanged, the expression of the non-classical ectonucleotidase CD38 was reduced, and its expression on T cells has also recently been shown to be negatively correlated with tumor control (39). CD38 regulates anti-tumor T cell responses, and genetic ablation or antibody-mediated targeting of CD38 on T cells improves tumor control. Furthermore, we show that T cells with reduced CD38 expression maintain high effector cytokine secretion and are not dysfunctional despite expressing PD-1. We also show that CD38 expression is not reprogrammable in non-reprogrammable PD-1 T cells with a fixed chromatin state. hi CD38 is highly expressed on dysfunctional T cells (40). Furthermore, combined PD-1 and CTLA-4 blockade eradicated CD38-deficient tumors in mice, and tumor-bearing mice treated with combined PD-1 and CTLA-4 blocking antibodies developed resistance through upregulation of CD38 (41). Therefore, reducing CD38 expression can rescue T cells from tumor-induced depletion.

[0043] Since calcium and NFAT signaling have been shown to regulate CD38 expression in various cell types (42), it is possible that its inhibition of SFRP2 leads to reduced Ca2+ / NFAT signaling in T cells, resulting in reduced CD38 expression. In B cells, NFATc1 has been reported to be essential for CD38 expression (43), which led the inventors to hypothesize that hSFRP2 mAb can reduce CD38 through its inhibitory effect on NFATc3 in T cells. However, the inventors' data support that inhibition of SFRP2 together with PD-1 results in better tumor control, which may be due to the simultaneous targeting of reduced immunosuppression due to CD38 in T cells and the Wnt signaling pathway in tumors. In the absence of the inventors' data, there is no reason to expect better tumor control with this combination.

[0044] In one embodiment, a subject has increased CD38 and / or PD-1 expression if any cell (e.g., T cell) in the subject has more CD38 and / or PD-1 expression compared to a corresponding healthy subject or a cancer subject that does not have increased CD38 and / or PD-1 expression.

[0045] definition

[0046] The articles "a" and "an" are used in this disclosure to refer to one or to more than one (ie, to at least one) of the grammatical object of the article.

[0047] A "subject" is a human, and the terms "subject" and "patient" are used interchangeably herein.

[0048] The term "treat" with respect to a subject encompasses, for example, inducing inhibition, regression or stagnation of a disease or disorder; or curing, ameliorating or at least partially ameliorating a condition; or alleviating, alleviating, suppressing, inhibiting a disease or disorder, reducing its severity, eliminating or substantially eliminating or ameliorating its symptoms. "Inhibiting" a subject's disease progression or disease complications means preventing or reducing a subject's disease progression and / or disease complications.

[0049] "Symptoms" associated with cancer include any clinical or laboratory manifestations associated with cancer, and are not limited to those that a subject can feel or observe.

[0050] "Administering to a subject" or "administering to a (human) patient" means giving, dispensing or applying an agent, drug or medicine to a subject / patient to alleviate, cure or reduce symptoms associated with a disorder (eg, a pathological disorder). Administration may be regular administration.

[0051] As used herein, "regular administration" means repeated / repeated administration at intervals of time. The time period between administrations is preferably the same each time. Regular administration may include, for example, administration once a day, twice a day, three times a day, four times a day, weekly, twice a week, three times a week, four times a week, etc.

[0052] As used herein, "unit dose," "unit doses," and "one or more unit dosage forms" mean one or more single pharmaceutical administration entities.

[0053] As used herein, when referring to the amount of a PD-1 antagonist and / or SFRP2 antagonist, "effective" or "therapeutically effective" refers to an amount of a PD-1 antagonist and / or SFRP2 antagonist sufficient to produce the desired therapeutic response. In certain embodiments, an effective amount refers to an amount that is effective to achieve the desired therapeutic or preventive result at the required dose and for the required period of time. The therapeutically effective amount of the SFRP2 antagonist and / or PD-1 / PD-L1 antagonist or inhibitor of the present invention may vary according to factors such as the disease state, the age, sex and weight of the individual, and the ability of one or more antibodies to induce the desired response of the individual. The therapeutically effective amount encompasses an amount in which the therapeutically beneficial effect outweighs any toxic or deleterious effects of one or more antibodies. In one embodiment, when administered in combination, the amount of the SFRP2 antagonist and the amount of the PD-1 antagonist are effective for treating the subject. According to certain embodiments, the antibody of the present invention is administered in an amount of 0.1 mg / kg body weight to 100 mg / kg body weight. According to other embodiments, the antibody of the present invention is administered in an amount of 0.5 mg / kg body weight to 20 mg / kg body weight. According to further embodiments, the antibody of the invention is administered in an amount of 1.0 mg / kg body weight to 10 mg / kg body weight.

[0054] The following abbreviations for general terms used in this specification apply regardless of whether the term in question appears alone or in combination with other groups:

[0055] Area under the curve (AUC); bovine serum albumin (BSA); calcium (Ca 2+ ); carboxyfluorescein succinimidyl ester (CFSE); clearance (CL); dissociation constant (Kd); enzyme-linked immunosorbent assay (ELISA); fetal bovine system (FBS); fluorescence-activated cell sorting (FACS); frizzled 5 (FZD5); humanized SFRP2 monoclonal antibody (hSFRP2 mAb); human recombinant secreted frizzled-related protein 2 (hrSFRP2); horseradish peroxidase (HRP); half-maximal effective concentration (EC50); intravenous (iv); intraperitoneal (ip); modified Eagle's basal medium (DMEM); mean fluorescence intensity (MIF); non-compartmental analysis (NCA); nuclear factor of activated T cells (NFAT); pharmacokinetics (PK); programmed cell death protein 1 (PD-1); secreted frizzled-related protein 2 (SFRP2); T cell receptor (TCR); terminal half-life (T1 / 2); and volume of distribution (Vd).

[0056] The combination of the present invention may be formulated with at least one pharmaceutically acceptable carrier, additive, adjuvant or vehicle as described herein for simultaneous, separate or sequential administration thereof. Thus, a combination of two active compounds may be administered:

[0057] a combination as part of the same pharmaceutical formulation, whereby both active compounds are administered simultaneously, or

[0058] As a combination of two units, each with one active substance, giving rise to the possibility of simultaneous, sequential or separate administration.

[0059] As used herein, "combination" refers to the collection of agents for therapy by simultaneous or concurrent administration. Simultaneous administration refers to the administration of a mixture of a PD-1 antagonist and SFRP2, CD38 and / or PD-1 antagonist (whether a true mixture, suspension, emulsion or other physical combination). In this case, the combination can be a mixture or separate container of a PD-1 antagonist and SFRP2, CD38 and / or PD-1 antagonist, which are combined just before administration. Concurrent administration or concurrent administration refers to the separate administration of a PD-1 antagonist and SFRP2, CD38 and / or PD-1 antagonist at the same time, or at a time close enough to observe synergistic activity relative to the activity of a single PD-1 antagonist or a single SFRP2, CD38 and / or PD-1 antagonist, or close enough in time to allow the individual therapeutic effects of each agent to be superimposed.

[0060] As used herein, "add-on" or "add-on therapy" means a collection of agents used for treatment, wherein a subject receiving treatment begins a first treatment regimen of one or more agents, and then begins a second treatment regimen of one or more different agents in addition to the first treatment regimen, such that all agents used in the therapy are not started at the same time. For example, adding PD-1 antagonist therapy to a patient who has already received SFRP2, CD38 and / or PD-1 antagonist therapy.

[0061] Any known PD-1 antagonist can be used in the practice of the present invention, and a variety of PD-1 antagonists are known and disclosed in the art. The PD-1 antagonist preferably neutralizes the biological function after binding. The PD-1 antagonist is preferably a human PD-1 antagonist. Optionally, the PD-1 antagonist can be an antibody, such as a monoclonal antibody or a fragment thereof; a chimeric monoclonal antibody (such as a human-mouse chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble PD-1 antagonist, including a small molecule PD-1 blocker. Optionally, the PD-1 antagonist is a functional fragment of a monoclonal antibody or a fusion protein comprising a functional fragment, such as Fab, F(ab')2, Fv, and preferably Fab. Preferably, the fragment is pegylated or encapsulated (e.g., for stability and / or sustained release). The PD-1 antagonist can also be a camelid antibody. As used herein, PD-1 antagonists include, but are not limited to, PD-1 receptor inhibitors.

[0062] The PD-1 antagonist may be selected from, for example, one or a combination of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab or atezolizumab or a functional fragment thereof.

[0063] Any known SFRP2 and / or CD38 antagonist can be used in the practice of the present invention, and a variety of SFRP2 and / or CD38 antagonists are known and disclosed in the art. SFRP2 and / or CD38 antagonists preferably neutralize biological functions after binding. SFRP2 and / or CD38 antagonists are preferably human SFRP2 and / or CD38 antagonists. Optionally, the SFRP2 and / or CD38 antagonist can be an antibody, such as a monoclonal antibody or a fragment thereof; a chimeric monoclonal antibody (such as a human-mouse chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble SFRP2 and / or CD38 antagonist, including a small molecule SFRP2 and / or CD38 blocker. Optionally, the SFRP2 and / or CD38 antagonist is a functional fragment of a monoclonal antibody or a fusion protein comprising a functional fragment, such as Fab, F(ab')2, Fv, and preferably Fab. Preferably, the fragment is pegylated or encapsulated (e.g., for stability and / or sustained release). The SFRP2 and / or CD38 antagonist may also be a camelid antibody. As used herein, SFRP2 and / or CD38 antagonists include, but are not limited to, SFRP2 and / or CD38 receptor inhibitors. For example, SFRP2 antagonists are disclosed in U.S. Pat. Nos. 8,734,789 and 9,073,982, the contents of which are hereby incorporated by reference.

[0064] The present invention will now be further described in the following examples, which are intended to be illustrative only and not to limit the scope of the present invention.

[0065] Example

[0066] The disclosure is further illustrated by the following examples, which should not be construed to limit the disclosure to the specific procedures described herein in scope or spirit. It should be understood that these examples are provided to illustrate certain embodiments and are not intended to limit the scope of the disclosure thereby. It should be further understood that, without departing from the spirit of the disclosure and / or the scope of the appended claims, various other embodiments, modifications and their equivalents that may be proposed by those skilled in the art may have to be resorted to.

[0067] Example 1

[0068] Humanized SFRP2 mAb rescues tumor cell inhibition of T cell proliferation. Since SFRP2 activates NFATc3, and NFAT proteins regulate T cell proliferation (28), the present inventors examined whether hSFRP2 mAb affects T cell proliferation (29) after activation with TCR stimulation (anti-CD3 / anti-CD28 antibodies). Figure 1 ). T cells were incubated alone, with TCR stimulation, with TCR stimulation + tumor cells, with TCR stimulation + tumor cells + IgG1 control, or with TCR stimulation + tumor cells + hSFRP2 mAb. TCR antigen (positive control) increased proliferation compared to that observed in the T cell population alone. In the presence of Hs578T (a human metaplastic breast cancer cell line), proliferation was reduced ( Figure 1 ). Addition of an IgG1 control to the co-culture had no effect. In contrast, addition of the hSFRP2 mAb to the co-culture partially rescued T cell proliferation. This effect was also seen when T cells were co-cultured in the presence of RF420 mouse osteosarcoma cells, where the presence of the hSFRP2 mAb significantly rescued tumor cell-mediated suppression of proliferation ( Figure 1 ). Likewise, addition of an IgG1 control did not affect proliferation compared to T cells treated with TCR in the presence of RF420 cells.

[0069] SFRP2 induces Wnt signaling in T cells. The FZD5 receptor binds SFRP2 in endothelial cells to stimulate NFATc3 activation and angiogenesis (23). However, the role of SFRP2 in T cell activation and Wnt signaling has not been previously evaluated. Western blot analysis of T cell lysates showed that FZD5 protein was present in T cells ( Figure 2 A). Mouse spleen T cells were stimulated with SFRP2 (30 nM) for 1 hour, and the nuclear and cytoplasmic fractions were separated. In the case of SFRP2 treatment, CD38 increased in the cytoplasmic fraction ( Figure 2 B) In the case of SFRP2 treatment, NFATc3 increased in the nuclear fraction ( Figure 2 C). Next, T cells were treated with cognate antigen for three days with or without hSFRP2 mAb, and the nuclear fraction was collected. NFATc3 in the nuclear fraction increased when stimulated with cognate antigen, and decreased in the case of hSFRP2 mAb treatment ( Figure 2 D).

[0070] hSFRP2 mAb inhibits PD-1 and CD38 in T cells and restores NAD. Next, we evaluated whether hSFRP2 mAb treatment of T cells in vitro inhibits CD38 in T cells exposed to TGFβ and restores NAD+ levels. TGFβ is a cytokine present in the tumor microenvironment that increases CD38 from T cells. Figure 3 A shows by Western blot that treatment of splenic T cells with IL2, TCR antigen and TGFβ resulted in an increase in SFRP2. FACS analysis shows a statistically significant increase in CD38+ cells with the addition of TCR / TGFβ, which was significantly inhibited by hSFRP2 mAb ( Figure 3 c, n = 3, p < 0.001). At the same time, NAD+ concentration increased inversely under hSFRP2 treatment ( Figure 3 b, n = 3, p = 0.02). In addition, we considered whether SFRP2 mAb directly inhibits PD-1 in splenic T cells. Treatment of CD8+ and CD4+ splenic T cells with IL2, TCR antigen, and TGFβ increased PD-1. This was reversed by the addition of SFRP2 mAb ( Figure 4 ).

[0071] Example 2

[0072] Prognosis and treatment options for osteosarcoma. Osteosarcoma (OS) is the most common primary bone malignancy, typically affecting adolescents and young adults. If feasible, the primary tumor is surgically removed, and neoadjuvant and adjuvant chemotherapy are delivered. However, even with chemotherapy, only two-thirds of patients with initially resectable disease are cured, while the long-term survival rate for patients with metastatic or recurrent tumors is <30%. The lungs are affected in approximately 80% of cases of metastatic disease, and subsequent respiratory distress is the cause of most deaths (29). Although immunotherapy has demonstrated efficacy in some tumor types, in a Phase II trial (SARC028), the administration of pembrolizumab resulted in a lack of efficacy in the treatment of osteosarcoma, in which only 5% of metastatic osteosarcoma patients had an objective response to pembrolizumab (30). For more than 30 years, the lack of new active agents has hampered any progress in increasing the survival rate of osteosarcoma patients, and new treatment methods are urgently needed (31).

[0073] Accumulating evidence strongly supports the contribution of secreted SFRP2 to osteosarcoma metastasis. SFRP2 is overexpressed in metastatic osteosarcoma compared with non-metastatic osteosarcoma (32). High expression of SFRP2 in OS patient samples is associated with poor survival, and SFRP2 overexpression suppresses normal osteoblast differentiation, promotes OS characteristics and favors angiogenesis (33). Functional studies have shown that stable overexpression of SFRP2 in localized human and mouse OS cells significantly increases cell migration and invasion in vitro and enhances metastatic potential in vivo. Additional studies knocking down SFRP2 in metastatic OS cells demonstrated reduced cell migration and invasion in vitro, thus confirming key biological phenotypes mediated by SFRP2 (12). Therefore, SFRP2 has become a potential therapeutic target for osteosarcoma. SFRP2 has also been shown to contribute to tumor growth in breast cancer (5, 8-11), angiosarcoma (9, 10), rhabdomyosarcoma (13), alveolar soft tissue sarcoma (14), malignant glioma (15), multiple myeloma (16), renal cell carcinoma (2), prostate cancer (17), lung cancer (18), and melanoma (19). Given the lack of efficacy of immunotherapy in osteosarcoma and the inventors' data detailed elsewhere in this application, the inventors investigated whether the combination of a humanized SFRP2 monoclonal antibody (hSFRP2mAb) would enhance the activity of PD-1 inhibitors.

[0074] Humanized SFRP2 mAb inhibits metastasis in vivo. To evaluate the anti-tumor activity of hSFRP2 mAb in immunocompetent mice, hSFRP2 mAb was tested in the RF420 murine osteosarcoma model of tumor metastasis in C57BL / 6 mice. RF420 cells were injected into the tail vein of C57BL / 6 mice. The presence of metastasis in the lungs was verified 7 days after the initial injection of tumor cells. In the first experiment, treatment with hSFRP2 mAb was started on day 10 after tumor injection (4 mg / kg iv injection every 3 days) and compared with treatment with IGg1 control. At the endpoint, the number of lung surface nodules was significantly reduced during hSFRP2 mAb treatment compared with the control (n=7, p<0.01, Figure 5 When evaluating depletion of cell surface markers, we noted that CD38, which has been shown to be tightly co-expressed with PD-1 (41), was significantly reduced in the spleens of mice treated with hSFRP2 mAb compared to mice treated with IgG1 control (n = 4, p < 0.01) ( Figure 5 ).

[0075] Administration of hSFRP2 mAb with a mouse PD-1 inhibitor effectively inhibited metastatic osteosarcoma growth in vivo. RF420 mouse osteosarcoma cells were injected into the tail vein of C57BL / 6 mice. After 7 days, the mice were treated with the following: 4 mg / kg IgG1 control iv weekly; 4 mg / kg hSFRP2 mAb iv every 3 days; mouse PD-1 mAb (200ug / mouse) every 3 days; or a combination of the two antibodies. After 21 days of treatment, the mice were euthanized and the lungs were harvested. The number of surface metastases in each group was counted. The combination of hSFRP2 mAb reduced the number of surface nodules by 75% (95% CI, 2.0-3.0) compared to the IgG1 control. Figure 6 ).

[0076] Method of Example 1-2

[0077] Antibodies and proteins. Control IgG1 omalizumab was purchased from Novartis (Basel, Switzerland). Human SFRP2 recombinant protein (SFRP2) was prepared as previously described (23) and provided by the Protein Expression and Purification Core Laboratory at the University of North Carolina at Chapel Hill. Humanized SFRP2 monoclonal antibody (hSFRP2 mAb) was prepared as previously described and as described in Example 4 and purified to remove endotoxin.

[0078] The following primary antibodies were used in Western blotting: rabbit anti-CD38 (#14637s) and rabbit anti-histone H3 antibody (#2650s) from Cell Signaling (Danvers, MA, USA); rabbit anti-FZD5 (#H00007855-D01P, Abnova, Taipei, Taiwan, China); mouse anti-PD1 (#66220-1, Proteintech, Rosemont, IL, USA); rabbit anti-NFATc3 (#SAB2101578); and rabbit anti-actin (#A2103, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were: horseradish peroxidase (HRP)-conjugated anti-mouse (#7076, Cell Signaling); HRP-conjugated anti-rabbit (#403005, Southern Biotech, Birmingham, AL, USA). For FACS analysis, rat anti-CD38-PE antibody (#102707) was from BioLegend (San Diego, CA, USA). Anti-mouse CD3 (#BE00011) and anti-mouse CD28 (#BE0015-1) were from BioXCell (West Lebanon, NH, USA). The following antibodies were purchased from Biolegend, San Diego, CA, and used for flow cytometry: anti-CD103 (clone 2E7, catalog number 121435), anti-CD5: gp100 antigen fragment from AnaSpec (#AS-62589).

[0079] Cell culture. RF420 and mouse osteosarcoma cells were obtained from a genetically engineered osteosarcoma mouse model (32). Cells were cultured at 37°C in a humidified 5% CO2-95% room air atmosphere. Cell lines were obtained by Mouse cells were certified and tested for rodent pathogens, including mycoplasma, by Charles River Research Animal (Wilmington, MA, USA) whenever they were used in vivo.

[0080] Fluorescence activated cell sorting (FACS) analysis of cell proliferation was performed by measuring the intensity of the carboxyfluorescein succinimidyl ester (CFSE) signal. Dilution of the CFSE signal correlates well with increased cell proliferation. TMThe instructions of the CFSE cell proliferation kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used to pre-label spleen T cells with CFSE dye. The cells were then kept untreated or activated for 3 days with soluble anti-CD3 (#BE0001-1, BioXCell, West Lebanon, New Hampshire, USA; 2 μg / ml) / anti-CD28 antibodies (#BE0015-1, BioXCell; 2 μg / ml) alone or in the presence of tumor cells (RF420 or Hs578T breast cancer-sarcoma) at a 2:1 ratio. In addition, some co-cultures were treated with control IgG1 (10 μM) or with hSFRP2 mAb (10 μM). After 3 days, CFSE intensity was measured using T cells of co-cultures. The mean fluorescence intensity (MFI) was measured by FACS and analyzed using FlowJo software.

[0081] Western blot. Splenic T cells were treated with or without SFRP2 (30nM) or hSFRP2 mAb (10μM) for 1 hour. For SFRP2, control cells received culture medium alone, and for hSFRP2 mAb experiments, control cells received 10μM IgG1. The cells were then centrifuged for 10min at 1000rpm. The culture medium was removed, and the cells were stored frozen at -80°C before treatment. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents as described in the manufacturer's manual (Pierce Biotechnology, Rockford, Illinois). Splenic T cells obtained from transgenic Pmel1 mice (The Jackson laboratory, Bar Harbor, Maine, USA) were treated with or without rhSFRP2 (30nM) or hSFRP2 mAb (10μM) for 1 hour. For rhSFRP2, control cells received culture medium alone, and for hSFRP2 mAb experiments, control cells received 10μM IgG1. The cells were then centrifuged for 10min at 1000rpm. Remove the culture medium, and before treatment, the cells were stored frozen at -80 ° C. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents as described in the manufacturer's manual (Pierce Biotechnology, Rockford, Illinois). Protein concentration was measured using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, California, the United States). Equal amounts of protein were loaded onto SDS-PAGE gels. Protein was transferred to polyvinylidene difluoride membranes, and Western blotting was performed using the following primary antibodies: rabbit anti-CD38 and rabbit anti-histone H3 antibodies, rabbit anti-FZD5, mouse anti-PD1, rabbit anti-NFATC3 and rabbit anti-actin. The following secondary antibodies were used: HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit. Visualization was performed using ECL Advance substrate (GE Healthcare Bio-Sciences, Piscataway, New Jersey, the United States).

[0082] Next, we evaluated whether in vitro hSFRP2 mAb treatment inhibits CD38 and restores NAD+ levels in TGFβ-exposed T cells. Spleens were obtained from C57 / BL6 mice, and single cell suspensions were prepared and resuspended in ACK lysis buffer with PBS for 1 minute. 1% FCS was added to stop the reaction. Once in single cell suspension, CD4 + and CD8 +Cells: TCR119, CD25, GR1, NK1.1, CD11C, CD11B, CD19, and the cells were incubated on ice for 15 minutes. The cells were incubated with 200 μL of streptavidin-bound bead solution. After separation, the cells were counted and 400,000 cells were plated on a plate pre-coated with anti-CD3 (2 μg / mL) and anti-CD28 (5 μg / mL). The negative control contained only isolated cells in a medium rich in IL-2, and no anti-CD3 / Cd28 (TCR) coating. Each experimental well with cells contained TCR and IL-2 (6,000 U / mL) and one of the following experimental conditions: hSFRP2 mAb (10 uM), with or without TGFβ (5 ng / ml). All conditions were performed in triplicate and cultured for three days. After the experiment, the cells were counted and stained for FACS or processed for NAD analysis using a NAD / NADH cell-based assay kit. For NAD analysis, at least 250,000 cells were required and processed immediately following the NAD protocol. The cells were centrifuged and incubated with permeabilization buffer under stirring. After another centrifugation, the samples and standards were incubated with reaction buffer under stirring for 1h 30min. Finally, the optical density was read at 450nm using a plate reader. For FACs analysis, 300,000 cells were resuspended in PBS and incubated in Live dead stain according to the manufacturer's protocol, then washed with PBS, spun down, and the supernatant removed. The cells were then suspended in a master mix of antibodies and staining buffer (50 μL / sample), which contained anti-CD38 PE / Cy5 (1 / 200), anti-CD4 FITC (1 / 100), anti-CD8 APC (1 / 200), and anti-PD1 PE (1 / 200), and incubated at room temperature and protected from light for 20 min. The cells were finally fixed in 4% paraformaldehyde for 10-15 minutes and then resuspended in 250 μL staining buffer.

[0083] In vivo metastatic osteosarcoma growth. In the first experiment, RF420 osteosarcoma cells (5 × 10 5) was injected iv via the tail vein of 6-8 week old C57BL / 6 mice (10 females and 13 males). On day 7, 2 mice were sacrificed, their lungs removed, fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin. Sections were screened under a microscope for the presence of metastases. Once the presence of metastases was confirmed, mice were treated with 4 mg / kg IgG1 control or 4 mg / kg hSFRP2 mAb on day 10 (n=10). After 3 weeks of treatment, animals were sacrificed and the lungs and spleens removed. The lung surface nodules were then counted and compared between treatment groups. Fresh spleens were collected for T cell isolation for flow cytometry.

[0084] Next, RF420 cells (5 × 10 5 ) was injected iv into the tail vein of 6-8 week old C57Bl / 6 male and female mice (strain code 044) purchased from Envigo (Indianapolis, IN, USA). The mice were randomly divided into 4 groups: control group (omalizumab, n=13); hSFRP2 mAb (n=11); mouse PD-1 MAb (n=12); PD-1m Ab + hSFRP2 mAb (n=12). Treatment was started 10 days after tumor cell inoculation. The dose, delivery route and frequency were as follows: control (omalizumab) 4 mg / kg iv once a week; hSFRP2 mAb 4 mg / kg iv every 3 days; pd-1 mab 8 mg / kg intraperitoneal (ip) every 3 days. After 23 days of treatment, the animals were sacrificed and their lungs were resected, and the surface nodules were counted. Surface nodules were counted from whole lung photographs taken immediately after resection. The lungs were fixed in formalin and embedded in paraffin. They were sectioned and stained with H&E.

[0085] Flow cytometry. Staining for CD38 was performed by incubating splenocytes from experiments injected intravenously with RF420 osteosarcoma with primary antibodies against CD38 in FACS buffer (PBS containing 0.1% bovine serum albumin (BSA)) for 30 min at 4°C. Samples were screened for mean fluorescence intensity (MFI) levels on the LSRFortessa and analyzed with FlowJo software (Tree Star, OR).

[0086] Statistics. All power and sample size calculations were performed using PASS version 08.0.13. In vitro experiments were performed in triplicate and repeated three times. Quantitative measurements were collected without the technician knowing the experimental conditions to reduce potential bias. For two or more group comparisons, group comparisons of continuous measurements were performed using a two-sample t-test or ANOVA, respectively.

[0087] Example 3

[0088] Humanization of SFRP2 mAb. Chimeric antibodies and combinations of composite heavy and light chains (a total of 16 antibodies) were tested for binding to SFRP2 in a competition ELISA assay. Specifically, a series of dilutions of purified fully human composite IgG variants were made to compete for binding to SFRP2 peptide B against a fixed concentration of biotinylated mouse antibody. Next, bound biotinylated mAb 80.8.6 (mouse SFRP2 mAb) was detected using streptavidin HRP and TMB substrate. This demonstrates that the binding efficiency of all composite antibodies to SFRP2 is roughly equivalent to that of the chimeric antibody, and all variants showed improvement when compared to the mouse antibody ( Fig.12 Chimeric antibodies and composite variants against SFRP2 were purified from cell culture supernatants on a protein A agarose column, buffer exchanged into pH 7.4 PBS and quantified based on the predicted amino acid sequence using the extinction coefficient (Ec( 0.1 %) = 1.76), by OD 280 The quantification was performed at 400 nm. Endotoxin testing of the lead hSFRP2 mAb showed endotoxin < 0.5 EU / m. SDS-PAGE of the lead hSFRP2 mAb showed two bands corresponding to the heavy chain and the light chain ( Fig.13 ).exist Fig.13 , SDS Page. 1 μg of purified lead hSFRP2 mAb was loaded on a 4-12% NuPAGE-SDS gel. A PageRuler Plus prestained ladder was loaded to allow for determination of band size. Lane 1 was reduced with β-mercaptoethanol; the sample showed two bands corresponding to the heavy and light chains. Lane 2 was not reduced.

[0089] Immunogenicity testing of hSFRP2 mAb using EpiScreen TM The time course T cell assay tested the lead fully humanized and chimeric anti-SFRP2 antibodies against a cohort of 22 healthy donors to determine the relative risk of immunogenicity. In the proliferation assay, the fully humanized anti-SFRP2 antibody did not induce a positive response in any donor, while the chimeric anti-SFRP2 antibody induced a positive T cell proliferation response in 23% of the donors using a SI ≥ 2.0, p < 0.05 threshold. The results with the control antigen KLH showed good correlation between positive and negative results in replicate studies (< 10% inter-assay variability), indicating that the assay has a high level of reproducibility ( Fig.14 ).exist Fig.14In the figure, PBMCs from bulk culture were sampled and evaluated for proliferation on days 5, 6, 7, and 8 after incubation with the three test samples. In this figure, significant (p<0.05) proliferation responses indicated by dashed lines with SI ≥ 2.0 (p<0.05) were considered positive using an unpaired two-sample student's t-test.

[0090] Humanized SFRP2 mAbs bind SFRP2 with high affinity. To determine the binding affinity of the lead hSFRP2 mAb to SFRP2, rhSFRP2 (1 μM) was incubated with increasing concentrations of hSFRP2 mAb in a microplate solid phase protein binding ELISA assay. The hSFRP2 mAb bound rhSFRP2 with an EC50 of 8.72 nM and a Kd of 74.1 nM. Figure 1 A shows that humanized SFRP2 mAb binds rhSFRP2 with high affinity and generates a concentration-response curve showing the absorbance at 480 nm measured after increasing concentrations of hSFRP2 mAb bind to a preset concentration of 1 μM rhSRP2 in an ELISA assay (n=16).

[0091] Humanized SFRP2 mAb inhibited endothelial tube formation, tumor cell proliferation and promoted tumor cell apoptosis. Consistent with previous reports; rhSFRP2 induced an increase in the number of branch points compared to control cells (n=4, p≤0.05). Figure 7 B is a bar graph showing the effect of rhSFRP2 and hSFRP2 mAb on 2H11 endothelial tube formation. To obtain this data, 2H11 cells were incubated with and treated with: IgG1 control only (5 μM), or IgG1 (5 μM) + rhSFRP2 protein (30 nM), or a combination of rhSFRP2 (30 nM) and hSFRP2 mAb (0.5 to 10 μM). n=4, *: p≤0.05; **: p≤0.001. In contrast, increasing concentrations of hSFRP2 mAb significantly counteracted the effect of rhSFRP2 on tube formation (n=4, p≤0.05). IC of hSFRP2 mAb inhibition of SFRP2-stimulated tube formation 50 It is 4.9±2μM.

[0092] The effects of rhSFRP2 mAb on tumor cell proliferation, apoptosis, and necrosis were evaluated in vitro in Hs578T human carcinoma / sarcoma breast cancer and mouse SVR angiosarcoma cells. Figure 7 C; p≤0.05 and p≤0.001 for 5 μM and 10 μM hSFRP2 mAb, respectively) and SVR angiosarcoma cells ( Figure 7F; p≤0.001 for both 5 μM and 10 μM hSFRP2 mAb), treatment with hSFRP2 mAb significantly increased tumor cell apoptosis, while necrosis remained unchanged. Treatment with hSFRP2 mAb had no effect on SVR proliferation ( Figure 7 H), but significantly reduced tumor cell proliferation in Hs578T breast cancer cells ( Figure 7 E, 5μM p≤0.05, 10μM p≤0.001).

[0093] Determination of the efficacy and toxicity of hSFRP2 mAb in vivo. Mice inoculated with SVR angiosarcoma cells were treated with 2, 4, 10 and 20 mg / kg hSFRP2 mAb doses or IgG1 control iv every three days for 21 days. There was no weight loss or lethargy in any of the antibody-treated mice. There were no pathological changes in the liver or lungs, even at the 20 mg / kg dose. At the end of the experiment, body weights remained similar between groups (control, 32.2±1.4 g; 2 mg / kg, 31.3±1.1 g; 4 mg / kg, 32.1±0.5 g; 10 mg / kg, 31.8±0.9 g; and 20 mg / kg, 32.7±1.0 g). The dose with the greatest effect was 4 mg / kg, where tumor volume was reduced by 69% (n=5 per group, p=0.05).

[0094] To investigate the pharmacokinetic properties of the antibody, nude mice were injected with a single dose of 4 mg / kg hSFRP2 mAb via the tail vein, and blood samples were collected at different time points ( Figure 8 ). Recombinant hSFRP2 treatment resulted in an increase in membrane CD38 and nuclear NFATc3 proteins, whereas hSFRP2 mAb inhibited the accumulation of nuclear NFATc3 in T cells. Figure 8 The data in A show that FZD5 protein exists in T cells. Figure 8 A is a pharmacokinetic curve showing that the hSFRP2 mAb concentration in mouse serum decreases over time after a single iv injection of 4 mg / kg. The half-life of the antibody in animal serum was 4.1±0.5 days, the maximum serum concentration (Cmax) was 7.8±1.0 mg / L, and the clearance (CL) was 13.0±0.6 mL / hour.

[0095] To confirm the efficacy of the dose identified in the MTD experiment, the inventors repeated the experiment on a larger number of animals (n=10 animals / group) with SVR angiosarcoma tumors and started treating them with 4 mg / kg hSFRP2 mAb. T cells were treated with rhSFRP2 (30 nM) for 1 h and processed using the NE-PER kit to separate the cytoplasmic and nuclear fractions ( Figure 8B). Samples were probed with antibodies against the indicated protein markers, and protein levels in treated cells were compared to those in untreated cells. After 3 weeks, tumors treated with hSFRP2 mAb were 43% smaller than those treated with IgG1 control (control, 1,631.3 ± 283 mm 3 ; hSFRP2 mAb, 928.5±148 mm 3 ; p≤0.05).

[0096] Next, the inventors considered whether hSFRP2 mAb could affect the growth of other tumor types. Mice bearing Hs578T breast cancer-sarcoma xenografts were treated with hSFRP2 mAb or IGg1 control. T cells were treated with antigen gp100 (0.87 μM) or hSFRP2 mAb (10 μM) alone or in combination for 60 min, and the nuclear fraction ( Figure 8 C). Protein levels of NFATc3 in rhSFRP2 treated cells were compared to those in untreated cells. Comparison between control and each treatment group at each time point showed that treatment was statistically significant (p=0.05) at day 22, day 25, and all time points from day 31 onwards from baseline. In fact, tumor volume was reduced by 61% in hSFRP2 mAb treated mice (n=11, *P<0.05). In addition, there was no weight loss or lethargy in either treated mouse.

[0097] Humanized SFRP2 mAb induces apoptosis of tumor cells in vivo. hSFRP2 mAb induces apoptosis in vitro and inhibits proliferation of breast cancer cells, and the inventors investigated whether these phenotypes are retained in vivo. Although the proportion of proliferative (Ki67-positive) cells was not affected by hSFRP2 mAb treatment compared with IgG1 control tumors (SVR tumors, 23±1.6% vs. 29±4.2%; Hs578T tumors, 18±2.7% vs. 18±2.8%, p=NS), the proportion of apoptotic cells increased by 188% in SVR tumors (8.4±0.9 for IgG1 control and 24.2±3.5 for hSFRP2 mAb tumors; n=10, p≤0.05) and by 181% in Hs578T tumors (15.1±4.9 for IgG1 control and 42.4±3.9 for hSFRP2 mAb tumors; n=10, p≤0.05) ( Fig. 9 ).

[0098] To evaluate the antitumor activity of hSFRP2 mAb in immunocompetent mice, the inventors tested hSFRP2 mAb in RF420 murine osteosarcoma in C57BL / 6 mice in a tumor metastasis model. RF420 osteosarcoma cells were injected into the tail vein of C57BL / 6 mice. Treatment with IgG1 control or hSFRP2 mAb was started on day 10. Mice were euthanized on day 21 of treatment, and surface nodules were counted. The number of surface nodules in mice treated with hSFRP2 mAb was significantly reduced compared with controls (n=7, p<0.01, Fig.10 A). When evaluating the depletion of cell surface markers, the inventors noted that CD38, which is tightly co-expressed with PD-1, was significantly reduced on splenocytes (n=4, p<0.01) and TILs (n=4, p<0.01) in mice treated with hSFRP2 mAb compared to IgG control mice, with no significant differences in PD-1, CD103, and CD5 (n=3) ( Figure 6 B). The expression of other consumption markers such as PD-1, CD103, TNFα or CD5 in splenocytes or TILs was not significant (n=4, p=NS).

[0099] In the second osteosarcoma experiment, osteosarcoma RF420 cells were injected intravenously into immunocompetent mice. The study was divided into four groups. The first group was treated with iv 4mg / kg hSFRP2 mAb every 3 days. There was also an IgG1 control group; a group that was administered the anti-PD-1 antibody nivolumab at 8mg / kg iv every 3 days; and a group that received both hSFRP2 mAb and anti-PD-1 antibody. Treatment began on the 10th day after injection, and the animals were euthanized 3 weeks later, their lungs were removed and the surface nodules were counted (*: p≤0.0001; **: p≤0.01, n=12). These groups were compared to measure the development of lung metastases. Each single treatment reduced the number of surface nodules compared to the IgG1 control (43.6±6.8 for IgG1 control, 18.3±3.4 for hSFRP2mAb, and 16.3±1.1 for nivolumab; p≤0.0001, Fig.11A). The incidence of metastatic lesions was reduced by 80% in mice treated with the combination of hSFRP2 mAb and nivolumab compared to mice treated with IgG1 control (IRR=0.20, 95% CI=0.13 to 0.32; p<0.0001). The incidence of metastatic lesions was reduced by 51% in mice treated with the combination of hSFRP2 mAb and nivolumab compared to mice treated with single-agent hSFRP2 mAb (IRR=0.49, 95% CI=0.31 to 0.77; p=0.0021). The incidence of metastatic lesions was reduced by 45% in mice treated with the combination of hSFRP2 mAb and nivolumab compared to mice treated with single-agent nivolumab (IRR=0.55, 95% CI=0.35 to 0.86; p=0.0084) ( Fig.11 A).

[0100] The inventors measured the effects of nivolumab and hSFRP2 mAb, given as single treatments or in combination, on CD38 levels in mouse T cells. Specifically, T cells were isolated from the spleens of C57BL / 6 mice injected with RF420 cells and treated with IgG1 control, hSFRP2 mAb, nivolumab, or a combination of hSFRP2 mAb and nivolumab. The cells were then stained with fluorescent dye-labeled CD38, and the mean fluorescence intensity (MFI) was analyzed by FACS. Nivolumab treatment alone had no effect on CD38 levels. However, hSFRP2 mAb reduced CD38 surface expression in T cells compared to T cells obtained from the group treated with control IgG antibody (p<0.001, Fig.11 B), indicating that targeting SFRP2 is sufficient to reduce CD38 expression on T cells. These results support the proposition that hSFPR2 mAb administration can restore T cell immune responses and prevent tumor growth. It should be noted that because nivolumab is a human antibody, it is not best suited for treatment in mouse models.

[0101] Humanization of SFRP2 monoclonal antibodies. The V region gene encoding the murine SFRP2 monoclonal antibody 80.8.6 (21) was first cloned and used to construct chimeric antibodies comprising the murine V region and the human IgG1 heavy chain constant region and the kappa light chain constant region. Chimeric antibodies and combinations of composite heavy and light chains (a total of 16 antibodies) were expressed in NS0 or HEK293 cells, purified, and tested for binding to SFRP2 peptide in a competitive ELISA assay.

[0102] Immunogenicity testing. Using EpiScreen TMA time-course T cell assay was performed to evaluate the immunogenic potential of the lead fully humanized anti-SFRP2 antibody (VH2 / VK5) and a reference chimeric anti-SFRP2 antibody using CD8 + Depleted PBMCs were cultured in bulk and after addition of samples, the cells were spiked with 3 H]-thymidine was used to measure T cell proliferation at various time points. TM Time-course T cell assay Lead fully humanized and chimeric anti-SFRP2 antibodies were tested against a cohort of 22 healthy donors to determine the relative risk of nonspecific immunogenicity. Samples were tested at a final concentration of 50 μg / ml based on previous studies by Antitope showing that this saturating concentration was sufficient to stimulate detectable antibody-specific T cell responses. To assess the immunogenic potential of each sample, Episcreen TM Time course T cell assays were used to measure T cell activation together with proliferation analysis. Since samples had not been previously evaluated in PBMC-based assays, an initial assessment of any overall toxic effects of the samples on PBMC viability was determined. After 7 days of culture with the test samples, trypan blue dye exclusion of PBMCs was used to calculate cell viability.

[0103] Antibodies and proteins. The following primary antibodies were used in Western blotting: rabbit anti-CD38 (#14637s) and rabbit anti-histone H3 antibody (#2650s) from Cell Signaling (Danvers, MA, USA); rabbit anti-FZD5 (#H00007855-D01P, Abnova, Taipei, Taiwan, China); mouse anti-PD1 (#66220-1, Proteintech, Rosemont, IL, USA); rabbit anti-NFATc3 (#SAB2101578); and rabbit anti-actin (#A2103, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were: HRP-conjugated anti-mouse (#7076, Cell Signaling); HRP-conjugated anti-rabbit (#403005, Southern Biotech, Birmingham, AL, USA). For ELISA, HRP-conjugated goat anti-human IgG was from Abcam, Cambridge, MA, USA. For FACS analysis, rat anti-CD38-PE antibody (#102707) was from BioLegend (San Diego, CA, USA). Anti-mouse CD3 (#BE00011) and anti-mouse CD28 (#BE0015-1) were from BioXCell (West Lebanon, NH, USA). Control IgG1 omalizumab was purchased from Novartis (Basel, Switzerland). Human SFRP2 protein (rhSFRP2) was prepared as previously described. gp100 antigen fragment was from AnaSpec (#AS-62589).

[0104] Microplate solid phase protein binding (ELISA) assay to determine the binding affinity of rhSFRP2 to hSFRP2 mAb. Microplate solid phase protein binding assay was used to determine the EC of rhSFRP2 and hSFRP2 mAb. 50 . 2+The coated 96-well microplate (#15442, Thermo Fisher Scientific, Waltham, MA, USA) was blocked overnight at 4°C with phosphate buffered saline (PBS, #BP399-1, Fisher Scientific, Waltham, MA, USA) containing 0.05% bovine serum albumin (BSA, #001-000-162, Jackson ImmunoResearch, West Grove, PA, USA). 1 μM his-tagged rhSFRP2 diluted in PBS (pH 7.4) was incubated overnight on the blocked plate at 37°C. The plate was washed 3 times with 250 μl / well of PBS. Increasing doses of hSFRP2 mAb (0pM, 100pM, 200pM, 400pM, 800pM, 1.6nM, 3.15nM, 6.3nM, 12.5nM, 25nM, 50nM, 100nM) in PBS were incubated with rhSFRP2 overnight at 37°C. The plates were washed 3 times, blocked in PBS containing 0.1% BSA for 1 hour at room temperature, and then incubated with 100μl / well of secondary antibody (HRP-conjugated goat anti-human IgG) diluted 1:40,000 in PBS for 1 hour at 37°C. After washing the plates 5 times, each well was incubated with 100μl K-Blue TMB substrate (#308176, Neogen, Lexington, KY, USA) for 5 minutes in the dark. The reaction was stopped with 100ul 2N H2SO4. The absorbance was read at 450 nm. EC was determined by nonlinear regression analysis and variable slope using GraphPad Prism log(inhibitor) versus normalized response - variable slope function. 50 The top constraint of the function is 100%. Using the Cheng-Prusoff equation, EC 50 Convert to Kd, where agonist concentration and EC 50 are equal (40). The results are expressed as mean ± standard error of the mean. Each data point is the result of 8 independent measurements (n=8).

[0105] Cell culture. 2H11 mouse endothelial cells (#CRL-2163, Manassas, VA, USA) were cultured in Opti-MEM (#22600134, Thermo Fisher Scientific, Waltham, MA, USA) with 5% heat-inactivated fetal bovine serum (FBS, #FB-12, Omega Scientific, Biel / Bienne, Switzerland) and 1% penicillin / streptomycin (v / v). Hs578T human breast cancer-sarcoma triple negative cells (#30-202, Manassas, VA, USA) in DMEM with 10% FBS, 0.01 mg / ml bovine insulin (#I0516, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin / streptomycin (#MT30009C, Thermo Fisher Scientific) SVR angiosarcoma cells were obtained from the American Type Culture Collection (#CRL-2280, ) and cultured in Opti-MEM (Thermo Fisher Scientific) with 8% FBS and 1% penicillin / streptomycin (v / v). RF420 mouse osteosarcoma cells (41) established from a genetically engineered osteosarcoma mouse model were obtained from Dr. Jason T. Yustein (Texas Children's Cancer and Hematology Centers, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA) and cultured in DMEM with 10% heat-inactivated FBS and 1% penicillin / streptomycin (v / v). All cell lines were cultured at 37°C in a humidified 5% CO2-95% room air atmosphere. Mouse cells were tested for rodent pathogens, including mycoplasma, by Charles River Research Animal (Wilmington, MA, USA) and whenever they were used in vivo. Murine T cells were isolated from C57BL / 6 mice, and Pmel transgenic mice carrying gp100-reactive TCRs on a C57BL / 6 background were obtained from Jackson Laboratory (Bar Harbor, ME, USA).

[0106] Endothelial tube formation assay. 2H11 endothelial cells were plated in Opti-MEM with 5% FBS and allowed to rest for 24 hours. Quiescence was induced by maintaining cells overnight in Opti-MEM with 2.5% FBS. Matrigel was polymerized in the wells of a 96-well plate according to the in vitro angiogenesis assay protocol. TM (#ECM625, Millipore, Bedford, MA, USA). In this assay, 9 treatment conditions were prepared: IgG1 alone (5 μM; omalizumab); rhSFRP2 protein (30 nM) and IgG1 (5 μM); or rhSFRP2 (30 nM) in combination with increasing concentrations of hSFRP2 mAb (0.5, 1, 5, 10, or 20 μM). Treatments resuspended in Opti-MEM with 2.5% FBS were preincubated on a shaker at 37°C, 5% CO2 for 90 min before they were added to the cells. 1.9×10 4 The cells were resuspended in 150 μL of pre-incubated treatment and then incubated for another 30 min at 37°C, 5% CO2 on a shaker. Finally, the cell suspension was added to each Matrigel coated TM . Each experiment was repeated 4 times independently, with n=4 for each condition. Fresh Opti-MEM with 2.5% FBS and 5 μM IgG1 was given to control cells. For each treatment condition, images were collected using a 4X objective lens of an EVOS FL digital imaging system (Thermo Fisher Scientific, Waltham, MA, USA) after incubation for 4 h at 37°C, 5% CO2. Branch points were counted using ImageJ angiogenesis analysis software (National Institutes of Health, Bethesda, MD, USA). Data were analyzed using nonlinear regression and dose-response-inhibition series equations in GraphPad Prism software to determine IC50.

[0107] Proliferation assay. Hs578T breast cancer-sarcoma and SVR angiosarcoma cells were plated at 3,000 cells / well in 96-well plates. After 4 hours, hSFRP2 mAb (1, 5 or 10 μM) was added to the growth medium at the indicated concentrations. The cells were allowed to incubate at 37°C, 5% CO2 for 72 hours. Proliferation was assessed using the Cyquant Direct Cell Proliferation Assay Kit (#C35011, ThermoFisher Scientific, Waltham, MA, USA). Images were acquired using the EVOS FLc Digital Imaging System (Thermo Fisher Scientific). Cells were counted using FIJI Cell Counting Software.

[0108] Apoptosis / necrosis. Hs578T breast cancer-sarcoma breast and SVR angiosarcoma cells were respectively 4 , 3×10 4 and 7.5×10 3 Cells / well were plated in 16-well chamber slides (#178599, Thermo Fisher Scientific, Waltham, MA, USA). The next day, cells were incubated in suspension with growth medium for 2 hours at 37°C, 5% CO2 with 1, 5 or 10 μM hSFRP2 mAb or 5 μM IgG1 control. Necrosis and apoptosis were determined according to the protocol of the Apoptosis / Necrosis Detection Kit (#PK-CA707-30017, PromoCell, GmbH, Heidelberg, Germany). Images were collected using the EVOS FLc digital imaging system (Thermo Fisher Scientific, Waltham, MA, USA). Cells were counted using ImageJ cell counting software. Each data point is the result of 2 independent experimental replicates, each replicate containing 4 individual wells (total n=8).

[0109] Western blot. Splenic T cells obtained from transgenic Pmel1 mice (The Jackson laboratory, Bar Harbor, Maine, USA) were treated for 1 hour with or without rhSFRP2 (30nM) or hSFRP2 mAb (10 μM). For rhSFRP2, control cells received culture medium alone, and for hSFRP2 mAb experiments, control cells received 10 μM IgG1. The cells were then centrifuged for 10 min at 1000 rpm. The culture medium was removed and the cells were stored frozen at -80 ° C before treatment. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents as described in the manufacturer's manual (Pierce Biotechnology, Rockford, Illinois). Protein concentrations were measured using Bio-Rad protein assays (Bio-Rad Laboratories, Hercules, California, USA). Equal amounts of protein were loaded onto SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes and Western blotted using the following primary antibodies: rabbit anti-CD38 and rabbit anti-histone H3 antibodies, rabbit anti-FZD5, mouse anti-PD1, rabbit anti-NFATc3, and rabbit anti-actin. The following secondary antibodies were used: HRP-conjugated anti-mouse and HRP-conjugated anti-rabbit. Visualization was performed using ECL Advance substrate (GE Healthcare Bio-Sciences, Piscataway, NJ, USA).

[0110] FACS analysis of cell proliferation was performed by measuring the CFSE signal intensity. Dilution of CFSE signal correlates well with increased cell proliferation. TM The instructions of CFSE cell proliferation kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were used to pre-mark the spleen T cells of Pmel1 transgenic mice with CFSE dye. The cells were then kept untreated or activated for 3 days with soluble anti-CD3 (#BE0001-1, BioXCell, West Lebanon, New Hampshire, USA; 2 μg / ml) / anti-CD28 antibodies (#BE0015-1, BioXCell; 2 μg / ml) alone or in the presence of tumor cells (SVR angiosarcoma or Hs578T breast cancer-sarcoma) at a 2:1 ratio. In addition, some co-cultures were treated with control IgG1 (10 μM) or with hSFRP2 mAb (10 μM). After 3 days, CFSE intensity was measured using T cells of co-culture. Mean fluorescence intensity (MFI) was measured by FACS and analyzed using FlowJo software.

[0111] The maximum tolerated dose (MTD) of hSFRP2 mAb in vivo. The animal experimental protocol was in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 6 SVR angiosarcoma cells were injected subcutaneously into the right flank of 6-week-old male and female nude mice obtained from Charles River (Wilmington, MA, USA). The next day, mice (n=5 per group) were treated iv with PBS control and various concentrations of purified hSFRP2 mAb (2, 4, 10 and 20 mg / kg) injected via the tail vein every 3 days. Animals were treated and tumor volumes were measured every three days until control tumors reached an average diameter of 2 cm, which was defined as the endpoint. After euthanasia, tumors, lungs, and livers were harvested and fixed in 10% formalin.

[0112] Pharmacokinetic study. Male and female C57BL / 6 mice were injected with 4 mg / kg of hSFRP2 mAb at different time points (0, 5 min, 1, 2, 7, 14, 21, 28, 35 and 42 days). Three mice (n=3) were used for each time point. At the end point, blood samples were obtained through the portal vein and placed in separation tubes (#367981, Becton Dickinson, Franklin Lakes, NJ, USA). The samples were centrifuged at 1300 x g for 15 min.

[0113] Microplate solid phase protein binding (ELISA) assay for the pharmacokinetics (PK) of hSFRP2 mAb. Flat bottom Ni plates were blocked with PBS containing 0.05% BSA at 4°C. 2+Coated 96-well microplate overnight. At 37°C, 1 μM his-tagged rhSFRP2 diluted in PBS (pH 7.4) was incubated overnight. The plate was washed 3 times with 250 μl / well PBS. Then, mouse serum diluted 1:50 was added to the plate and incubated overnight with gentle shaking at 37°C. The plate was washed 3 times, blocked in PBS containing 0.1% BSA for 1 hour at room temperature, and then incubated with 100 μl / well secondary antibody (HRP-conjugated goat anti-human IgG) diluted 1:40,000 in PBS at 37°C for 1 hour. After the plate was washed 5 times, each well was incubated with 100 μl K-Blue TMB substrate for 5 minutes in the dark. The reaction was stopped with 100 μl 2N H2SO4. The absorbance was read at 450nm. For PK estimation of AUC, t1 / 2, CL, Vd, Tmax, and Cmax were determined using non-compartmental analysis (NCA) in EXCEL and (42). NCA used the linear trapezoidal rule to determine the area under the plasma concentration-time curve (AUC). 1 / 2 For AUC calculations, nM concentrations from ELISA were converted to mg / L.

[0114] In vivo angiosarcoma allografts. 6 SVR angiosarcoma cells were injected subcutaneously into the right flank of 6-week-old male and female nude mice obtained from Charles River (Wilmington, MA, USA). The next day, mice (n=10 animals / group) were injected iv with hSFRP2 mAb (4 mg / kg) or IgG1 control (omalizumab 4 mg / kg) via the tail vein and treated every 3 days. Tumor volume was calculated using the following formula using serial caliper measurements of perpendicular diameters performed twice a week: [(L (mm) × W (mm) × H (mm)) × 0.5]. The body conditioning scores and body weights of mice were monitored daily. Mice were sacrificed when the control reached a diameter of 2 cm, and tumors were excised and fixed in formalin and embedded in paraffin.

[0115] In vivo Hs578T breast cancer-sarcoma xenografts. Hs578T xenografts were established in 5- to 6-week-old female nude mice from Charles River (Wilmington, MA, USA). 6 cells, and when the average tumor size is approximately 100 mm 3Treatment was started at 40 days (day 30). Animals were treated with 4 mg / kg hSFRP2 mAb (n=11) or 4 mg / kg IgG1 control (n=11) injected iv every 3 days until endpoint, when the diameter of the control tumors reached 2 cm. Tumors were measured twice a week using calipers and tumor volumes were calculated as described above. Tumors were excised, fixed in formalin and embedded in paraffin.

[0116] In vivo RF420 metastatic osteosarcoma. In the first experiment, RF420 osteosarcoma cells (5 × 10 5 ) was injected iv via the tail vein of 6-8 week old C57BL / 6 mice (10 females and 13 males). On day 7, 2 mice were sacrificed, their lungs removed, fixed in 10% formalin, embedded in paraffin and stained with hematoxylin and eosin. Sections were screened under a microscope for the presence of metastases. Once the presence of metastases was confirmed, mice were treated with 4 mg / kg IgG1 control or 4 mg / kg hSFRP2 mAb on day 10 (n=10). After 3 weeks of treatment, animals were sacrificed and lungs removed. Surface nodules were counted. In a second experiment, RF420 cells (5×10 5 ) was injected iv into the tail vein of 6-8 week old C57Bl / 6 male and female mice (strain code 044) purchased from Envigo (Indianapolis, IN, USA). The mice were randomly divided into 4 groups: control group (omalizumab, n=13); hSFRP2 mAb (n=11); nivolumab (NDC#0003-3772-11, Bristol-Meyers Squibb, Princeton, NJ) (n=12); nivolumab + hSFRP2 mAb (n=12). Treatment was started 10 days after tumor cell inoculation. The dose, delivery route and frequency were as follows: control (omalizumab) 4 mg / kg iv once a week; hSFRP2 mAb 4 mg / kg iv every 3 days; nivolumab 8 mg / kg ip every 3 days. After 23 days of treatment, the animals were sacrificed and their lungs were removed, and the surface nodules were counted. Surface nodules were counted from whole lung photographs taken immediately after resection. Fresh spleens were collected for T cell isolation, immunohistochemistry, and tunnel assays.

[0117] Immunohistochemistry. Formalin-fixed, paraffin-embedded tumor sections were deparaffinized twice in xylene for 10 minutes and hydrated twice in absolute ethanol, twice in 95% ethanol, and then in tap water. The slides were incubated in 3% hydrogen peroxide for ten minutes at room temperature, followed by two washes in PBS1X. The citrate buffer antigen retrieval step was performed in a vegetable steamer using the kit carrier antigen retrieval citrate buffer pH6 (H-3300) for 40 minutes and cooled for 10 minutes. The slides were incubated in the blocking serum provided in the carrier rabbit IMPRES HRP kit (MP-4100) for 1 hour at room temperature in a humidified slide chamber. The blocking serum was then drained and the slides were incubated overnight at 4°C with a 1:40 dilution of the Ki67 antibody (PA1-21520). The next day, the slides were rinsed 3 times in PBS for 5 minutes each wash. Secondary antibodies from the Vector Rabbit MPRESS HRP Kit were added and slides were incubated at RT for 30 min, then rinsed 3 times in PBS for 5 min each wash. DAB solution was prepared and added to the slides as directed in the Vector DAB Kit (SK-4100) for 5 min, rinsed in PBS, and counterstained with hematoxylin for 30 seconds. The slides were then washed in distilled water, followed by washing in amino alcohol, dehydrated twice in 95% ethanol, twice in 100% ethanol, and twice in xylene before coverslipping. Tumor proliferation was quantified as the number of positively stained cells / unit area, using an average of 3 fields per section.

[0118] TUNEL assay. Sections from Hs578T and SVR tumors were stained for apoptotic cells using the manufacturer's protocol for the peroxidase in situ apoptosis detection kit (#S7100). All sections were deparaffinized using (#HS-200, National Diagnostics, Atlanta, Georgia, USA). The following materials were not supplied with the TUNEL kit and were purchased separately: 30% hydrogen peroxide (#5155-01, JTBaker, Phillipsburg, New Jersey, USA), proteinase K (#21627, Millipore, Burlington, Massachusetts, USA), metal-enhanced DAB substrate kit (#34065, Thermoscientific, Waltham, Massachusetts, USA), stable peroxidase substrate buffer 1X (#1855910, Thermoscientific, Waltham, Massachusetts, USA) and 1-butanol (#B7908, Sigma-Aldrich, St. Louis, Missouri, USA). Five fields were randomly selected in each sample and photographed using an EVOS FLc microscope (Life Technologies Inc., Waltham, MA, USA). In each field, tumor cell apoptosis was quantified as the number of apoptotic nuclei / HPF.

[0119] Flow cytometry. Staining for CD38 surface expression was performed by incubating splenocytes from experiments with intravenously injected RF420 osteosarcomas with rat anti-CD38-PE antibody (1:200; #102707, Biolegend, San Diego, CA, USA) in FACS buffer (PBS containing 0.1% BSA) for 30 min at 4°C. Samples were screened for CD38 mean fluorescence intensity (MFI) levels on the LSRFortessa and analyzed with FlowJo software (Tree Star, OR).

[0120] Statistics. For in vitro assays, statistical differences between IgG1 and hSFRP2 mAb treatment were calculated using a two-tailed student's t-test, with p≤0.05 considered significant. For in vivo tumor studies of angiosarcoma (where treatment was initiated one day after tumor inoculation), a two-tailed student's t-test was used. For Hs578T, where treatment was initiated on day 30 when the tumor was palpable, data were normalized to adjust for differences in baseline tumor volume by dividing the tumor volume from day 34 to day 82 by the baseline (day 30) tumor volume of each group. A two-sample t-test was used for each time point, and tumor volumes were compared between treated and control animals. In order to meet the normality assumption of the t-test, normalized tumor volumes were log-transformed. For multiple comparisons in osteosarcoma studies, the inventors modeled the counts of macrometastatic lesions as a function of treatment group using a negative binomial generalized linear model (NBGLM). Treatment group comparisons were performed using a model-based linear contrast. All analyses were performed using R version 3.2.3. The inventors summarize the incidence rate ratios (IRRs) and corresponding 95% confidence intervals (CIs) comparing IgG1, hSFRP2 mAb, and nivolumab to the combination of hSFRP2 mAb and nivolumab. The inventors hypothesize that the combination therapy will reduce the incidence of macrometastatic lesions relative to single-agent therapy, so constructing the IRR with the treatment represented in the denominator (IgG1, hSFRP2 mAb, or nivolumab) helps explain the impact of the combination therapy relative to the single agents.

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[0173] 50.U.S.Patents Nos.8,734,789,and 9,073,982

[0174] ***

[0175] It should be understood that the present invention is not limited to the specific embodiments of the invention described above, as variations may be made thereto and still fall within the scope of the appended claims.

[0176] The present invention will be further described by, but not limited to, the following numbered paragraphs:

[0177] 1. A pharmaceutical combination comprising a therapeutically effective amount of a SFRP2 antagonist, a CD38 antagonist and / or a PD-1 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0178] 2. The drug combination according to paragraph 1, wherein the SFRP2, CD38 and / or PD-1 antagonist is:

[0179] a. an antibody or antigen-binding fragment of an antibody that specifically binds to SFRP2, CD38 and / or PD-1 receptor and inhibits their activation, or

[0180] b. A soluble form of the SFRP2, CD38 and / or PD-1 receptor that specifically binds to the SFRP2, CD38 and / or PD-1 ligand and inhibits the binding of the SFRP2, CD38 and / or PD-1 ligand to the SFRP2, CD38 and / or PD-1 receptor.

[0181] 3. The pharmaceutical combination according to any one of paragraphs 1-2, wherein the SFRP2, CD38 and / or PD-1 antagonist is a SFRP2, CD38 and / or PD-1 monoclonal antibody (mAb).

[0182] 4. The pharmaceutical combination according to paragraph 3, wherein the SFRP2 monoclonal antibody is a human antibody or a humanized antibody.

[0183] 5. The pharmaceutical combination according to any one of paragraphs 1 to 4, wherein the PD-1 antagonist is:

[0184] a. an antibody or an antigen-binding fragment of an antibody that specifically binds to the PD-1 receptor and inhibits its activation, or

[0185] b. A soluble form of the PD-1 receptor that specifically binds to a PD-1 ligand and inhibits the binding of the PD-1 ligand to the PD-1 receptor.

[0186] 6. The pharmaceutical combination according to paragraph 5, wherein the PD-1 antagonist is a soluble form of the PD-1 receptor and the PD-1 ligand is PD-L1 or PD-L2.

[0187] 7. The pharmaceutical combination according to any one of paragraphs 1 to 5, wherein the PD-1 antagonist is a PD-1 monoclonal antibody.

[0188] 8. The pharmaceutical combination according to any one of paragraphs 1-5, wherein the PD-1 antagonist is nivolumab.

[0189] 9. The pharmaceutical combination according to any one of paragraphs 1-5, wherein the PD-1 antagonist is pembrolizumab, avelumab, durvalumab, cemiplimab or atezolizumab.

[0190] 10. The pharmaceutical combination according to any one of paragraphs 1-9, wherein the therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonist is 0.1 mg / kg body weight to 100 mg / kg body weight.

[0191] 11. The pharmaceutical combination according to any one of paragraphs 1-9, wherein the therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonist is 0.2-3, 0.27-2.70, 0.27, 0.54, 1.35 or 2.70 mg / kg body weight.

[0192] 12. The pharmaceutical combination according to any one of paragraphs 1-11, wherein the therapeutically effective amount of SFRP2, CD38 and / or PD-1 antagonist is 10 mg-200 mg, 17 mg, 33 mg, 84 mg or 167 mg.

[0193] 13. The pharmaceutical combination according to any one of paragraphs 1-12, wherein the therapeutically effective amount of the PD-1 antagonist is 0.1 mg / kg body weight to 100 mg / kg body weight.

[0194] 14. The pharmaceutical combination according to any one of paragraphs 1-12, wherein the therapeutically effective amount of the PD-1 antagonist is 0.02-1.2, 0.027-1.08, 0.027 or 1.08 mg / kg body weight.

[0195] 15. The pharmaceutical combination according to any one of paragraphs 1-14, wherein the therapeutically effective amount of the PD-1 antagonist is 1-80, 1.6-67, 1.6 or 67 mg / kg body weight.

[0196] 16. A method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2, CD38 and / or PD-1 antagonist and a therapeutically effective amount of a PD-1 antagonist.

[0197] 17. The method according to paragraph 16, wherein the administration is simultaneous administration or sequential administration.

[0198] 18. The method according to paragraph 16 or 17, wherein the SFRP2, CD38 and / or PD-1 antagonist is:

[0199] a. an antibody or antigen-binding fragment of an antibody that specifically binds to SFRP2, CD38 and / or PD-1 receptor and inhibits their activation, or

[0200] b. A soluble form of the SFRP2, CD38 and / or PD-1 receptor that specifically binds to the SFRP2, CD38 and / or PD-1 ligand and inhibits the binding of the SFRP2, CD38 and / or PD-1 ligand to the SFRP2, CD38 and / or PD-1 receptor.

[0201] 19. The method according to any of paragraphs 16-18, wherein the SFRP2, CD38 and / or PD-1 antagonist is a SFRP2, CD38 and / or PD-1 monoclonal antibody (mAb).

[0202] 20. The method according to paragraph 19, wherein the SFRP2 monoclonal antibody is a human antibody or a humanized antibody.

[0203] 21. The method according to any one of paragraphs 16-20, wherein the PD-1 antagonist is:

[0204] a. an antibody or an antigen-binding fragment of an antibody that specifically binds to the PD-1 receptor and inhibits its activation, or

[0205] b. A soluble form of the PD-1 receptor that specifically binds to a PD-1 ligand and inhibits the binding of the PD-1 ligand to the PD-1 receptor.

[0206] 22. The method according to paragraph 21, wherein the PD-1 antagonist is a soluble form of the PD-1 receptor and the PD-1 ligand is PD-L1 or PD-L2.

[0207] 23. The method according to any of paragraphs 16-22, wherein the PD-1 antagonist is a PD-1 monoclonal antibody.

[0208] 24. The method according to any of paragraphs 16-23, wherein the PD-1 antagonist is nivolumab.

[0209] 25. The method according to any of paragraphs 16-23, wherein the PD-1 antagonist is pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

[0210] 26. The method according to any one of paragraphs 16-25, wherein the cancer is breast cancer.

[0211] 27. The method according to any one of paragraphs 16-26, wherein the cancer is angiosarcoma, lung cancer, osteosarcoma, melanoma, non-small cell lung cancer or renal cancer.

[0212] 28. The method according to any of paragraphs 16-27, wherein administration of the SFRP2, CD38 and / or PD-1 antagonist precedes administration of the PD-1 antagonist.

[0213] 29. The method according to any of paragraphs 16-27, wherein administration of the PD-1 antagonist precedes administration of the SFRP2, CD38 and / or PD-1 antagonist.

[0214] 30. The method according to any of paragraphs 16-29, wherein the SFRP2, CD38 and / or PD-1 antagonist is administered adjunctively to the PD-1 antagonist.

[0215] 31. The method according to any of paragraphs 16-29, wherein the PD-1 antagonist is administered adjunctively to the SFRP2, CD38 and / or PD-1 antagonist.

[0216] 32. The method according to any of paragraphs 16-31, wherein the SFRP2, CD38 and / or PD-1 antagonist is administered daily, more than once daily, or less than once daily.

[0217] 33. The method according to any of paragraphs 16-31, wherein the SFRP2, CD38 and / or PD-1 antagonist is administered once every 3 days, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks.

[0218] 34. The method according to any of paragraphs 16-33, wherein the PD-1 antagonist is administered daily, more than once daily, or less than once daily.

[0219] 35. The method according to any of paragraphs 16-33, wherein the PD-1 antagonist is administered once every 3 days, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks.

[0220] 36. The method according to any of paragraphs 16-35, wherein the PD-1 antagonist is nivolumab and the amount of nivolumab administered to the subject is 3 mg / kg body weight every 3 weeks, 240 mg every 2 weeks, or 480 mg every 4 weeks.

[0221] 37. The method according to any of paragraphs 16-35, wherein the PD-1 antagonist is pembrolizumab and the amount of pembrolizumab administered to the subject is 200 mg every 3 weeks.

[0222] 38. The method of any of paragraphs 16-35, wherein the PD-1 antagonist is avelumab and the amount of avelumab administered to the subject is 800 mg every 2 weeks.

[0223] 39. The method according to any of paragraphs 16-35, wherein the PD-1 antagonist is durvalumab and the amount of durvalumab administered to the subject is 10 mg / kg body weight every 2 weeks.

[0224] 40. The method according to any of paragraphs 16-35, wherein the PD-1 antagonist is cemiprimab and the amount of cemiprimab administered to the subject is 250 mg every 3 weeks.

[0225] 41. The method according to any of paragraphs 16-35, wherein the PD-1 antagonist is atezolizumab and the amount of atezolizumab administered to the subject is 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks.

[0226] 42. The method according to any of paragraphs 16-41, wherein the subject is receiving PD-1 antagonist therapy prior to initiating SFRP2, CD38 and / or PD-1 antagonist therapy.

[0227] 43. The method according to any of paragraphs 16-41, wherein the subject is receiving SFRP2, CD38 and / or PD-1 antagonist therapy prior to initiating PD-1 antagonist therapy.

[0228] 44. The method according to paragraph 42 or 43, wherein the subject is receiving the first therapy for at least 8 weeks, at least 10 weeks, at least 24 weeks, at least 28 weeks, at least 48 weeks or at least 52 weeks prior to starting the second therapy.

[0229] 45. The method according to any of paragraphs 16-44, wherein the regular administration of the SFRP2, CD38 and / or PD-1 antagonist and / or the PD-1 antagonist continues for at least 3 days, at least 30 days, at least 42 days, at least 8 weeks, at least 12 weeks, at least 24 weeks or at least 6 months.

[0230] 46. ​​The method according to any of paragraphs 16-45, wherein the amount of SFRP2, CD38 and / or PD-1 antagonist when taken alone and the amount of PD-1 antagonist when taken alone are each effective to treat the subject.

[0231] 47. The method according to any of paragraphs 16-45, wherein the amount of SFRP2, CD38 and / or PD-1 antagonist when taken alone, the amount of PD-1 antagonist when taken alone, or each such amount when taken alone, is ineffective in treating the subject.

[0232] 48. The method according to any of paragraphs 16-45, wherein the amount of SFRP2, CD38 and / or PD-1 antagonist when taken alone, the amount of PD-1 antagonist when taken alone, or each such amount when taken alone, is less effective in treating the subject.

[0233] 49. The method according to any one of paragraphs 16-48, wherein the subject is a human patient.

[0234] 50. The method according to any of paragraphs 16-49, wherein the patient previously received PD-1 antagonist therapy and stopped receiving PD-1 antagonist therapy prior to receiving the combination therapy.

[0235] 51. The method according to any one of paragraphs 16-50, wherein the patient has previously failed to respond to PD-1 antagonist therapy, or administration of a PD-1 antagonist fails to treat the subject.

[0236] 52. A kit for treating a patient suffering from cancer, the kit comprising a therapeutically effective amount of an SFRP2, CD38 and / or PD-1 antagonist, a therapeutically effective amount of a PD-1 antagonist, and an insert comprising instructions for use of the kit.

[0237] 53. A pharmaceutical composition comprising an amount of a PD-1 antagonist and an amount of a SFRP2, CD38 and / or PD-1 antagonist.

[0238] 54. The pharmaceutical composition according to paragraph 53, which essentially comprises an amount of a PD-1 antagonist and an amount of a SFRP2, CD38 and / or PD-1 antagonist.

[0239] 55. The pharmaceutical composition according to paragraph 53 or 54, for treating a subject suffering from cancer, wherein said amount of said PD-1 antagonist and an amount of said SFRP2, CD38 and / or PD-1 antagonist are administered simultaneously, concurrently or concurrently.

[0240] 56. A therapeutic package for distribution to or for distribution to a subject having cancer, the therapeutic package comprising: a) one or more unit doses, each such unit dose comprising: i) an amount of a PD-1 antagonist and ii) an amount of a SFRP2, CD38 and / or PD-1 antagonist, wherein the respective amounts of the PD-1 antagonist and the SFRP2, CD38 and / or PD-1 antagonist in the unit dose are effective for treating the subject when administered concurrently to the subject, and b) a finished pharmaceutical container thereof, the pharmaceutical container containing the one or more unit doses, the container further containing or comprising a label directing the use of the package in the treatment of the subject.

[0241] 57. An SFRP2, CD38 and / or PD-1 antagonist for use as an add-on therapy or in combination with a PD-1 antagonist in treating a subject suffering from cancer.

[0242] 58. A PD-1 antagonist for use as an add-on therapy or in combination with an SFRP2, CD38 and / or PD-1 antagonist in treating a subject suffering from cancer.

[0243] 59. Use of an amount of SFRP2, CD38 and / or PD-1 antagonist and an amount of PD-1 antagonist in the preparation of a combination for treating a subject with cancer, wherein the SFRP2, CD38 and / or PD-1 antagonist and the PD-1 antagonist are prepared for simultaneous, concurrent or concurrent administration.

[0244] 60. A combination of a SFRP2, CD38 and / or PD-1 antagonist and a PD-1 antagonist for use in the manufacture of a medicament.

[0245] 61. A combination according to paragraph 60, wherein the medicament is for the treatment, prevention or alleviation of symptoms of cancer.

[0246] 62. A method for treating cancer, the method comprising administering a therapeutically effective amount of a SFRP2 monoclonal antibody (mAb) to a subject in need thereof, wherein the subject has increased expression of CD38 and / or PD-1.

[0247] 63. The method according to paragraph 62, wherein the subject's T cells have increased expression of CD38 and / or PD-1.

[0248] 64. The method according to paragraph 62 or 63, wherein the SFRP2 monoclonal antibody is a human antibody or a humanized antibody.

[0249] 65. The method according to any of paragraphs 62-64, wherein the cancer is breast cancer.

[0250] 66. The method according to any of paragraphs 62-64, wherein the cancer is angiosarcoma, lung cancer, osteosarcoma, melanoma, non-small cell lung cancer or renal cancer.

[0251] 67. The method according to any of paragraphs 62-66, wherein the SFRP2 monoclonal antibody is administered daily, more than once daily, or less than once daily.

[0252] 68. The method according to any of paragraphs 62-67, wherein the SFRP2 monoclonal antibody is administered once every 3 days, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks.

[0253] 69. The method according to any of paragraphs 62-67, wherein the regular administration of the SFRP2 monoclonal antibody continues for at least 3 days, at least 30 days, at least 42 days, at least 8 weeks, at least 12 weeks, at least 24 weeks, or at least 6 months.

[0254] 70. The method according to any of paragraphs 62-69, wherein the subject is a human patient.

Claims

1. A method for treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a SFRP2, CD38 and / or PD-1 antagonist and a therapeutically effective amount of a PD-1 antagonist.

2. The method of claim 1, wherein the administration is simultaneous administration or sequential administration.

3. The method of claim 1, wherein the SFRP2, CD38 and / or PD-1 antagonist is: a. an antibody or antigen-binding fragment of an antibody that specifically binds to SFRP2, CD38 and / or PD-1 receptor and inhibits their activation, or b. A soluble form of the SFRP2, CD38 and / or PD-1 receptor that specifically binds to the SFRP2 and / or CD38 ligand and inhibits the binding of the SFRP2, CD38 and / or PD-1 ligand to the SFRP2, CD38 and / or PD-1 receptor.

4. The method of claim 1, wherein the SFRP2, CD38 and / or PD-1 antagonist is a SFRP2, CD38 and / or PD-1 monoclonal antibody (mAb). The method of claim 4 , wherein the SFRP2 monoclonal antibody is a human antibody or a humanized antibody.

6. The method of claim 1, wherein the PD-1 antagonist is: a. an antibody or an antigen-binding fragment of an antibody that specifically binds to the PD-1 receptor and inhibits its activation, or b. A soluble form of the PD-1 receptor that specifically binds to a PD-1 ligand and inhibits the binding of the PD-1 ligand to the PD-1 receptor.

7. The method of claim 6, wherein the PD-1 ligand is PD-L1 or PD-L2. The method of claim 1 , wherein the PD-1 antagonist is a PD-1 monoclonal antibody.

9. The method of claim 1, wherein the PD-1 antagonist is nivolumab.

10. The method of claim 1, wherein the PD-1 antagonist is pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

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