Methods and Compositions for the Treatment of Cancer

Abstract

Standard treatment for cancer patients includes administration of broadly cytotoxic drugs that do not exhibit direct antitumor activity. Such treatments exhibit debilitating side effects and are prone to developing resistance. Thus, there is a need for improved cancer therapeutics that are directly antitumor and antimetastatic. The present technology relates to methods and compositions for improved cancer therapy. Specifically, the present technology relates to determining the levels of CD36 and CD47 in cancers that exhibit elevated CD36 and CD47 levels or activity and administering agents that modulate their activity.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 290,834, filed December 17, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present technology relates to methods and compositions for improved cancer therapy. Specifically, the present technology relates to determining levels of CD36 and CD47 in cancers and administering agents that modulate their activity, where the cancer exhibits elevated levels or activity of CD36 and CD47. [Background technology]

[0003] Standard treatment for cancer patients involves the administration of broadly cytotoxic agents that do not exhibit direct antitumor activity. Such treatments exhibit debilitating side effects and are prone to the development of resistance. Thus, there is a need for improved cancer therapeutics that are directly antitumor and antimetastatic.

[0004] One of the main barriers to effective cancer treatment is the immunosuppressive nature of the tumor microenvironment (TME), mediated by myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells (Tregs). High levels of the cell surface receptors CD36 and CD47 are associated with poor prognostic outcomes in many types of cancer, including pancreatic cancer and glioblastoma (Enciu et al., 2018; Huang et al., 2020). However, therapeutic agents that simultaneously target both molecules are now known.

[0005] It has previously been shown that thrombodospondin-1 (TSP-1) binds to CD36 and CD47 to induce apoptosis in tumor and endothelial cells, increase the M1:M2 macrophage ratio, and activate cytotoxic T lymphocytes (CTLs) (Dawson et al. 1996, Martin-Manso et al. 2008, Russell et al. 2015). Thus, therapeutic agents that induce TSP-1 may be useful in treating cancers associated with dually elevated CD36 and CD47 levels. Summary of the Invention

[0006] In one aspect, the disclosure provides a method for assessing a subject's responsiveness to cancer treatment with a Tsp-1 inducer, the method comprising determining levels of CD36 and CD47 in a sample obtained from the subject, wherein doubly elevated levels of CD36 and CD47 in the sample compared to control levels indicate that the subject will respond, or is likely to respond, to cancer treatment with a Tsp-1 inducer.

[0007] In some embodiments, the levels of CD36 and CD47 in the sample are determined in vitro. In some embodiments, the method further comprises selecting a subject having doubly elevated levels of CD36 and CD47 in the sample compared to control levels for treatment with a Tsp-1 inducer. In some embodiments, the method further comprises administering to the subject an effective amount of a Tsp-1 inducer to treat cancer.

[0008] In one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising determining levels of CD36 and CD47 in a sample obtained from the subject, wherein doubly elevated levels of CD36 and CD47 in the sample compared to control levels indicate that the subject will respond or is likely to respond to cancer treatment with a Tsp-1 inducer, and further comprising administering to a subject having doubly elevated levels of CD36 and CD47 in the sample compared to control levels an effective amount of a Tsp-1 inducer to treat the cancer.

[0009] In one aspect, the disclosure provides a method for treating a subject having cancer, the method comprising: (a) selecting a subject having cancer based on the subject being known to have doubly elevated levels of CD36 and CD47 in a sample compared to control levels; and (b) administering to the subject an effective amount of a Tsp-1 inducer to treat the cancer.

[0010] In some embodiments, the control level is a CD36 and CD47 level in a non-cancerous cell or tissue obtained from the subject. In some embodiments, the control level is a CD36 and CD47 level in a cell or tissue obtained from a healthy subject or a population of healthy subjects. In some embodiments, the control level is a predetermined level. In some embodiments, the CD36 and CD47 level comprises CD36 and CD47 protein levels, CD36 and CD47 mRNA levels. In some embodiments, the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

[0011] In some embodiments, the Tsp-1 inducer comprises a Psap peptide having the amino acid sequence CDWLPK (SEQ ID NO:1), DWLPK (SEQ ID NO:2), or DWLP (SEQ ID NO:3), or an amino acid substitution variant thereof, wherein the amino acid substitution is a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof, for leucine (L); c) arginine (R) for lysine (K); d) the D isomer of aspartic acid (D) versus the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) versus the L isomer of leucine (L); e) the L-isomer of tryptophan (W) versus the D-isomer of tryptophan (W), and / or the L-isomer of proline (P) versus the D-isomer of proline (P), or a combination thereof.

[0012] In some embodiments, the Psap peptide is 50 amino acids or less in length. In some embodiments, the Psap peptide is 30 amino acids or less in length. In some embodiments, the Psap peptide is 15 amino acids or less in length. In some embodiments, the Psap peptide is 6 amino acids or less in length. In some embodiments, the Psap peptide is a cyclic peptide. In some embodiments, the non-standard amino acid of similar size is methylvaline, methylleucine, or sarcosine. In some embodiments, the Tsp-1 inducer is cyclic DWLPK (SEQ ID NO:2).

[0013] In one aspect, the disclosure provides a composition for use in treating a subject having a cancer characterized by doubly elevated levels of CD36 and CD47 in a sample compared to control levels, the composition comprising a Tsp-1 inducer.

[0014] In one aspect, the disclosure provides the use of a composition in the manufacture of a medicament for treating a subject having a cancer characterized by doubly elevated levels of CD36 and CD47 in a sample compared to control levels, wherein the composition comprises a Tsp-1 inducer.

[0015] In some embodiments, the control level is a CD36 and CD47 level in a non-cancerous cell or tissue obtained from the subject. In some embodiments, the control level is a CD36 and CD47 level in a cell or tissue obtained from a healthy subject or a population of healthy subjects. In some embodiments, the control level is a predetermined level. In some embodiments, the CD36 and CD47 level comprises CD36 and CD47 protein levels, CD36 and CD47 mRNA levels. In some embodiments, the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

[0016] In some embodiments, the Tsp-1 inducer comprises a Psap peptide having the amino acid sequence CDWLPK, DWLPK, or DWLP, or an amino acid substitution variant thereof, wherein the amino acid substitution is a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof, for leucine (L); c) arginine (R) for lysine (K); d) the D isomer of aspartic acid (D) versus the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) versus the L isomer of leucine (L); e) the L-isomer of tryptophan (W) versus the D-isomer of tryptophan (W), and / or the L-isomer of proline (P) versus the D-isomer of proline (P), or a combination thereof.

[0017] In some embodiments, the Psap peptide is 50 amino acids or less in length. In some embodiments, the Psap peptide is 30 amino acids or less in length. In some embodiments, the Psap peptide is 15 amino acids or less in length. In some embodiments, the Psap peptide is 6 amino acids or less in length. In some embodiments, the Psap peptide is a cyclic peptide. In some embodiments, the non-standard amino acid of similar size is methylvaline, methylleucine, or sarcosine. In some embodiments, the Tsp-1 inducer is cyclic DWLPK (SEQ ID NO: 2). In some embodiments, the sample is a tumor sample. [Brief description of the drawings]

[0018] [Figure 1] 1 is a chart showing the modulation of the tumor microenvironment (TME) by VT1021 via CD36 and CD47. VT1021 binds to its receptor on myeloid-derived suppressor cells (MDSCs) and activates a signaling pathway that leads to increased expression of thrombospondin-1 (Tsp-1). Tsp-1 then executes a myriad of anti-tumor activities via binding to its two major cell surface receptors, CD36 and CD47. [Diagram 2] Figure 1 shows the pharmacokinetic profile of VT1021 concentration in plasma over time in human patients with glioblastoma (red), pancreatic cancer (green), ovarian cancer (blue), other (purple), or unreported (red) following the first dose at time 0. "Other" and "unreported" refer to subjects enrolled in clinical trials with solid tumor indications other than GBM, pancreatic cancer, and ovarian cancer. [Diagram 3] Representative immunohistochemical staining images for CD36 and CD47 are shown, showing features of high, moderate, and low staining intensity. [Figure 4] 1 is a bar graph showing the correlation between CD36 and CD47 expression in patient tumor tissue, as measured by immunohistochemical staining, and the length of time each patient remained on the clinical trial. [Figure 5A]VT1021 regulates the TME in pancreatic cancer. Metal ion immunostaining of patient tumor tissues before (pre) and during VT1021 treatment shows overall histology based on vimentin, keratin, double-stranded (ds)DNA, thrombospondin-1, CD11b, and keratin staining in MDSCs, levels of monocytic MDSCs based on CD14, CD11b, and keratin, levels of cytotoxic T cells (CTLs) based on CD3 and CD8, and levels of regulatory T cells (Tregs) based on CD3 and FoxP3. [Figure 5B] VT1021 modulates the TME in pancreatic cancer. Metal ion immunostaining to identify and quantitate M1 macrophages using cell surface markers iNOS and CD68, and M2 macrophages using cell surface markers CD163 and CD68 in patient tumor tissue samples obtained before (pre) and during (on) VT1021 treatment (left panel), and graphical depiction of Tsp-1 protein levels, CTL to Treg ratio, M1:M2 macrophage ratio, and fold change of macrophage subtypes before and during VT1021 treatment as measured by metal ion immunostaining (right panel). [Figure 6] FIG. 1 shows immunohistochemical staining of CD36 and CD47 protein expression in tumor tissue microarrays composed of tissue from patients with pancreatic cancer. [Figure 7] FIG. 1 shows the pharmacokinetic profile of VT1021 plasma concentrations over time in male (solid line) and female (dotted line) glioblastoma patients after the first dose at time point 0. [Figure 8]1 is a swimmer plot showing the days on study of glioblastoma patients treated with VT1021, with the bar colors indicating the expression levels of CD36 and CD47 in patients as determined by immunohistochemistry analysis of tumor tissue taken before the start of treatment with VT1021. Purple bars indicate high expression of both proteins, light blue bars indicate high expression of either CD36, CD47 or neither, and grey bars indicate that the expression level could not be determined. [Figure 9] Figure 1 shows that VT1021 induces complete responses in rGBM subjects. (A) MRI image of a glioblastoma lesion in a patient treated with VT1021 showing the decrease in lesion size over time during treatment, (B) graphical depiction of the change in lesion area measured over time using MRI imaging during treatment with VT1021, (C) immunohistochemical staining of CD36 and CD47 levels in this patient showing high levels of expression of each protein. [Figure 10] Figure 1: VT1021 induces TSP-1 in the circulation and TME: (A) Bar graph of Tsp-1 protein levels measured by ELISA in circulating MDSCs taken before (baseline) and after (induction) treatment with VT1021 from patients who experienced a complete or partial response, stable disease, or disease progression with VT1021, (B) H&E and immunohistochemistry analysis of patient tumor tissues before the start of treatment (pre) or during treatment with VT1021, (C) Immunohistochemistry analysis showing representative images of high levels of CD36 and CD47. [Figure 11A] Figure 1 shows the modulation of the immune system by VT1021 in the circulation and TME. Box plots of fold changes in: (top right) proliferating (Ki67+) cytotoxic T lymphocytes, (top left) proliferating (Ki67+) helper (CD4+) T lymphocytes, (bottom right) monocytic (CD11b / CD14+) MDSCs, and (bottom left) activated MDSCs in patients who experienced complete or partial response (CR / PR), stable disease (SD), or progressive disease (PD) after treatment with VT1021. [Figure 11B]Figure 1 shows the modulation of the immune system by VT1021 in the circulation and TME. Metal ion immunostaining of iNOS, CD68, and DNA to identify M1 macrophages, and metal ion immunostaining of CD163, CD68, and DNA to identify M2 macrophages in patient tumor tissue. [Figure 11C] Figure 1 shows modulation of the immune system by VT1021 in the circulation and TME.Bar graph of the change in the percentage of M1 and M2 macrophages in patient tumor tissue as determined by metal ion immunostaining. [Figure 11D] Figure 1 shows the modulation of the immune system by VT1021 in the circulation and TME. Metal ion immunostaining of patient tumor tissues to examine the overall histology based on vimentin, CD56, and double-stranded (ds) DNA, the levels of cytotoxic T cells (CTL) based on CD3, CD8, and CD56, the total levels of MDSC based on CD11b and CD56, and the levels of monocytic MDSC based on CD11b, CD14, and CD56. [Figure 11E] Figure 1 shows modulation of the immune system by VT1021 in the circulation and TME.Bar graph showing fold change in cytotoxic T lymphocytes (CTLs) in patients before initiation of treatment with VT1021 (pretreatment) and during treatment with VT1021 (on-study) as determined by metal ion immunostaining. [Figure 11F] Figure 1 shows modulation of the immune system by VT1021 in the circulation and TME.Bar graphs depicting fold change in total monocytic myeloid-derived suppressor cells and monocytic myeloid-derived suppressor cells (MDSC) in patients before initiation of treatment with VT1021 (pretreatment) and during treatment with VT1021 (on-study) as determined by metal ion immunostaining. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Provided herein is a novel cancer therapeutic strategy that has both anti-cancer activity and the ability to target the cancer microenvironment to prevent cancer recurrence and / or metastasis. The anti-cancer strategy described herein relies on stimulating the activity of thrombospondin-1 (Tsp-1), a potent anti-angiogenic and anti-tumor protein. "Tsp-1" is a subunit of a disulfide-linked homotrimeric protein. Tsp-1 is an adhesive glycoprotein that mediates cell-cell and cell-matrix interactions. Tsp-1 binds to fibrinogen, fibronectin, laminin, type V collagen, and integrin alpha-V / beta-1, and has been shown to play a role in platelet aggregation, angiogenesis, and tumor development. For purposes of this disclosure, Tsp-1 is a potent anti-tumor and anti-angiogenic factor, the activation of which suppresses tumor growth and metastasis and blocks angiogenesis in the tumor microenvironment.

[0020] As described herein, tumor cells from several different types of cancer that are responsive to Psap peptides have been found to exhibit doubly elevated levels of CD36 and CD47.

[0021] Prosaposin or prosaposin-derived peptides have previously been shown to be capable of stimulating the activity of Tsp-1 and to be effective in treating several types of cancer (see, e.g., PCT publications WO2009002931, WO / 2011 / 084685 and WO / 2013 / 096868, WO2015148801, and U.S. patent application Ser. Nos. 12 / 640,788 and 13 / 516,511, all of which are incorporated by reference in their entireties).

[0022] The progression of cancer to the metastatic stage is a major factor in its lethality. For a tumor to form lethal metastases, it must gain access to the vascular or lymphatic system (intravasation), survive during migration, exit the blood or lymphatic vessels (extravasation), and grow at the metastatic site [1]. In this process, heterotypic signaling between the tumor and its microenvironment can affect tumor growth by regulating the production and secretion of factors that mediate tumor growth, angiogenesis, and immune responses. Two proteins, prosaposin and PRSS2, were identified through a functional proteomics screen designed to identify secreted proteins that regulate Tsp-1 in the microenvironment [2]. Prosaposin is preferentially expressed by weakly metastatic tumors and stimulates Tsp-1 in the tumor microenvironment. Conversely, PRSS2 is preferentially expressed by highly metastatic cells and inhibits Tsp-1 expression in the tumor microenvironment. Tsp-1 inhibits tumor growth and progression through multimodal activity, specifically by (1) being a broadly acting antiangiogenic factor, (2) having direct antitumor activity against tumors expressing CD36, and (3) promoting macrophage phagocytosis and T cell activation through binding to CD47 [3-5]. Both the Tsp-1 stimulating activity of prosaposin and the Tsp-1 suppressing activity of PRSS2 have been determined to be mediated through binding to LRP1. Provided herein are antibodies that mimic the Tsp-1 stimulating activity of prosaposin and block the Tsp-1 suppressing activity of PRSS2.

[0023] Prosaposin was first identified as a novel suppressor of tumor metastasis, and it was demonstrated that such inhibition was achieved by stimulating p53 and subsequently Tsp-1 in the tumor microenvironment [2]. A 5-amino acid cyclic peptide derived from prosaposin with potent antitumor and antimetastatic activity has subsequently been identified and is referred to herein as VT1021. This peptide has been shown to inhibit metastasis by stimulating Tsp-1 in bone marrow-derived cells in the tumor microenvironment [6]. Therefore, the hypothesis was tested that PSAP would have efficacy in the treatment of metastatic pancreatic cancer, a cancer in which the microenvironment constitutes the majority of the tumor mass [7]. Thus, 1 × 10 6 AsPc1 human pancreatic cancer cells were injected into the pancreas of SCID mice. Tumors were allowed to grow for 25 days, at which point luciferase intensity was >1 × 10 in all tumors. 8 Treatment was then initiated with psap peptide at doses of 20 mg / kg and 40 mg / kg QD. After 21 days of treatment, all mice were sacrificed when control (vehicle) treated mice became moribund.

[0024] Figure 1 shows the regulation of the tumor microenvironment (TME) by VT1021 via CD36 and CD47. VT1021 reprograms the TME from immunosuppressive to immune-enhancing. The immunosuppressive tumor immune microenvironment is characterized by high levels of Tregs, M2 tumor-associated macrophages (M2 TAMs), and MDSCs. VT1021 binds to receptors on MDSCs and induces the expression of Tsp-1. Tsp-1 binding to one of the receptors, CD36, inhibits angiogenesis, induces apoptosis in tumor cells, endothelial cells, and Tregs, and increases adhesion, survival, and the M1 / M2 ratio of M1 macrophages. Tsp-1 binding to the other major receptor, CD47, blocks the "do not eat me" signal, allowing phagocytosis of tumor cells by macrophages, and increases CTL infiltration and activity, resulting in tumor cell death. Therefore, VT1021 is an effective agent for co-regulating CD36 and CD47 for the treatment of cancer.

[0025] The present disclosure is based on the finding that doubly elevated levels of CD36 and CD47 predict a subject's responsiveness to cancer treatment using Psap peptides, including CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), and DWLP (SEQ ID NO: 3), amino acid substitution variants thereof, and cyclized versions thereof. Accordingly, aspects of the present disclosure relate to methods for assessing a subject's responsiveness to treatment with Psap peptides by determining the levels of CD36 and CD47 in a sample, such as a tumor sample. In some embodiments, the methods described herein relate to identifying or selecting a subject for treatment with Psap peptides based on the levels of CD36 and CD47 in a sample, such as a tumor sample. Other aspects of the present disclosure relate to compositions and methods for treating a subject with a cancer characterized by elevated levels of CD36 and CD47 (e.g., the cancer is selected or identified based on having elevated levels of CD36 and CD47 in the sample compared to control levels).

[0026] Provided herein are methods and compositions for the treatment of cancer by administering an agent that modulates Tsp-1 activity. In some embodiments, the methods include determining levels of CD36 and CD47 in a subject having cancer and administering a Tsp-1 inducer, wherein the subject exhibits a cancer with elevated CD36 and CD47 levels or activity compared to a control.

[0027] Aspects of the present disclosure relate to performing an assay to determine the levels of CD36 and CD47 in a sample. Any assay known in the art can be used to measure CD36 and CD47 levels (see, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley&Sons, Inc., New York. Microarray technology is described in Microarray Methods and Protocols, R. Matson, CRC Press, 2009, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley&Sons, Inc., New York). The levels of CD36 and CD47 can be mRNA levels and / or protein levels. In some embodiments, the levels are protein levels. Assays for detecting mRNA include, but are not limited to, Northern blot analysis, RT-PCR, sequencing techniques, RNA in situ hybridization (e.g., using a DNA or RNA probe to hybridize to RNA molecules present in a sample), in situ RT-PCR (e.g., as described in GJ, et al. Am J Surg Pathol. 1993, 17:683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190:1017-25), and oligonucleotide microarrays (e.g., by hybridization of polynucleotide sequences derived from the sample to oligonucleotides attached to a solid surface (e.g., a glass wafer) containing addressable locations, such as Affymetrix microarrays (Affymetrix.RTM., Santa Clara, Calif.)).Methods for designing nucleic acid binding partners, such as probes, are well known in the art. In some embodiments, the nucleic acid binding partner binds to a portion or the entire nucleic acid sequence of CD36 or CD47, which sequence is distinguishable from CD36 or CD47 as known in the art.

[0028] Assays for detecting CD36 and CD47 protein levels include, but are not limited to, immunoassays (also referred to herein as immune-based or immuno-based assays, e.g., Western blots, immunohistochemistry, and ELISA assays), mass spectrometry, and multiplexed bead-based assays. Such assays for protein level detection are well known in the art. Binding partners for protein detection are known in the art and can be designed using the methods described herein. In some embodiments, CD36 and CD47 protein binding partners, e.g., anti-CD36 and CD47 antibodies, bind to part or the entire amino acid sequence of CD36 and CD47 proteins. Other examples of protein detection and quantification methods include multiplexed immunoassays, e.g., as described in U.S. Pat. Nos. 6,939,720 and 8,148,171, and published U.S. Patent Application No. 2008 / 0255766, and protein microarrays, e.g., as described in published U.S. Patent Application No. 2009 / 0088329.

[0029] In some embodiments, the sample obtained from the subject is a tumor biopsy and the assays to detect CD36 and CD47 protein levels are immuno-based assays performed on the tumor biopsy.

[0030] In some embodiments, CD36 and CD47 are measured in cancer cells or tumors, in some embodiments, CD36 and CD47 are measured in the tumor microenvironment, and in some embodiments, CD36 and CD47 are measured in the circulation.

[0031] Any suitable binding partner of CD36 or CD47 is contemplated for detecting CD36 or CD47 levels. In some embodiments, the binding partner is any molecule that specifically binds to CD36 or CD47 protein. As described herein, "specifically binds to CD36 or CD47 protein" means that the molecule is more likely to bind to a portion or the whole of the CD36 or CD47 protein, rather than a portion or the whole of a non-CD36 or non-CD47 protein. In some embodiments, the binding partner is an antibody or an antigen-binding fragment thereof, such as a Fab, F(ab)2, Fv, single chain antibody, Fab and sFab fragments, F(ab')2, Fd fragment, scFv, or dAb fragment. Methods for producing antibodies and antigen-binding fragments thereof are well known in the art (see, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990); and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989); WO2006 / 040153, WO2006 / 122786, and WO2003 / 002609). Binding partners also include other peptide molecules and aptamers that specifically bind to CD36 or CD47. Methods for producing peptide molecules and aptamers are well known in the art (see, e.g., Published U.S. Patent Application No. 2009 / 0075834, U.S. Patent Nos. 7,435,542, 7,807,351, and 7,239,742).

[0032] In some embodiments, the binding partner is any molecule that specifically binds to CD36 or CD47 mRNA. As described herein, "specifically binds to CD36 or CD47 mRNA" means that the molecule is more likely to bind (e.g., by complementary base pairing) to a portion or the entirety of the mRNA, rather than to a portion or the entirety of a non-CD36 or non-CD47 mRNA nucleic acid. In some embodiments, the binding partner that specifically binds to CD36 or CD47 mRNA is a nucleic acid, e.g., a probe. The binding partner can be designed using the nucleotide and amino acid sequences of CD36 or CD47 provided herein. In some embodiments, the CD36 or CD47 binding partner can include a detectable label, such as an enzymatically active group, a fluorescent molecule, a chromophore, a luminescent molecule, a ligand capable of specific binding, or a radioisotope, etc. In some embodiments, a second binding partner specific to the CD36 or CD47 binding partner, e.g., a secondary antibody, is also contemplated.

[0033] Aspects of the present disclosure relate to determining the level of CD36 or CD47 in a sample obtained from a subject. In some embodiments, the sample obtained from the subject is a tumor sample. As used herein, a tumor sample may include, for example, tumor cells, a population of tumor cells, a fragment of a tumor (e.g., a biopsy), or an entire tumor. In some embodiments, the tumor sample is a tumor biopsy. In some embodiments, the tumor sample includes circulating tumor cells. In some embodiments, the tumor sample includes ascites fluid. In some embodiments, the tumor sample includes pleural effusion. The tumor sample may include non-tumor cells or non-tumor tissue (e.g., a biopsy including normal tissue surrounding a tumor fragment). In some embodiments, the sample may be a tissue or fluid sample obtained from the subject. Examples of liquid samples are blood, plasma, serum, and urine.

[0034] As used herein, "stimulate" refers to activating or increasing the level or activity of a biological molecule (e.g., a protein). For example, an agent of the disclosure "stimulates Tsp-1" means that in the presence of the agent, the expression or activity level of Tsp1 is increased by at least 30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more) compared to the absence of the agent.

[0035] As used herein, "inhibit" means to prevent expression, to reduce the level of a protein (e.g., CD36 or CD47), or to reduce the activity of a biological molecule (e.g., a protein). For example, an agent that inhibits expression of CD36 or CD47 may prevent expression of CD36 or CD47 or may reduce the level of CD36 or CD47 by at least 30% (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more) compared to the absence of the agent.

[0036] In some embodiments, the Tsp-1 inducer is a Psap peptide. In some embodiments, the Psap peptide is a Psap peptide comprising the sequence CDWLPK (SEQ ID NO:1), DWLPK (SEQ ID NO:2), or DWLP (SEQ ID NO:3), or an amino acid substitution variant thereof, wherein the amino acid substitution is a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution for leucine (L) selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof; c) arginine (R) to lysine (K); d) the D isomer of aspartic acid (D) versus the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) versus the L isomer of leucine (L); e) D isomer of tryptophan (W) versus L isomer of tryptophan (W), and / or D isomer of proline (P) versus L isomer of proline (P), or combinations thereof. In some embodiments, the Psap peptide is 50 amino acids or less in length. In some embodiments, the Psap peptide is 30 amino acids or less in length. In some embodiments, the Psap peptide is 15 amino acids or less in length. In some embodiments, the Psap peptide is 6 amino acids or less in length. In some embodiments, the Psap peptide is a cyclic peptide. In some embodiments, the non-standard amino acid of similar size is methylvaline, methylleucine, or sarcosine. In some embodiments, the peptide is cyclic DWLPK (SEQ ID NO: 2). As used herein, "VT1021" refers to cyclic DWLPK (SEQ ID NO: 2).

[0037] In some embodiments, the Psap peptides may be modified, for example, by oligomerization or polymerization (e.g., dimers, trimers, multimers, etc.), modification of amino acid residues or the peptide backbone, crosslinking, cyclization, conjugation, pegylation, glycosylation, acetylation, phosphorylation, fusion to additional heterologous amino acid sequences (e.g., antibodies or antibody Fc domains, serum transferrin or portions thereof, albumin, or transthyretin), or other modifications that substantially alter the stability, solubility, or other properties of the peptide while substantially retaining or enhancing therapeutic activity. Conjugation may, for example, be to a polymer. Suitable polymers include, for example, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyols, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives (including methylcellulose and carboxymethylcellulose), starch and starch derivatives, polyalkylene glycol and its derivatives, copolymers of polyalkylene glycol and its derivatives, polyvinyl ethyl ether, and alpha, beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. Conjugation may be via a linker, for example, a peptide or chemical linker. Methods for modifying peptides are well known in the art (see, e.g., U.S. Pat. Nos. 5,180,816, 5,596,078, 5,990,273, 5,766,897, 5,856,456, 6,423,685, 6,884,780, 7,610,156, 7,256,258, 7,589,170, and 7,022,673, and PCT publication WO2010 / 014616, which are incorporated herein by reference).

[0038] In some embodiments, the TSP-1 inducer comprises a mixture of TSP-1 inducers disclosed herein. As used herein, "Tsp-1 inducer" refers to an agent that promotes an increase in Tsp-1 levels or activity. In some embodiments, promoting Tsp-1 activity comprises increasing Tsp-1 expression or the half-life of Tsp-1 protein. In some embodiments, promoting Tsp-1 activity comprises restoring wild-type levels of Tsp-1 levels or activity. In some embodiments, promoting Tsp-1 activity comprises increasing Tsp-1 levels or activity to above wild-type levels. In some embodiments, promoting Tsp-1 levels or activity comprises increasing Tsp-1 levels or activity that remain lower than wild-type but are increased compared to a control.

[0039] The term "binding" refers to the association of two substances (e.g., two proteins) when the affinity (KD) between them is <10 -4 M, <10 -5 M, <10 -6 M, <10 -7 M, <10 -8 M, <10 -9 M, <10 -10 M, <10 -11 M or <10 -12 They are considered to bind to each other when M. Those of skill in the art are familiar with methods for assessing the affinity of two substances (e.g., two proteins).

[0040] The terms "protein", "peptide" and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification. A protein, peptide, or polypeptide may also be a single molecule or a multi-molecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0041] A peptide "derived from" a protein means that the peptide is obtained from a protein and has an amino acid sequence that shares homology with the corresponding fragment of the protein. The amino acid sequence of the peptide can be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of the fragment of the protein to which it corresponds. A peptide derived from a protein can also include chemical modifications, amino acid substitutions, and / or unnatural amino acids.

[0042] An "antibody" or "immunoglobulin (Ig)" is a large Y-shaped protein produced primarily by plasma cells that is used by the immune system to neutralize foreign substances (e.g., pathogens such as bacteria and viruses). Antibodies are classified as IgA, IgD, IgE, IgG, and IgM. "Antibody" and "antigen-binding fragment" include whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. "Antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, from amino to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody can be a polyclonal or monoclonal antibody.

[0043] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical L chains and two H chains (IgM antibodies consist of five basic heterotetrameric units with an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units with the J chain). For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. At the N-terminus, each H chain has a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are predicted to form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of a VH and a VL together forms a single antigen-binding site. The structure and properties of different classes of antibodies are described in (e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6 (incorporated herein by reference)).

[0044] The L chains of any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domain. Immunoglobulins can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in the sequence and function of the CH; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0045] The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed across the 110 amino acid length of the variable domain. Rather, the V region consists of relatively invariant stretches of 15-30 amino acids, called framework regions (FRs), separated by shorter regions of extreme variability, called "hypervariable regions", each 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs, mainly adopting a β-sheet structure, connected by three hypervariable regions (forming loops that connect and sometimes form part of the β-sheet structure). The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0046] An "antigen-binding fragment" for use according to the present disclosure includes an antigen-binding portion of an antibody. An antigen-binding portion of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (e.g., as described in Ward et al., (1989) Nature 341:544-546, which is incorporated herein by reference); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombinant techniques as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fvs (scFvs), see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as full-length antibodies.

[0047] In some embodiments, the antigen-binding fragment may be an Fc fragment, an Fv fragment, or a single chain Fv fragment. The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences within the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells.

[0048] The Fv fragment is the smallest antigen-binding fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0049] Single-chain Fv, also abbreviated as "sFv" or "scFv", is an antigen-binding fragment comprising the VH and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding (e.g., as described in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, which are incorporated herein by reference).

[0050] The antibody may be isolated. An isolated antibody is an antibody that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, and most preferably greater than 99% by weight, as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue staining, or preferably silver staining. An isolated antibody includes the antibody in situ within a recombinant cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0051] In some embodiments, the antibody of the present disclosure is a monoclonal antibody. A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Moreover, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the technology of the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), which are incorporated herein by reference.

[0052] Monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0053] In some embodiments, the antibody of the present technology is a polyclonal antibody. "Polyclonal antibody" refers to a mixture of different antibody molecules that react with more than one immunogenic determinant of an antigen. Polyclonal antibodies can be isolated or purified from mammalian blood, secretions, or other fluids, or from eggs. Polyclonal antibodies can also be recombinant. Recombinant polyclonal antibodies are polyclonal antibodies produced by the use of recombinant technology. Recombinantly produced polyclonal antibodies usually contain a high concentration of different antibody molecules, all or most of which (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or more) exhibit the desired binding activity against an antigen consisting of more than one epitope.

[0054] Methods for producing antibodies (e.g., monoclonal or polyclonal antibodies) are known in the art. For example, polyclonal antibodies may be prepared by immunizing an animal, preferably a mammal, with a selected allergen, followed by isolating antibody-producing B lymphocytes from the blood, bone marrow, lymph nodes, or spleen. Alternatively, antibody-producing cells may be isolated from an animal and exposed in vitro to the allergen against which the antibodies are produced. The antibody-producing cells may then be cultured to obtain a population of antibody-producing cells, optionally after fusion with an immortalized cell line such as a myeloma. In some embodiments, B lymphocytes may be isolated from tissues of allergic patients as starting material to produce fully human polyclonal antibodies. Antibodies may be produced in mice, rats, porcine (pig), ovine, bovine material, or other animals transgenic for human immunoglobulin genes as starting material for producing fully human polyclonal antibodies. In some embodiments, mice or other animals transgenic for human immunoglobulin genes (e.g., as disclosed in U.S. Pat. No. 5,939,598) may be immunized to stimulate the in vivo production of specific antibodies and antibody-producing cells prior to preparation of polyclonal antibodies from the animal by extraction of B lymphocytes or purification of polyclonal sera.

[0055] Monoclonal antibodies are typically produced by cell culture involving fusing myeloma cells with mouse spleen cells immunized with the desired antigen (i.e., hirubidoma technology). The mixture of cells is diluted and clones are grown from a single parent cell on microtiter wells. The antibodies secreted by the different clones are then assayed for their ability to bind the antigen (in tests such as ELISA or antigen microarray assays) or in immunodot blots. The most productive and stable clones are then selected for future use.

[0056] In some embodiments, the antibodies described herein are "humanized" for use in humans (e.g., as therapeutics). "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0057] As used herein, "small molecule" refers to a molecule of low molecular weight (e.g., <900 Daltons) organic or inorganic compound that may function in regulating biological processes. Non-limiting examples of small molecules include lipids, simple sugars, second messengers, other natural products and metabolites, as well as drugs and other xenobiotics.

[0058] "Lipid" refers to a group of naturally occurring molecules including fats, waxes, sterols, fat-soluble vitamins (vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and the like. "Monosaccharide" refers to a class of sugars (e.g., glucose) that cannot be hydrolyzed to yield simpler sugars. Non-limiting examples of monosaccharides include glucose (dextrose), fructose (levulose), and galactose. "Second messengers" are molecules that relay signals received at receptors on the cell surface (e.g., from protein hormones, growth factors, etc.) to target molecules in the cytoplasm and / or nucleus. Non-limiting examples of second messenger molecules include cyclic AMP, cyclic GMP, inositol triphosphate, diacylglycerol, and calcium. "Metabolites" are molecules formed as intermediates in metabolism. Non-limiting examples of metabolites include ethanol, glutamic acid, aspartic acid, 5' guanylic acid, isoascorbic acid, acetic acid, lactic acid, glycerol, and vitamin B2. A "xenobiotic" is a foreign chemical found in an organism that is not normally produced naturally by the organism or expected to be present in the organism. Non-limiting examples of xenobiotics include drugs, antibiotics, carcinogens, environmental pollutants, food additives, hydrocarbons, and pesticides.

[0059] The therapeutic agents described herein can be formulated into pharmaceutical compositions. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject, e.g., a human. A pharma- ceutically acceptable carrier is "acceptable" in the sense of being compatible with the other components of the formulation and not deleterious to the patient's tissues (e.g., physiologically compatible, sterile, physiological pH, etc.). The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.Some examples of materials which can function as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) cellulose acetate, cellulose acetate, cellulose ether, cellulose acetate esters ... 1) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG), (12) esters, such as ethyl oleate and ethyl laurate, (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffers, (21) polyesters, polycarbonates, and / or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, (22) C2-C12 alcohols, such as ethanol, and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.

[0060] The pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. The term "unit dose", when used in reference to the pharmaceutical compositions of the present disclosure, refers to physically discrete units suitable as unitary administration for subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent (i.e., carrier or vehicle).

[0061] The formulation of the pharmaceutical composition may depend on the route of administration. Suitable injectable preparations for parenteral administration, or intratumoral, peritumoral, intralesional, or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, which may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3 propanediol or 1,3 butanediol. Acceptable vehicles and solvents that may be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are useful in the preparation of injectables. Injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water, or other sterile injectable medium prior to use.

[0062] For topical administration, the pharmaceutical composition may be formulated into ointments, salves, gels, or creams as generally known in the art. Topical administration may utilize transdermal delivery systems, which are well known in the art. One example is a skin patch.

[0063] Compositions suitable for oral administration may be presented as discrete units such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or emulsion.

[0064] Other delivery systems can include sustained release, delayed release or sustained release delivery systems. Such systems can avoid repeated administration of anti-inflammatory agents, enhancing the convenience of subjects and physicians. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the aforementioned polymers containing drugs are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, i.e. lipids including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats, e.g., mono-, di-, and triglycerides; hydrogel release systems; silastic systems, peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants, and the like. Specific examples include, but are not limited to, (a) erosion systems in which the anti-inflammatory agent is contained in some form within a matrix, as described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034, and 5,239,660, and (b) diffusion systems in which the active ingredient permeates through the polymer at a controlled rate, as described in U.S. Patent Nos. 3,832,253 and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0065] The use of long-term sustained release implants may be particularly suitable for treating chronic diseases.Long-term release as used herein means that the implant is constructed and arranged to deliver therapeutic levels of active ingredient for at least 30 days, preferably 60 days.Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

[0066] In some embodiments, pharmaceutical compositions used for therapeutic administration must be sterile. Sterilization is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. A variety of preservatives are well known and include, for example, phenol and ascorbic acid. The cyclic Psap peptides and / or pharmaceutical compositions are usually stored in lyophilized form or as an aqueous solution, where they are highly stable against thermal and oxidative denaturation. The pH of the preparations is typically about 6-8, although higher or lower pH values ​​may be appropriate in certain cases.

[0067] Another aspect of the present disclosure provides a method of treating cancer using the agents and pharmaceutical compositions described herein. In some embodiments, the method comprises administering to a subject in need thereof an effective amount of an agent that stimulates Tsp-1. In some embodiments, the method comprises administering to a subject in need thereof one or more additional agents for inducing Tsp-1 or suppressing CD36 or CD47. When more than one agent is administered, they may be administered simultaneously or sequentially. The mode of administration can be determined by one of skill in the art (e.g., a physician).

[0068] "Treating" cancer or "treatment" of cancer includes, but is not limited to, preventing, reducing, or halting the onset of cancer, reducing or eliminating symptoms of cancer, suppressing or inhibiting the growth of cancer, preventing or reducing metastasis and / or invasion of an existing cancer, promoting or inducing regression of cancer, inhibiting or suppressing the growth of cancer cells, reducing angiogenesis, and / or increasing the amount of apoptotic cancer cells.

[0069] An "effective amount" is the administration of an agent sufficient to bring about a medically desirable result, such as the treatment of cancer. Effective amounts will vary depending on the particular disease or disorder being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of any concomitant therapy, the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. For administration to subjects, such as humans, dosages of about 0.001, 0.01, 0.1, or 1 mg / kg to 50, 100, 150, or 500 mg / kg or more may typically be used.

[0070] In some embodiments, the method further comprises identifying a subject having elevated levels of CD36 and CD47 in the sample as being responsive or likely to be responsive to treatment with a Psap peptide, relative to control levels. In some embodiments, the method further comprises administering to the subject identified as being responsive or likely to be responsive to treatment with a Psap peptide an effective amount of a Psap peptide described herein to treat the cancer. In some embodiments, the sample obtained from the subject with cancer is a tumor sample.

[0071] In some embodiments, elevated levels of CD36 and CD47 in the sample compared to control levels indicate that the cancer will regress or is likely to regress in response to treatment with the Psap peptide. In some embodiments, the method further comprises identifying a subject having elevated levels of CD36 and CD47 in the sample compared to control levels as having a cancer that will regress or is likely to regress in response to treatment with the Psap peptide. In some embodiments, the method further comprises administering to a subject identified as having a cancer that will regress or is likely to regress in response to treatment with the Psap peptide an effective amount of a Psap peptide as described herein to cause regression of the cancer.

[0072] As used herein, "elevated levels of CD36 and CD47" means that the level of CD36D47 is above a control level, such as a predetermined threshold or level, a level measured in a non-cancerous sample from the same subject, or a level measured in a sample from a healthy subject or population of subjects.

[0073] Control levels are described in detail herein. Elevated levels of CD36 and CD47 include, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above control levels. Elevated levels of CD36 and CD47 also include increasing the phenomenon from a zero state (e.g., no or undetectable expression in a control) to a non-zero state (e.g., some expression or detectable expression in a sample).

[0074] As used herein, "treatment with a Psap peptide" refers to administering a Psap peptide to a subject. Psap peptides are described herein. It is understood that treatment with a Psap peptide can include treatment with only a Psap peptide, or can include treatment with multiple agents or therapies, for example, a Psap peptide and another chemotherapeutic agent and / or another form of therapy, such as surgery, radiation therapy, or chemotherapy.

[0075] As used herein, "responsive to treatment with a Psap peptide" includes, but is not limited to, preventing or alleviating the onset of cancer, alleviating symptoms of cancer, suppressing or inhibiting the growth of cancer, preventing metastasis and / or invasion of an existing cancer, promoting or inducing regression of cancer, inhibiting or suppressing the growth of cancerous cells, reducing angiogenesis, and / or increasing the amount of apoptotic cancer cells in response to treatment with a Psap peptide.

[0076] As used herein, "not responding to treatment with a Psap peptide" includes, but is not limited to, a lack of prevention or alleviation of the onset of cancer, a lack of alleviation of symptoms of cancer, a lack of suppression or inhibition of cancer growth, a lack of prevention of metastasis and / or invasion of an existing cancer, a lack of promotion or induction of regression of cancer, a lack of inhibition or suppression of cancer cell growth, a lack of reduction in angiogenesis, and / or a reduction in the amount of apoptotic cancer cells in response to treatment with a Psap peptide.

[0077] In some embodiments, an effective amount is an amount of an agent that does not cause toxicity to a subject. In some embodiments, an effective amount is an amount of an agent that results in reduced toxicity to a subject. Methods for measuring toxicity are well known in the art (e.g., liver, spleen, and / or kidney biopsy / histology, alanine transferase, alkaline phosphatase, and bilirubin assays for hepatotoxicity, and creatinine levels for nephrotoxicity).

[0078] The agents and pharmaceutical compositions described herein can be formulated for various modes of administration, including systemic, topical, or local administration. Various routes of administration are available. The particular mode selected depends on the type of cancer being treated and the dosage required for therapeutic efficacy. Generally speaking, the methods of the present disclosure can be carried out using any mode of administration that is medically acceptable, i.e., any mode that produces an effective level of the active compound without causing clinically unacceptable adverse effects. Such modes of administration include, but are not limited to, oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. The pharmaceutical compositions described herein are also suitably administered by intratumoral, peritumoral, intralesional, intratracheal, intraventricular, intraperitoneal, or perilesional routes to exert local and systemic effects.

[0079] Techniques and formulations can generally be found in Remington: The Science and Practice of Pharmacy, Pharmaceutical Press; 22nd edition and other similar references. When administered, Psap peptides can be applied in pharma- ceutically acceptable amounts and in pharma- ceutically acceptable compositions. Pharmaceutical compositions and pharma- ceutically acceptable carriers are also described herein. Such preparations may routinely include salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharma- ceutically acceptable, but salts that are not pharma- ceutically acceptable may be conveniently used to prepare pharma- ceutically acceptable salts thereof and are not excluded from the scope of this disclosure. Such pharmacologically and pharma- ceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Also, pharma- ceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

[0080] In some embodiments, the treatment of cancer with the described agents or pharmaceutical compositions may be combined with another therapy, such as chemotherapeutic agents, radiation, cytostatic agents, anti-VEGF agents, anti-angiogenic factors, p53 reactivators and / or surgery.

[0081] A subject refers to a human or vertebrate or mammal, including but not limited to rodents, such as rats or mice, dogs, cats, horses, cows, pigs, sheep, goats, turkeys, chickens, and primates, such as monkeys. The methods of the present disclosure are useful for treating a subject in need thereof. A subject in need thereof may be a subject at risk of developing cancer (i.e., via genetic testing) or a subject with cancer.

[0082] A subject with cancer can be identified using any method known in the art (e.g., blood test, histology, CT scan, X-ray, MRI, physical examination, cytogenetic analysis, urine test, or genetic test). A subject suspected of having cancer may exhibit one or more symptoms of the disease. Signs and symptoms of cancer are well known to those skilled in the art. Some exemplary laboratory tests include, but are not limited to, tests for cancer biomarkers such as cancer antigen (CA) 15-3, carcinoembryonic antigen (CEA) and HER-2 for breast cancer, human papillomavirus (HPV) E6 and E7 oncoproteins for cervical cancer, alpha-fetoprotein (AFP), AFP fraction L3, P4 / 5, and +II bands for hepatocellular carcinoma, and ultrasound, prostate specific antigen (PSA) for prostate cancer, and serum CA-125 for ovarian and HCC.

[0083] Cancers may be benign or malignant, and may or may not have metastasized. Any type of cancer is contemplated herein, including, but not limited to, leukemia, lymphoma, myeloma, carcinoma, metastatic carcinoma, sarcoma, adenoma, nervous system cancer, and genitourinary cancer. Exemplary cancer types include, but are not limited to, adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, biliary tract cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, glioblastoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoid tumor. , cancer of unknown etiology, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, acute lymphocytic and myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumors, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, Gestational trophoblastic neoplasia, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, pancreatic islet tumor, Kaposi's sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom's hypergammaglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous cell cervical cancer, multiple endocrine neoplasia syndrome, Multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous cell cervical cancer,These include supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, choriocarcinoma, hematological tumors, adult T-cell leukemia, lymphoma, lymphocytic lymphoma, stromal tumor, and germ cell tumor, or Wilms' tumor. In some embodiments, the cancer is melanoma or ovarian cancer. EXAMPLES

[0084] Example 1: Doubly elevated CD36 and CD47 levels correlate with responsiveness to VT1021 treatment in subjects with pancreatic cancer. This example demonstrates that dually elevated CD36 and CD47 levels correlate with responsiveness to VT2021 treatment in pancreatic cancer.

[0085] Preclinical studies of VT1021 have demonstrated robust antitumor activity in multiple animal models of ovarian, pancreatic, and breast cancer. VT1021 was evaluated in a recently completed Phase I / II open-label, multicenter, dose-escalation (part 1) and expansion (part 2) clinical trial (NCT03364400) in advanced, refractory, solid tumors, including pancreatic cancer. Seven subjects with pancreatic cancer were dosed in part 1 and 32 in part 2, of which 17 were considered evaluable because they had completed at least one cycle of VT1021 treatment and had completed tumor imaging during cycle 2. VT1021 had no serious adverse events (AEs) and had a predictable pharmacokinetic profile.

[0086] The "expansion phase" of the VT1021-01 study enrolled subjects diagnosed with recurrent GBM, pancreatic cancer, ovarian cancer, and other solid tumors. They were treated with VT1021 at 11.8 mg / kg twice weekly by IV infusion. Blood samples were taken from the clinical trial before treatment and at 0, 2, 4, and 6 hours after treatment to measure the levels of VT1021 (Figure 2). Tumor biopsy samples were taken from the clinical trial before and during treatment to measure the expression levels of biomarkers including CD36 and CD47, as well as cell surface markers of immune cell populations in the TME, by immunohistochemistry assays (Figures 3, 4, and 5). The correlation of study days and tumor shrinkage to the expression profiles of CD36 and CD47 was analyzed (Figure 4). The general expression profiles of CD36 and CD47 were evaluated by IHC on tumor tissue microarrays (TMA) containing biopsy samples taken from pancreatic cancer patients. TMA was purchased from a commercial source unrelated to the VT1021-01 clinical trial.

[0087] Common adverse events (AEs) in accrual subjects with pancreatic cancer (Part 1) are shown in Table 1. AEs shown occurred in ≥2 subjects, in all cycles, and in the highest grade, regardless of toxicity.

[0088] Of the 39 pancreatic cancer subjects enrolled and dosed, 7 subjects were enrolled in the escalation cohort in Part 1. All 7 subjects reported at least one AE (regardless of causality). Four subjects had at least one AE that was probably or possibly related to the study drug. There were no dose-limiting toxicities. Five subjects reported at least one serious adverse event (SAE) (regardless of causality). Two subjects had SAEs definitely or possibly related to the study drug: infusion-related reaction (grade 3), mental status change (grade 2).

[0089] Thirty-two subjects were enrolled in the Part 2 escalation cohort. Thirty subjects reported at least one AE (regardless of causality). Eighteen subjects had at least one AE that was definitely, probably, or possibly related to the study drug. Twenty subjects reported at least one SAE (regardless of causality). Two subjects had SAEs possibly related to the study drug: thrombosis (grade 3), pyrexia (grade 1). [Table 1]

[0090] FIG. 2 shows the pharmacokinetics of VT1021 in subjects with glioblastoma, pancreatic cancer, and ovarian cancer.

[0091] Figure 3 shows the expression intensity of CD36 and CD47 in subjects with pancreatic cancer. Double high expression of both CD36 and CD47 in evaluable subjects is shown in part A. Non-double high expression of CD36 and CD47 in evaluable subjects is shown in part B. Non-double high expression of CD36 and CD47 in non-evaluable subjects is shown in part C.

[0092] Figure 4 showed that dual high expression of CD36 and CD47 correlated with longer study days and decreased tumor burden. Waterfall plots show the change in tumor burden for 14 pancreatic cancer subjects in part 2 with measurable disease. Three of the three subjects (100%) with stable disease showed dual high CD36 and CD47 levels. Of the 14 subjects with measurable disease, all five subjects with decreased tumor burden showed dual high levels and remained on the study for an average of 105 days.

[0093] Figures 5A and 5B show that VT1021 modulates the TME in pancreatic cancer. Modulation of the TME by VT1021 in paired biopsies from subjects with pancreatic cancer was assessed by multiple ion beam image analysis. Representative images from one of three regions of interest of tumor biopsies from subjects obtained before treatment (pre) and during the trial (on). Figure 5B quantifies the fold change in mean intensity of TSP-1+, CTL to Treg ratio, M1 to M2 macrophage ratio, and percentage of M1 macrophages compared to total macrophages for eight paired biopsies from parts 1 and 2. TSP-1+ increased more than 2-fold in biopsies during the trial compared to before treatment, both the CTL / Treg ratio and the M1 / M2 macrophage ratio increased, and the percentage of M1 macrophages increased more than 3-fold in the TME after treatment with VT1021.

[0094] Figure 6 shows that double high expression of CD36 and CD47 is a predictive biomarker for pancreatic cancer. A high percentage of double high CD36 and CD47 is observed in pancreatic cancer. The figure shows a representative image of a commercially available pancreatic tumor tissue microarray stained and scored for double high CD36 and CD47. Table 2 below shows that subjects in part 2 with double high CD36 and CD47 were more likely to be considered evaluable in phase 1 trials with VT1021. [Table 2]

[0095] These data indicate that pancreatic cancer subjects with doubly high CD36 and CD47 levels have reduced tumor burden and are more likely to continue in clinical trials than subjects without doubly high CD36 and CD47 levels. Increased TSP-1 expression was observed in the TME of subjects administered VT1021, and VT1021 remodels the TME to be more immune-sensitive through increased CTL / Treg and M1 macrophage accumulation. Therefore, doubly high CD36 and CD47 levels are predictive biomarkers for the responsiveness of pancreatic cancer subjects to treatment with VT1021.

[0096] These results indicate that doubly elevated CD36 and CD47 levels are robust biomarkers of cancer responsiveness to treatment with VT1021, including pancreatic cancer responsiveness. The results further indicate that peptides of the present technology, including VT1021, are useful in treating cancers that exhibit doubly elevated CD36 and CD47 levels compared to control levels, including pancreatic cancer.

[0097] Example 2: Doubly elevated CD36 and CD47 levels correlate with responsiveness to VT1021 treatment in subjects with glioblastoma. This example demonstrates that dually elevated CD36 and CD47 levels correlate with responsiveness to VT2021 treatment in glioblastoma.

[0098] Preclinical studies of VT1021 have demonstrated robust antitumor activity in multiple animal models of ovarian, pancreatic, and breast cancer. VT1021 was evaluated in a recently completed Phase I / II open-label, multicenter, dose escalation (part 1) and expansion (part 2) clinical trial (NCT03364400) in advanced, refractory, solid tumors, including recurrent GBM (rGBM). Thirty-two subjects with rGBM were dosed in part 2, of which 22 were considered evaluable because they had completed at least one cycle of VT1021 treatment and had completed tumor imaging during cycle 2. VT1021 had no significant adverse events (AEs) and a predictable pharmacokinetic profile. Table 3 below shows the subject demographics. Table 4 shows the incidence of adverse effects.

[0099] Blood samples were collected from subjects with expanding GBM treated with VT1021 at 11.8 mg / kg twice weekly by IV infusion. VT1021 levels were measured and analyzed between male and female subjects (Figure 7). Study days were collected from the same group of subjects (Figure 8). One subject achieved a "complete response" as indicated by MRI scans and measurement of lesion size over the course of treatment with VT1021 (Figure 9). CD36 and CD47 levels were measured by immunohistochemistry assays in pre-treatment tumor biopsies of GBM subjects (Figures 9, 10). Tsp-1 levels were measured by ELISA for circulating Tsp-1 levels in collected blood samples and by immunohistochemistry for accumulated Tsp-1 levels in tumor biopsies (Figure 10). Infiltration of immune and inflammatory cells, specifically MDSC, T cells and macrophages, was determined by immunohistochemistry analysis of cell surface marker levels in tumor biopsies of subjects (Figure 11). [Table 3] [Table 4]

[0100] FIG. 7 shows the pharmacokinetics of VT1021 in subjects with glioblastoma.

[0101] Figure 8 shows that double high expression of CD36 and CD47 correlates with clinical response and duration of treatment. Of 22 evaluable rGBM subjects, 3 had a complete response (CR), 1 had a partial response (PR), and 7 had stable disease (SD) with a median duration of study of 203 days or more. The overall disease control rate (DCR) was 50%. Nine of 20 evaluable subjects (45%) with available biopsy samples showed high expression levels of both CD36 and CD47. Of the nine double high subjects, three achieved a CR giving an overall response rate of 33.3%, and another three subjects achieved SD with a DCR of 67%. Of the 11 CR / PR / SD, six were double high (55%).

[0102] Figure 9 shows that VT1021 induces complete response in rGBM subjects. Complete response scans and lesion shrinkage of one of three subjects who achieved CR. Part A shows lesion measurements and scans of one CR subject who was in the study for 476 days and is still continuing on the study. Part B shows that after nine cycles of treatment, the lesion steadily decreased until it was no longer measurable. Part C shows dual high expression of CD36 and CD47 by IHC analysis of pre-treatment biopsy.

[0103] Figure 10 shows that VT1021 induces TSP-1 in the circulation and TME. Part A shows that TSP-1 protein measured from peripheral blood mononuclear cells (PBMCs) was induced by VT1021 in all evaluable subjects with rGBM. Part B shows TSP-1 induction in on-treatment (on) biopsies from subjects with rGBM who achieved CR. Pathological examination of on-treatment biopsies did not detect tumor cells. Part C shows the intensity of double high expression of CD36 and CD47 in pre-treatment (pre) biopsies from the same subjects in part B.

[0104] Figures 11A, 11B, 11C, 11D, 11E, and 11F show the modulation of the immune system by VT1021 in the circulation and in the TME. Figure 11A shows immune cells isolated from whole blood collected 6 hours before and after treatment with VT1021 on day 1 of cycle 1 were analyzed by flow cytometry. Proliferative CTLs increased by 1.5-fold or more regardless of clinical response, and proliferative helper T cells increased by 1.5-fold or more in CR / PR and PD subjects, while monocytic MDSCs (mMDSCs) and activated MDSCs showed a decrease of 0.5-fold or less in all evaluable subjects. Figure 11B shows IHC of M1 and M2 macrophages in tumor biopsies obtained before (pre) and during (on) treatment from subjects with rGBM who achieved CR. Figure 11C shows the fold change of M1 and M2 macrophages quantified for the biopsy pairs in Figure 11B. After treatment, the proportion of M1 increased 1.9-fold, whereas M2 decreased 0.6-fold. Figure 11D shows representative tumor biopsies obtained pre-treatment and during the study from the subject in Figure 11B, as assessed by multiple ion beam imaging. Figure 11E shows the fold change in CTL. Figure 11F shows total and mMDSC quantified for the biopsy pair in Figure 11D. In the biopsies during the study, CTL increased more than 4-fold compared to pre-treatment, and total and mMDSC increased more than 15-fold.

[0105] These data indicate that VT1021 is effective as a single agent clinical activity in rGBM, especially in subjects with high expression levels of CD36 and CD47. Subjects with rGBM that exhibit doubly high CD36 and CD47 levels had reduced tumor burden and were more likely to continue in clinical trials longer than subjects that do not exhibit doubly high CD36 and CD47 levels. Increased TSP-1 expression was observed in circulating PBMC and TME, and VT1021 remodeled the TME to be more immune-enhanced through increased M1 macrophages and CTLs. Thus, doubly high expression of CD36 and CD47 is a predictive biomarker for the responsiveness of GBM subjects to treatment with VT1021.

[0106] These results indicate that doubly elevated CD36 and CD47 levels are robust biomarkers of cancer responsiveness to treatment with VT1021, including glioblastoma responsiveness. The results further indicate that peptides of the present technology, including VT1021, are useful in treating cancers that exhibit doubly elevated CD36 and CD47 levels compared to control levels, including glioblastoma.

[0107] Example 3: Treatment of pancreatic cancer with doubly elevated CD36 and CD47 levels using VT1021. This example demonstrates the efficacy of VT1021 for the treatment of pancreatic cancers that exhibit doubly elevated levels of CD36 and CD47.

[0108] Subjects with pancreatic cancer are identified using standard clinical protocols. Samples are obtained and the levels of CD36 and CD47 are measured using methods known in the art. Subjects that show doubly elevated CD36 and CD47 levels compared to suitable controls are selected for administration with VT1021.

[0109] VT1021 is administered to subjects who have progressed to the treatment stage.Frequency and dosage are determined according to standard methods in the art according to the stage and severity of disease.Tumor burden and other disease indicators are measured using methods known in the art, and data is compiled.

[0110] The results are expected to show a reduction in tumor burden in subjects receiving VT1021 compared to control subjects.

[0111] The results will show that doubly elevated CD36 and CD47 levels are robust biomarkers of cancer responsiveness to treatment with VT1021, including pancreatic cancer responsiveness. The results will further show that peptides of the present technology, including VT1021, are useful in treating cancers that exhibit doubly elevated CD36 and CD47 levels compared to control levels, including pancreatic cancer.

[0112] Example 4: Treatment of GBM with doubly elevated CD36 and CD47 levels with VT1021. This example demonstrates the efficacy of VT1021 for the treatment of GBM exhibiting doubly elevated levels of CD36 and CD47.

[0113] Subjects with GBM are identified using standard clinical protocols. Samples are obtained and the levels of CD36 and CD47 are measured using methods known in the art. Subjects that show doubly elevated CD36 and CD47 levels compared to suitable controls are selected for administration with VT1021.

[0114] VT1021 is administered to subjects who have progressed to the treatment stage.Frequency and dosage are determined according to standard methods in the art according to the stage and severity of disease.Tumor burden and other disease indicators are measured using methods known in the art, and data is compiled.

[0115] The results are expected to show a reduction in tumor burden in subjects receiving VT1021 compared to control subjects.

[0116] The results will show that doubly elevated CD36 and CD47 levels are robust biomarkers of cancer responsiveness to treatment with VT1021, including GBM responsiveness. The results will further show that peptides of the present technology, including VT1021, are useful in treating cancers that exhibit doubly elevated CD36 and CD47 levels compared to control levels, including GBM. References 1. Fidler, IJ, The pathogenesis of cancer metastasis: the'seed and soil'hypothesis revisited. Nat Rev Cancer, 2003.3(6):p.453-8. 2.Kang,S.Y.,et al.,Prosaposin inhibits tumor metastasis via paracrine and endocrine stimulation of stromal p53 and Tsp-1.Proc Natl Acad Sci U S A,2009.106(29):p.12115-20. 3.Lamy,L.,et al.,Interactions between CD47 and thrombospondin reduce inflammation.Journal of Immunology(Baltimore,Md.:1950),2007.178(9):p.5930-9. 4.Salajegheh,M.,et al.,Upregulation of thrombospondin-1(TSP-1)and its binding partners,CD36 and CD47,in sporadic inclusion body myositis.J Neuroimmunol,2007.187(1-2):p.166-74. 5.Vallejo,A.N.,et al.,Central role of thrombospondin-1 in the activation and clonal expansion of inflammatory T cells.Journal of Immunology(Baltimore,Md.:1950),2000.164(6):p.2947-54. 6.Catena,R.,et al.,Bone marrow-derived Gr1+ cells can generate a metastasis-resistant microenvironment via induced secretion of thrombospondin-1.Cancer Discov,2013.3(5):p.578-89. 7.Feig, C., et al., The pancreas cancer microenvironment. Clin Cancer Res, 2012.18(16):p.4266-76.

[0117] Without further elaboration, a person skilled in the art can make full use of the present disclosure based on the above description. The embodiments disclosed herein should be construed as merely illustrative and not limiting in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referred to herein.

[0118] As used herein, the indefinite articles "a" and "an" as used in the specification and claims should be understood to mean "at least one," unless expressly indicated otherwise.

Claims

1. 1. A method for assessing a subject's responsiveness to cancer treatment with a Tsp-1 inducer, the method comprising determining the levels of CD36 and CD47 in a sample obtained from the subject, wherein a doubling of CD36 and CD47 levels in the sample compared to control levels indicates that the subject will respond or is likely to respond to cancer treatment with a Tsp-1 inducer.

2. The method of claim 1, wherein the levels of CD36 and CD47 in the sample are determined in vitro.

3. 3. The method of claim 1 or 2, further comprising selecting the subject having levels of CD36 and CD47 in the sample that are doubly elevated compared to control levels for treatment with a Tsp-1 inducer.

4. 4. The method of claim 3, further comprising administering to the subject an effective amount of a Tsp-1 inducer to treat the cancer.

5. 1. A pharmaceutical composition for use in a method for treating a subject having cancer, comprising: the method comprising determining levels of CD36 and CD47 in a sample obtained from the subject, wherein a doubling of CD36 and CD47 levels in the sample compared to control levels indicates that the subject will respond or is likely to respond to cancer treatment with a Tsp-1 inducer, and further comprising administering to a subject having doubling of CD36 and CD47 levels in the sample compared to control levels an effective amount of a Tsp-1 inducer to treat the cancer; A pharmaceutical composition comprising a Tsp-1 inducer.

6. 1. A pharmaceutical composition for use in a method for treating a subject having cancer, comprising: The method comprises: (a) selecting a subject having cancer based on the subject being known to have levels of CD36 and CD47 in a sample that are doubly elevated compared to control levels; and (b) administering to the subject an effective amount of a Tsp-1 inducer to treat the cancer; A pharmaceutical composition comprising a Tsp-1 inducer.

7. 2. The method of claim 1, wherein the control levels are CD36 and CD47 levels in non-cancerous cells or tissues obtained from the subject.

8. The pharmaceutical composition described in claim 5 or 6, wherein the control levels are CD36 and CD47 levels in non-cancerous cells or tissues obtained from the subject.

9. 2. The method of claim 1, wherein the control levels are CD36 and CD47 levels in cells or tissues obtained from a healthy subject or a population of healthy subjects.

10. A pharmaceutical composition described in claim 5 or 6, wherein the control levels are CD36 and CD47 levels in cells or tissues obtained from a healthy subject or a population of healthy subjects.

11. The method of claim 1 , wherein the control level is a predetermined level.

12. A pharmaceutical composition described in claim 5 or 6, wherein the control level is a predetermined level.

13. The method of claim 1, wherein the levels of CD36 and CD47 include CD36 and CD47 protein levels, CD36 and CD47 mRNA levels.

14. The pharmaceutical composition described in claim 5 or 6, wherein the levels of CD36 and CD47 include CD36 and CD47 protein levels, CD36 and CD47 mRNA levels.

15. 10. The method of claim 1, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

16. The pharmaceutical composition of claim 5 or 6, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

17. The Tsp-1 inducer comprises a Psap peptide having the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitution variant thereof, wherein the amino acid substitution is a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution for leucine (L) selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof; c) arginine (R) to lysine (K); d) the D isomer of aspartic acid (D) relative to the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) relative to the L isomer of leucine (L); e) the D-isomer of tryptophan (W) relative to the L-isomer of tryptophan (W), and / or the D-isomer of proline (P) relative to the L-isomer of proline (P), or a combination thereof.

18. The Tsp-1 inducer comprising a Psap peptide having the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substituted variant thereof, wherein the amino acid substitution is: a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution for leucine (L) selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof; c) arginine (R) to lysine (K); d) the D isomer of aspartic acid (D) relative to the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) relative to the L isomer of leucine (L); e) The pharmaceutical composition according to claim 5 or 6, wherein the D-isomer of tryptophan (W) is present relative to the L-isomer of tryptophan (W), and / or the D-isomer of proline (P) is present relative to the L-isomer of proline (P), or a combination thereof.

19. 18. The method of claim 17, wherein the Psap peptide is 50 amino acids or less in length.

20. The pharmaceutical composition of claim 18, wherein the Psap peptide is 50 amino acids or less in length.

21. 20. The method of claim 19, wherein the Psap peptide is 30 amino acids or less in length.

22. The pharmaceutical composition of claim 20, wherein the Psap peptide is 30 amino acids or less in length.

23. 22. The method of claim 21, wherein the Psap peptide is 15 amino acids or less in length.

24. The pharmaceutical composition of claim 22, wherein the Psap peptide is 15 amino acids or less in length.

25. 24. The method of claim 23, wherein the Psap peptide is six amino acids or less in length.

26. The pharmaceutical composition of claim 24, wherein the Psap peptide is 6 amino acids or less in length.

27. 18. The method of claim 17, wherein the Psap peptide is a cyclic peptide.

28. The pharmaceutical composition described in claim 18, wherein the Psap peptide is a cyclic peptide.

29. 18. The method of claim 17, wherein the similarly sized non-standard amino acid is methylvaline, methylleucine, or sarcosine.

30. The pharmaceutical composition of claim 18, wherein the non-standard amino acid of similar size is methylvaline, methylleucine, or sarcosine.

31. 2. The method of claim 1, wherein the Tsp-1 inducer is cyclic DWLPK (SEQ ID NO: 2).

32. The pharmaceutical composition of claim 5 or 6, wherein the Tsp-1 inducer is cyclic DWLPK (sequence number 2).

33. A composition for use in treating a subject having a cancer characterized by doubly elevated levels of CD36 and CD47 in the sample compared to control levels, the composition comprising a Tsp-1 inducer.

34. Use of a composition in the manufacture of a medicament for treating a subject having cancer characterized by doubly elevated levels of CD36 and CD47 in the sample compared to control levels, wherein the composition comprises a Tsp-1 inducer.

35. 34. The composition of claim 33, wherein the control levels are CD36 and CD47 levels in non-cancerous cells or tissue obtained from the subject.

36. The use described in claim 34, wherein the control levels are CD36 and CD47 levels in non-cancerous cells or tissues obtained from the subject.

37. 34. The composition of claim 33, wherein the control levels are CD36 and CD47 levels in cells or tissues obtained from a healthy subject or a population of healthy subjects.

38. The use described in claim 34, wherein the control levels are CD36 and CD47 levels in cells or tissue obtained from a healthy subject or a population of healthy subjects.

39. The composition of claim 33, wherein the control level is a predetermined level.

40. The use described in claim 34, wherein the control level is a predetermined level.

41. The composition of claim 33, wherein the levels of CD36 and CD47 include CD36 and CD47 protein levels, CD36 and CD47 mRNA levels.

42. The use described in claim 34, wherein the levels of CD36 and CD47 include CD36 and CD47 protein levels, CD36 and CD47 mRNA levels.

43. 34. The composition of claim 33, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

44. The use described in claim 34, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

45. The Tsp-1 inducer comprises a Psap peptide having the amino acid sequence CDWLPK, DWLPK, or DWLP, or an amino acid substitution variant thereof, wherein the amino acid substitution is a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution for leucine (L) selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof; c) arginine (R) to lysine (K); d) the D isomer of aspartic acid (D) relative to the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) relative to the L isomer of leucine (L); e) the D-isomer of tryptophan (W) versus the L-isomer of tryptophan (W), and / or the D-isomer of proline (P) versus the L-isomer of proline (P), or a combination thereof.

46. ​​The Tsp-1 inducer comprises a Psap peptide having the amino acid sequence CDWLPK, DWLPK, or DWLP, or an amino acid substituted variant thereof, wherein the amino acid substitution is: a) tyrosine (Y) to tryptophan (W); b) an amino acid substitution for leucine (L) selected from valine (V), alanine (A) or glycine (G), or a non-standard amino acid of similar size, or a derivative thereof; c) arginine (R) to lysine (K); d) the D isomer of aspartic acid (D) relative to the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) relative to the L isomer of leucine (L); e) The use according to claim 34, wherein the D isomer of tryptophan (W) is used in contrast to the L isomer of tryptophan (W), and / or the D isomer of proline (P) is used in contrast to the L isomer of proline (P), or a combination thereof.

47. 46. ​​The composition of claim 45, wherein the Psap peptide is 50 amino acids or less in length.

48. The use of claim 46, wherein the Psap peptide is 50 amino acids or less in length.

49. 48. The composition of claim 47, wherein the Psap peptide is 30 amino acids or less in length.

50. The use of claim 48, wherein the Psap peptide is 30 amino acids or less in length.

51. 50. The composition of claim 49, wherein the Psap peptide is 15 amino acids or less in length.

52. The use of claim 50, wherein the Psap peptide is 15 amino acids or less in length.

53. 52. The composition of claim 51, wherein the Psap peptide is six amino acids or less in length.

54. The use of claim 52, wherein the Psap peptide is six amino acids or less in length.

55. 46. ​​The composition of claim 45, wherein the Psap peptide is a cyclic peptide.

56. The use described in claim 46, wherein the Psap peptide is a cyclic peptide.

57. 46. ​​The composition of claim 45, wherein the similarly sized non-standard amino acid is methylvaline, methylleucine, or sarcosine.

58. The use described in claim 46, wherein the non-standard amino acid of similar size is methylvaline, methylleucine, or sarcosine.

59. 34. The composition of claim 33, wherein the Tsp-1 inducer is cyclic DWLPK (SEQ ID NO: 2).

60. The use of claim 34, wherein the Tsp-1 inducer is cyclic DWLPK (sequence number 2).

61. The method of claim 1 , wherein the sample is a tumor sample.

62. The composition described in claim 5, 6 or 33, wherein the sample is a tumor sample.

63. The use described in claim 34, wherein the sample is a tumor sample.

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