Use of CD36 to identify cancer subjects for treatment

Abstract

To provide compositions for use in the treatment of subjects with cancer, characterized by elevated CD36 levels in their samples compared to the control level.SOLUTION: A composition comprises cisplatin and a Psap peptide, where the Psap peptide comprises the amino acid sequence dWlP.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of the filing date of U.S. Provisional Application No. 61 / 782,850, filed on March 14, 2013, the entire content of which is incorporated herein by reference.

[0002] Federally Sponsored Research or Development This invention was made with government support under R01CA135417 awarded by the National Cancer Institute. The U.S. government has certain rights in this invention.

Background Art

[0003] Background of the Invention Cancer remains a major public health priority. For example, in 2008, an estimated 7.6 million people died of cancer. Treatments for cancer have been continuously improved with the progress of science and technology. Unfortunately, many cancer treatments have been shown to be effective only in a subset of cancer patients and, further, only in a subset of patients having the same type of cancer. As a result, finding ways to identify patients who are likely to respond to treatment has become increasingly important.

Summary of the Invention

[0004] Aspects of the present disclosure are based in part on the discovery that elevated levels of CD36 in tumor cells indicate that a subject is responsive or potentially responsive to treatment with a Psap peptide. Accordingly, aspects of the present disclosure relate to methods for assessing a subject's responsiveness to treatment with a Psap peptide by determining the level of CD36 in a sample such as a tumor sample. In some embodiments, the methods described herein relate to the identification or selection of a subject for treatment with a Psap peptide based on the level of CD36 in a sample. Other aspects of the present disclosure relate to compositions and methods for treating a subject having cancer and characterized by elevated levels of CD36.

[0005] In some aspects, the present disclosure is a method for evaluating the responsiveness of a subject to treatment with a Psap peptide, the method comprising determining the level of CD36 in a sample obtained from a subject having cancer, wherein an elevated level of CD36 in the sample as compared to a control level indicates that the subject is responsive or potentially responsive to treatment with the Psap peptide. In some embodiments, the level of CD36 in the sample is determined by performing an assay. In some embodiments, the method further comprises identifying a subject having an elevated level of CD36 in the sample as compared to the control level as being responsive or potentially responsive to treatment with the Psap peptide. In some embodiments, the method further comprises administering an effective amount of the Psap peptide for treating cancer to a subject identified as being responsive or potentially responsive to treatment with the Psap peptide.

[0006] Another aspect of the present disclosure is a method for treating a subject having cancer, the method comprising administering an effective amount of the Psap peptide for treating cancer to a subject having cancer and characterized by an elevated level of CD36 in a sample as compared to a control level. In some embodiments, the control level is the level of CD36 from non-cancerous cells or tissue obtained from a subject having cancer. In some embodiments, the control level is the level of CD36 in cells 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 level is the CD36 protein level.

[0007] A further aspect of the present disclosure is a method for treating a subject having cancer, the method comprising: (a) selecting a subject having cancer based on the knowledge that the subject has an elevated level of CD36 in a sample as compared to a control level; and (b) administering to the subject an effective amount of a Psap peptide since the subject has an elevated level of CD36 in the sample as compared to the control level. In some embodiments, the control level is the level of CD36 from non-cancerous cells or tissue obtained from a subject having cancer. In some embodiments, the level is the level of CD36 in cells 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 level is the CD36 protein level. In some embodiments of any of the methods provided herein, the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

[0008] In some embodiments of any of the methods provided herein, the Psap peptide comprises 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) for 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) for the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) for the L isomer of leucine (L); e) The D-isomer of tryptophan (W) with respect to the L-isomer of tryptophan (W), and / or the D-isomer of proline (P) with respect to the L-isomer of proline (P); or a combination 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, non-standard amino acids of similar size are methylvaline, methyll leucine, or sarcosine.

[0009] In yet another aspect, the present disclosure relates to a composition containing a Psap peptide for use in treating a subject having cancer and characterized by an elevated level of CD36 in a sample as compared to a control level. In another aspect, the present disclosure relates to the use of a composition in the manufacture of a medicament for treating a subject having cancer and characterized by an elevated level of CD36 in a sample as compared to a control level, wherein the composition contains a Psap peptide. In some embodiments of the uses or compositions provided herein, the control level is the level of CD36 from non-cancerous cells or tissues obtained from a subject having cancer. In some embodiments of the uses or compositions provided herein, the control level is the level of CD36 in cells or tissues obtained from a healthy subject or a population of healthy subjects. In some embodiments of the uses or compositions provided herein, the control level is a predetermined level. In some embodiments of the uses or compositions provided herein, the CD36 level is the CD36 protein level.

[0010] In some embodiments of the uses or compositions provided herein, the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma. In some embodiments of the uses or compositions provided herein, the Psap peptide comprises the amino acid sequences CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or amino acid substitution variants thereof, where the amino acid substitutions are a) tyrosine (Y) for 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) for the L-isomer of aspartic acid (D), and / or the D-isomer of leucine (L) for the L-isomer of leucine (L);

[0011] e) the D-isomer of tryptophan (W) for the L-isomer of tryptophan (W), and / or the D-isomer of proline (P) for the 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 acids of similar size are methylvaline, methyleucine, or sarcosine. In some embodiments of the methods, compositions or uses provided herein, the sample is a tumor sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

[0013]

Figure 5

Figure 6

Figure 7

Figure 8

[0014] The Psap peptide is a therapeutic peptide that contains an amino acid sequence originally derived from a fragment of the known anti-angiogenic protein, saposin A. The Psap peptide generally contains a core sequence of CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), or an amino acid substitution variant thereof, and can be of a relatively short length of about 4 amino acids (e.g., a peptide consisting of DWLP (SEQ ID NO: 3) or an amino acid substitution variant). Such Psap peptides have previously been shown to be effective in treating multiple types of cancer (see, for example, PCT Publications WO2009002931 and WO / 2011 / 084685; PCT Application PCT / US2012 / 71424, published as PCT Publication WO / 2013 / 096868, and U.S. Patent Applications 12 / 640788 and 13 / 516511, all of which are hereby incorporated by reference in their entirety). Administration of the Psap peptide was previously thought to stimulate thrombospondin (Tsp-1) in vivo, which then acts on endothelial cells to cause an anti-angiogenic effect that indirectly inhibits the growth of cancer and / or metastasis.

[0015] As described herein, tumor cells from several different types of cancer responsive to the Psap peptide were found to express CD36. CD36 is a member of the class B scavenger receptor family of cell surface proteins and has many ligands including oxidized low density lipoprotein, oxidized phospholipids, long chain fatty acids, collagen, and Tsp-1. Without wishing to be bound by any theory or mechanism, administration of the Psap peptide is thought to stimulate Tsp-1, which then acts directly on the tumor cells by interacting with CD36 on the tumor cells. The interaction between Tsp-1 and CD36 in tumor cells can result in inhibition of tumor cell proliferation and / or induction of apoptosis of the tumor cells. Thus, the Psap peptide appears to treat cancer via two different independent mechanisms, i.e., indirectly via an anti-angiogenic effect and directly via the interaction of CD36 and Tsp-1 on the tumor cells. Thus, the responsiveness of a subject with cancer to treatment with the Psap peptide may depend on the level of CD36 expressed by the cancer.

[0016] Accordingly, aspects of the present disclosure relate to methods for assessing the responsiveness of a subject to treatment with the Psap peptide by determining the level of CD36 in a sample such as a tumor sample. In some embodiments, the methods described herein relate to the identification or selection of a subject for treatment with the Psap peptide based on the level of CD36 in a sample such as a tumor sample. Other aspects of the present disclosure relate to compositions and methods for treating a subject having cancer and characterized by elevated levels of CD36 (e.g., the cancer is selected or identified based on having a high level of CD36 in a sample compared to a control level). As used herein, "responsive to treatment with a Psap peptide" includes, but is not limited to, prevention or reduction of cancer development, reduction of cancer symptoms, suppression or inhibition of cancer growth, prevention of metastasis and / or invasion of existing cancer, promotion or induction of cancer regression, inhibition or suppression of cancer cell proliferation, reduction of angiogenesis, and / or increase in the amount of apoptotic cancer cells in response to treatment with a Psap peptide.

[0017] As used herein, "not responsive to treatment with a Psap peptide" includes, but is not limited to, the absence of prevention or reduction of cancer development, the absence of reduction of cancer symptoms, the absence of suppression or inhibition of cancer growth, the absence of prevention of metastasis and / or invasion of existing cancer, the absence of promotion or induction of cancer regression, the absence of inhibition or suppression of cancer cell proliferation, the absence of reduction of angiogenesis, and / or reduction in the amount of apoptotic cancer cells in response to treatment with a Psap peptide.

[0018] Diagnostic and seranostic methods Aspects of the present disclosure relate to diagnostic and seranostic methods useful for evaluating the responsiveness of a subject to treatment with a Psap peptide. In some embodiments, the method includes determining the level of CD36 in a sample obtained from a subject having cancer, where an elevated level of CD36 in the sample, compared to a control level, indicates that the subject is responsive or likely to be responsive to treatment with a Psap peptide (i.e., if the level of CD36 in the sample is higher than the control level, the subject is identified as being responsive or likely to be responsive to treatment with a Psap peptide). In some embodiments, the method further includes identifying a subject having an elevated level of CD36 in the sample, compared to a control, as being responsive or likely to be responsive to treatment with a Psap peptide. In some embodiments, the method further includes administering to a subject identified as being responsive or likely to be responsive to treatment with a Psap peptide an effective amount of the Psap peptide described herein for treating cancer. In some embodiments, the sample obtained from a subject having cancer is a tumor sample.

[0019] In some embodiments, an elevated level of CD36 in the sample, compared to a control level, indicates that the cancer regresses or is likely to regress in response to treatment with a Psap peptide. In some embodiments, the method further includes identifying a subject having an elevated level of CD36 in the sample, compared to a control, as having cancer that regresses or is likely to regress in response to treatment with a Psap peptide. In some embodiments, the method further includes administering to a subject identified as having cancer that regresses or is likely to regress in response to treatment with a Psap peptide an effective amount of the Psap peptide described herein for causing regression of the cancer.

[0020] As used herein, an "elevated level of CD36" means that the level of CD36 is above a control level, such as a predetermined threshold or the level of CD36 in a control sample. Control levels are described in detail herein. An elevated level of CD36 includes a CD36 level that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or more higher than the control level. An elevated level of CD36 also includes an increase from a zero state (e.g., no CD36 expression or undetectable CD36 expression in the control) to a non-zero state phenomenon (e.g., some CD36 expression or detectable CD36 expression in the sample). As used herein, "treatment with a Psap peptide" means including administration of a Psap peptide to a subject. The Psap peptide is described herein. Treatment with a Psap peptide may include treatment with only the Psap peptide, or may include treatment with multiple agents or therapies, such as treatment with the Psap peptide and other chemotherapeutic agents, and / or treatment with other modalities such as surgery, radiation therapy, or chemotherapy.

[0021] Treatment Another aspect of the disclosure relates to a method for treating a subject having cancer. In some embodiments, the method comprises administering to a subject having cancer and characterized by an elevated level of CD36 in a sample obtained from the subject as compared to a control level, an effective amount of the Psap peptide described herein for treating cancer. In some embodiments, the method comprises (a) selecting a subject having cancer based on the knowledge that the subject has an elevated level of CD36 in a sample as compared to a control level; and (b) administering to the subject an effective amount of the Psap peptide because the subject has an elevated level of CD36 in a sample as compared to a control level, comprising. Other aspects of the present disclosure relate to a composition in the manufacture of a medicament for treating a subject having cancer and characterized by elevated levels of CD36 in a sample as compared to control levels, and to the use of the composition. In some embodiments, the composition comprises a Psap peptide as described herein. In some embodiments, the sample is a tumor sample.

[0022] As used herein, "treating" or "treatment" includes, but is not limited to, preventing or reducing the development of cancer, alleviating the symptoms of cancer, suppressing or inhibiting the growth of cancer, preventing the metastasis and / or invasion of existing cancer, promoting or inducing the regression of cancer, inhibiting or suppressing the proliferation of cancer cells, reducing angiogenesis and / or increasing the amount of apoptotic cancer cells. In some embodiments, treating cancer is directly inhibiting or suppressing the proliferation of cancer cells and does not involve inhibiting or suppressing angiogenesis, which indirectly results in inhibiting or suppressing the proliferation of cancer cells. An effective amount is an amount of Psap peptide sufficient to provide a medically desirable result, such as the treatment of cancer. The effective amount will vary depending on the particular cancer being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of any concurrent therapy, the particular route of administration, and similar factors within the knowledge and experience of the physician. For administration to a subject such as a human, dosages generally from about 0.001, 0.01, 0.1 or 1 mg / kg to 50, 100, 150 or 500 mg / kg or more can be used.

[0023] The Psap peptides and their compositions can be formulated for various modes of administration, including systemic, topical, or local administration. The techniques and dosage forms can generally be found in the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. When administered, the Psap peptides can be applied in a pharmaceutically acceptable amount and in a pharmaceutically acceptable composition. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but salts that are not pharmaceutically acceptable may also be used to prepare their pharmaceutically acceptable salts and are not excluded from the scope of the present disclosure. Such pharmacologically and pharmaceutically 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, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth salts such as sodium, potassium, or calcium salts.

[0024] The Psap peptide can be optionally combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. The term "carrier" represents natural or synthetic organic or inorganic components that facilitate application in combination with the active ingredient. The components of the pharmaceutical composition can also be co-mixed in a manner such that there is no interaction that substantially impairs the desired pharmaceutical effect between the molecules of the present disclosure and among themselves. Some examples of materials that can serve as pharmaceutically acceptable carriers include the following: (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 carboxymethyl cellulose, methyl cellulose, ethyl cellulose, 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) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, 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 buffer solutions; (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; (24) C2-C22 alcohols, such as ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations.Humectants, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrance agents, preservatives, and antioxidants can also be present in the formulation.

[0025] The pharmaceutical composition can conveniently be provided in unit dosage form and can be prepared by any method well known in the art of pharmacy. The term "unit dose" as used in connection with the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable as unit dosage amounts for a subject, each unit containing a predetermined quantity of the active substance calculated to produce the desired therapeutic effect in association with the required diluents such as carriers or vehicles. A variety of routes of administration are available. The particular mode selected depends on the type of cancer being treated and the dosage required for the therapeutic effect. The methods of the present disclosure can generally be carried out using any medically acceptable mode of administration, i.e., any mode that produces an effective level of the active compound without causing clinically unacceptable side effects. Such modes of administration include oral, rectal, topical, nasal, intradermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion.

[0026] In some embodiments, the administration is parenteral. Injectable formulations suitable for parenteral administration include, for example, sterile aqueous or oily suspensions and can be formulated according to well-known techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable formulations can also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-propanediol or 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils have been conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0027] For topical administration, the pharmaceutical composition can be formulated into ointments, plasters, gels, or creams, as is generally known in the art. Topical administration can utilize transdermal delivery systems known in the art. An example is a skin patch. Alternatively, a biolistic gene gun delivery method can be used. A gene gun is a device designed initially for plant transformation for injecting genetic information into cells. The payload is elemental particles of heavy metal coated with plasmid DNA. This technique is often simply referred to as biolistic. Another device using biolistic technology is the PDS-1000 / He particle delivery system. The compositions described herein can be coated onto fine gold particles, and these coated particles are "driven" into living tissues such as hemangiomas and melanomas under high pressure. An example of a gene gun-based method is described for bovine DNA-based vaccination in Loehr B. I. et al., J. Virol. 2000, 74:6077-86.

[0028] The pharmaceutical compositions described in this specification are also administered by routes within, around, into, or around tumors, lesions, or pathological lesions in order to exert local and systemic effects. The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors. For these uses, additional conventional pharmaceutical preparations, such as tablets, granules, powders, capsules, and sprays, may be preferably required. In such preparations, additional conventional additives, such as binders, wetting agents, propellants, lubricants, and stabilizers, may also be required. Compositions suitable for oral administration can be provided as individual units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous or non-aqueous liquids, such as syrups, elixirs, or emulsions.

[0029] Other delivery systems can include time-release, delayed-release or sustained-release delivery systems. Such systems can avoid repeated dosing of the anti-inflammatory drug and increase convenience for the subject and the physician. Many types of release delivery systems are available and are known to those skilled in the art. They include polymer-based systems such as poly(lactide-glycolide), copolyoxalate, copolycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyrate, and polyanhydrides. Microcapsules of the aforementioned polymers containing the drug are described, for example, in U.S. Patent 5,075,109. Delivery systems also include non-polymeric systems such as lipids containing sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; hydrogel release systems; elastomeric systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants, etc. Specific examples include, but are not limited to: (a) erosion systems in which the anti-inflammatory agent is contained in matrix form, as described, for example, in U.S. Patents Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660; (b) diffusion systems in which the active ingredient penetrates from the polymer at a controlled rate, as described, for example, in U.S. Patents Nos. 3,832,253 and 3,854,480. Further, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0030] The use of long-term sustained-release implants can be particularly appropriate for the treatment of chronic conditions. As used herein, long-term release means that the implant is constructed and configured to deliver a therapeutic level of the 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.

[0031] In some embodiments, the pharmaceutical composition for therapeutic administration must be sterile. Sterility is achieved by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The active ingredient and / or pharmaceutical composition are usually stored in lyophilized form or as an aqueous solution if it is very stable against heat and oxidative denaturation. The pH of the formulation is typically about 6 - 8, although higher or lower pH values may also be appropriate in certain cases. In some embodiments, administration of the Psap peptide may be combined with other therapies, such as chemotherapy, radiation, and / or surgery.

[0032] CD36 CD36 (cluster of differentiation 36) is an integral membrane protein found on the surface of many cell types in vertebrates and is also known as FAT, GP4, GP3B, GPIV, CHDS7, PASIV, SCARB3, and BDPLT10. The Entrez gene ID for human CD36 is 948. Exemplary human CD36 transcripts and proteins are as follows: [Table 1-1] [Table 1-2]

[0033] [Table 2]

[0034] [Table 3]

[0035] [Table 4]

[0036]

Table 5

[0037]

Table 6

[0038] Psap peptide Pro-saposin (Psap) is a precursor protein of saposin, which is composed of about 524 - 527 amino acids including a 16 - amino acid signal peptide. The full - length precursor polypeptide undergoes co - translational glycosylation and modification in the endoplasmic reticulum and Golgi system to produce a precursor protein of 70 - 72 kDa. After transport to the lysosome, cathepsin D is involved in proteolytic processing to produce an intermediate molecular form of 35 - 53 kDa, which then becomes a 13 - kDa glycoprotein and finally the mature 8 - 11 kDa partially glycosylated form of individual saposin molecules (O’Brien J. S., and Kishimoto Y, The FASEB J., 5: 301 - 8, 1991; Kishimoto Y. et al., J. Lipid Res. 33:1255 - 67, 1992). Pro - saposin is converted into four cleavage products, saposin A, B, C, and D. The amino acid sequences of Psap pre - proprotein isoforms A, B, and C, as well as the amino acid sequence of the saposin A cleavage product, are as follows:

[0039]

Table 7

[0040]

Table 8

[0041]

Table 9

Table 10

[0042] Aspects of the present disclosure relate to Psap peptides and their use. The Psap peptides include sequences originally derived from fragments of saposin A. Fragments of saposin A consisting of only four amino acids and variants of these fragments have previously been shown to have anti-angiogenic and anti-cancer activity. Psap peptides and methods of making Psap peptides are known in the art (see, for example: PCT publications WO2009002931 and WO / 2011 / 084685; PCT application PCT / US2012 / 71424 published as PCT publication WO / 2013 / 096868, and US patent applications 12 / 640,788 and 13 / 516,511; all of these are hereby incorporated by reference in their entirety).

[0043] In some embodiments, the Psap peptide includes 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, where the amino acid substitution is a) tyrosine (Y) for 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) for the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) for the L isomer of leucine (L); e) the D isomer of tryptophan (W) for the L isomer of tryptophan (W), and / or the D isomer of proline (P) for the L isomer of proline (P); or combinations thereof. In some embodiments, the Psap peptide includes the amino acid sequence CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3).

[0044] It should be understood that the Psap peptide can be of any length. In some embodiments, the Psap peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more amino acids in length. In some embodiments, the Psap peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, or less amino acids in length. In some embodiments, the Psap peptide is 4 - 500, 4 - 400, 4 - 300, 4 - 200, 4 - 100, 4 - 90, 4 - 80, 4 - 70, 4 - 60, 4 - 50, 4 - 40, 4 - 30, 4 - 25, 4 - 20, 5 - 500, 5 - 400, 5 - 300, 5 - 200, 5 - 100, 5 - 90, 5 - 80, 5 - 70, 5 - 60, 5 - 50, 5 - 40, 5 - 30, 5 - 25, 5 - 20, 6 - 500, 6 - 400, 6 - 300, 6 - 200, 6 - 100, 6 - 90, 6 - 80, 6 - 70, 6 - 60, 6 - 50, 6 - 40, 6 - 30, 6 - 25, or 6 - 20 amino acids in length.

[0045] The amino acids adjacent to CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) may be the naturally adjacent amino acids present in saposin A or prosaposin (e.g., LEKTC DWLP KPNMS(SEQ ID NO: 14), the underlined amino acids are the amino acids that are naturally adjacent to DWLP (SEQ ID NO: 3) of saposin A). Thus, in some embodiments, the Psap peptide comprises the amino acid sequences DWLPKPNMS (SEQ ID NO: 15), CDWLPKPNM (SEQ ID NO: 16), TCDWLPKPN (SEQ ID NO: 17), KTCDWLPKP (SEQ ID NO: 18), EKTCDWLPK (SEQ ID NO: 19), LEKTCDWLP (SEQ ID NO: 20), or amino acid substitution variants thereof, where the substitution occurs at CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3). Other examples of the Psap peptide include, without limitation: DWLPKPNMS (SEQ ID NO: 21), CDWLPKPNM (SEQ ID NO: 22), TCDWLPKPN (SEQ ID NO: 23), KTCDWLPKP (SEQ ID NO: 24), EKTCDWLPK (SEQ ID NO: 25), and LEKTCDWLP (SEQ ID NO: 26). Another example of the Psap peptide includes, without limitation: DWLPKPNM (SEQ ID NO: 27), CDWLPKPN (SEQ ID NO: 28), TCDWLPKP (SEQ ID NO: 29), KTCDWLPK (SEQ ID NO: 30), EKTCDWLP (SEQ ID NO: 31), DWLPKPN (SEQ ID NO: 32), CDWLPKP (SEQ ID NO: 33), TCDWLPK (SEQ ID NO: 34), KTCDWLP (SEQ ID NO: 35), DWLPKP (SEQ ID NO: 36), CDWLPK (SEQ ID NO: 1), TCDWLP (SEQ ID NO: 37), DWLPK (SEQ ID NO: 2), CDWLP (SEQ ID NO: 38), and DWLP (SEQ ID NO: 3).

[0046] The amino acids adjacent to CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) need not be the amino acids that are naturally adjacent to prosaposin A or prosaposin, but rather can be any amino acid, and this should be understood. Thus, the Psap peptide can include any number and identity of amino acids adjacent to the amino acids. In some embodiments, the adjacent amino acids can include an antibody or the Fc domain of an antibody, serum transferrin or a portion thereof, albumin, or transthyretin (see, e.g., G. M. Subramanian, (2007), Nature Biotechnology 25, 1411-141).

[0047] The Psap peptide can be synthesized using any method known in the art. Examples of synthetic methods include, but are not limited to, recombinant synthesis, liquid-phase synthesis, solid-phase synthesis, chemical ligation (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, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York; Schnolzer, M. A., P.; Jones, A.; Alewood, D.; Kent, S.B.H. (2007). "In Situ Neutralization in Boc-chemistry Solid Phase Peptide Synthesis". Int. J. Peptide Res. Therap. 13 (1-2): 31-44; Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press. p. 848; and Nilsson BL, Soellner MB, Raines RT (2005). "Chemical Synthesis of Proteins". Annu. Rev. Biophys. Biomol. Struct. 34: 91-118; and U.S. Patent Nos. 4,749,742, 4,794,150, 5,552,471, 5,637,719, 6,001,966, 7,038,103, 7,094,943, 7,176,282, and 7,645,858; the entireties of which are hereby incorporated by reference herein).

[0048] In some embodiments, the Psap peptide can be modified, for example, by: oligomerization or polymerization (e.g., dimer, trimer, multimer, 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., an antibody or the Fc domain of an antibody, serum transferrin or a portion thereof, albumin, or transthyretin), or other modifications that substantially change the stability, solubility, or other properties of the peptide while substantially retaining or improving its therapeutic activity. Conjugation can be, for example, 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 glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives, polyvinyl ethyl ether, and α,β-poly[(2-hydroxyethyl)-DL-aspartamide], or mixtures thereof. Conjugation can be, for example, via a linker such as a peptide or a chemical linker. Methods for modifying peptides are known in the art (see, for example: U.S. Patent 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 WO 2010 / 014616; the contents of which are incorporated herein by reference).

[0049] In some embodiments, the Psap peptide is a cyclic peptide. A cyclic peptide is a polypeptide chain in which its amino terminus and carboxyl terminus are linked by a peptide bond or other covalent bond to form a circular chain. In one embodiment, the peptide contains amino and carboxyl terminal cysteine amino acid residues. Cysteine promotes the formation of S-S disulfide bonds. In one embodiment, the peptide contains additional cysteine amino acid residues, where the cysteine amino acid residues are near the termini but not necessarily at the very termini. In some embodiments, the cysteine amino acid residues are within the range of 5 amino acid residues from the peptide termini. Methods for the design and synthesis of cyclic peptides are well known in the art and are described, for example, in U.S. Patent Nos. 5,596,078, 5,990,273, 7,589,170, and U.S. Patent Application No. 20080287649.

[0050] In some embodiments, the Psap peptide is functionally modified to enhance stability. In some embodiments, the Psap peptide contains an N-terminal acetyl group and / or a C-terminal amide group. In some embodiments, the Psap peptide contains an N-terminal acetyl group and a C-terminal amide group. In some embodiments, the Psap peptide is Ac-dWlP-amide or Ac-DWLP-amide (Ac = acetyl group, the lowercase D and L represent the D-amino acids of SEQ ID NOs: 39 and 40, respectively). In some embodiments, chemical modifications of the Psap peptide include, but are not limited to, the following: alkyl, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino, alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom, carbocycle, carbocyclic, carbocyclo, carbocyclic, aryl, aralkyl, aralkoxy, aryloxyalkyl, heterocycle, heterocyclic, heterocyclic, heteroaryl, and / or aliphatic group.

[0051] The Psap peptide also includes peptidomimetics (e.g., D-peptides, β-peptides, and peptoids). The peptidomimetics utilized can include the full length of the Psap peptide, or only a portion of the Psap peptide. Peptidomimetics can include, for example, D-amino acids, reduced amide bonds for the peptide backbone, and non-peptide bonds for linking side chains, pyrrolinones, and sugar mimetics. The design and synthesis of glycan scaffold peptidomimetics are described by Hirschmann et al. (J. Med. Chem., 1996, 36, 2441-2448; incorporated herein by reference in its entirety). Additionally, pyrrolinone-based peptidomimetics have been described (see, for example, Smith et al., J. Am. Chem. Soc. 2000, 122, 11037-11038; incorporated herein by reference in its entirety). In some embodiments, the Psap peptide is in the form of a peptoid (U.S. Patent No. 5,811,387; Simon et al. Proceedings of the National Academy of Sciences USA, (1992), 89(20), 9367-9371). In some embodiments, the peptoid is poly-N-substituted glycine. In a peptoid, the side chains are attached to the nitrogen of the peptide backbone rather than to the α-carbon as in a peptide. In some embodiments, the peptoid contains nitroaromatic monomer units (Fowler et al., J Org Chem. 2009 Feb 20;74(4):1440-9). In some embodiments, the peptoid is N-substituted with an α-chiral aromatic side chain at one or more residues (Gorske et al., J Am Chem Soc. 2006 Nov 8;128(44):14378-87). In some embodiments, the Psap peptide includes a peptoid region (i.e., including one or more side chains attached to the nitrogen of the peptide backbone) and a peptide region (i.e., including one or more side chains attached to the α-carbon).

[0052] Psap Amino Acid Substitutions In some embodiments, the Psap peptide comprises an amino acid substitution variant of CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3), where the amino acid substitution is a) tyrosine (Y) for 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) for the L isomer of aspartic acid (D), and / or the D isomer of leucine (L) for the L isomer of leucine (L); e) the D isomer of tryptophan (W) for the L isomer of tryptophan (W), and / or the D isomer of proline (P) for the L isomer of proline (P); or combinations thereof.

[0053] Conservative amino acid substitutions can be substitutions of one amino acid residue by one amino acid residue having a side chain with similar charge, size, polarity, hydrophobicity, or combinations thereof. Families of amino acid residues having side chains with similar charge are defined in the art. These families include amino acids having: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0054] Conservative amino acid substitutions typically do not change the overall structure of the peptide and / or the types of amino acid side chains available to form van der Waals bonds with binding partners. In some embodiments, a conservative substitution for leucine is valine. In some embodiments, a conservative substitution for leucine is valine or alanine. In some embodiments, conservative or non-conservative substitutions for leucine are contemplated. In some embodiments, the substitution for leucine is valine, glycine or alanine. In some embodiments, the substitution for leucine is glycine. In some embodiments, the substitution for leucine is glycine or valine. In some embodiments, the amino acid substitution is tyrosine (Y) for tryptophan (W). Exemplary amino acid substitution mutants include, but are not limited to, DWAP (SEQ ID NO: 41), DYLPK (SEQ ID NO: 42), DWVPK (SEQ ID NO: 43), DWLPR (SEQ ID NO: 44), DWAPK (SEQ ID NO: 45), and DYLP (SEQ ID NO: 46).

[0055] Substitutions with non-standard amino acids are also contemplated herein. In some embodiments, leucine is substituted with a non-standard amino acid. In some embodiments, the non-standard amino acid substituent for leucine has a size similar to leucine, valine, alanine, or glycine. Examples of non-standard amino acids include azidoalanine, azidohomoalanine, azidonorvaline, azidonorleucine, azidonorvaline, homoallylglycine, homopropargylglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine, allylglycine, 3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine, p-ethynyl-phenylalanine, p-propargyl-oxy-phenylalanine, m-ethynyl-phenylalanine, 3-(6-chloroindolyl)alanine, 3-(6-bromoindolyl)alanine, 3-(5-bromoindolyl)alanine, azidohomoalanine, homopropargylglycine, p-chlorophenylalanine, α-aminocaprylic acid, methylvaline, methyleucine, or sarcosine. In some embodiments, leucine is substituted with a non-standard amino acid selected from methylvaline, methyleucine, or sarcosine. Non-standard amino acids and methods for their synthesis are well known in the art (see, e.g., U.S. Patent Publication Nos. 2010-0247433, 2008-0214439, 2004-0053390, and 2004-0058415; PCT Publication WO 03 / 073238; and U.S. Patent No. 6,586,207; all of which are incorporated herein by reference).

[0056] Amino acid substitutions can be achieved by adding the desired alternative amino acid in the appropriate sequence of the synthesis process during chemical synthesis of the peptide. Alternatively, molecular biology methods can be used. Non-conservative substitutions are also included to the extent that they substantially retain the activity of the peptides described herein. As described above, Psap peptides containing CDWLPK (SEQ ID NO: 1), DWLPK (SEQ ID NO: 2), or DWLP (SEQ ID NO: 3) and having D - amino acid substitutions have also been shown to have desired therapeutic activities (see PCT application PCT / US2012 / 71424, published as PCT publication WO / 2013 / 096868). Thus, amino acid substitution variants obtained from one or more D - amino acid substitutions for similar L - amino acids are also contemplated herein. In some embodiments, there is one D - amino acid substitution. In some embodiments, there are two or more D - amino acid substitutions. In some embodiments, there are 3, 4, or 5 D - amino acid substitutions. In some embodiments, the D - amino acid substitutions are evenly spaced, for example, placed at every other amino acid in a 4 - to 6 - mer. In some embodiments, the D - amino acid substitutions are for tryptophan (W) and / or proline (P). In some embodiments, the D - amino acid substitutions are for aspartic acid (D) and / or leucine (L). The L and D designations of amino acid configuration refer not to the optical activity of the amino acid itself, but to the optical activity of the isomer of glyceraldehyde from which the amino acid could theoretically be synthesized (D - glyceraldehyde is dextrorotatory; L - glyceraldehyde is levorotatory). Exemplary D - amino acid substitutions include dWlP and DwLp (lower case D and L denote the D - amino acids of SEQ ID NOs: 47 and 48, respectively).

[0057] Assay Aspects of the present disclosure relate to performing an assay to determine the level of CD36 in a sample. Any assay known in the art can be used to measure the level of CD36 (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, F.M. Ausubel, 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, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. The level of CD36 may be at the mRNA level and / or the protein level. In some embodiments, the level of CD36 is at the protein level.Assays for detecting CD36 mRNA include, but are not limited to, Northern blot analysis, RT-PCR, sequencing techniques, RNA in situ hybridization (e.g., hybridizing to RNA molecules present in a sample using a DNA or RNA probe), in situ RT-PCR (performed as described in Nuovo 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., hybridization of polynucleotide sequences from a sample to oligonucleotides attached to a solid surface (e.g., a glass wafer) having addressable locations such as Affymetrix microarrays (Affymetrix®, Santa Clara, CA)). Methods for designing nucleic acid binding partners, such as probes, are known in the art. In some embodiments, the nucleic acid binding partner binds to a partial or full nucleic acid sequence of CD36, which sequence is distinguishable using the CD36 sequences provided herein.

[0058] Assays for detecting CD36 protein levels include, but are not limited to, immunoassays (also referred to herein as immune-based or immuno-based assays, such as Western blot, immunohistochemistry, and ELISA assays), mass spectrometry, and multiplex bead-based assays. Such assays for detecting protein levels are well known in the art. Binding partners for protein detection can be designed using methods known in the art as described herein. In some embodiments, a CD36 protein binding partner, such as an anti-CD36 antibody, binds to a portion or the entire amino acid sequence of the CD36 protein. Other examples of methods for detecting and quantifying proteins include multiplex immunoassays, which are described, for example, in U.S. Pat. Nos. 6,939,720 and 8,148,171, and published U.S. patent application 2008 / 0255766, and also include protein microarrays as described, for example, in U.S. patent application 2009 / 0088329. In some embodiments, the sample obtained from the subject is a tumor biopsy material, and the assay for detecting CD36 protein levels is an immune-based assay performed on the tumor biopsy material.

[0059] Any suitable binding partner for CD36 is contemplated for detecting levels of CD36. In some embodiments, the binding partner is any molecule that specifically binds to the CD36 protein. As used herein, "specifically binds to the CD36 protein" means that the molecule is more likely to bind to a portion or the whole of the CD36 protein than to a portion or the whole of a non-CD36 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 or 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). The binding partner also includes other peptide molecules and aptamers that specifically bind to CD36. Methods for producing peptide molecules and aptamers are well known in the art (see, e.g., published US patent application No. 2009 / 0075834, US Patent Nos. 7435542, 7807351, and 7239742).

[0060] Examples of commercially available CD36 antibodies include, for example, N-15, SMφ, L-17, ME542, H300, 185-1G2, and V-19 from Santa Cruz Biotechnology (catalog numbers sc-5522, sc-7309, sc-13572, sc-5523, SC-9154, sc-21772, and sc-7641, respectively), and JC63.1, FA6-152, and anti-CD36 from Abcam (catalog numbers ab23680, ab17044, and ab78054, respectively). In some embodiments, the binding partner is any molecule that specifically binds to CD36 mRNA. As used herein, "specifically binds to CD36 mRNA" means that the molecule is more likely to bind (e.g., by complementary base pairing) to a part or the whole of CD36 mRNA than to a part or the whole of non-CD36 mRNA or other non-CD36 nucleic acids. In some embodiments, the binding partner that specifically binds to CD36 mRNA is a nucleic acid, such as a probe. The binding partner can be designed using the nucleotide and amino acid sequences of CD36 provided herein. In some embodiments, the CD36 binding partner may include a detectable label, such as an enzymatically active group, a fluorescent molecule, a chromophore, a luminescent molecule, a specifically bindable ligand, or a radioisotope. In some embodiments, a second binding partner specific for the CD36 binding partner is also contemplated, such as a secondary antibody.

[0061] Sample Aspects of the present disclosure relate to determining the level of CD36 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 can include, for example, tumor cells, a population of tumor cells, a fragment of a tumor (e.g., a biopsy specimen), or an entire tumor. In some embodiments, the tumor sample is a tumor biopsy specimen. In some embodiments, the tumor sample includes circulating tumor cells. In some embodiments, the tumor sample includes ascites. 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 specimen that includes normal tissue surrounding a tumor fragment, etc.). In some embodiments, the sample can be a tissue or body fluid sample obtained from the subject. Examples of body fluid samples include blood, plasma, serum, and urine.

[0062] subject Aspects of the present disclosure relate to a subject, such as a human, having cancer. Any type of cancer is contemplated herein, including but not limited to leukemia, lymphoma, myeloma, carcinoma, metastatic carcinoma, sarcoma, adenoma, cancers of the nervous system and urogenital cancers. Exemplary types of cancer are as follows: adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrosarcoma, brain cancer, brainstem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenoma, Burkitt lymphoma, carcinoid tumor, cancer of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, pediatric cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and optic pathway glioma, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, renal cell carcinoma, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer,

[0063] Non-small cell lung cancer, primary central nervous system lymphoma, Waldenström macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell cancer, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorder, chronic myeloproliferative disorder, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary cancer, plasmacytoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck carcinoma, supratentorial primitive neuroectodermal tumor, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Wilms tumor. In some embodiments, the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma or melanoma. In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, leukemia, pancreatic cancer, glioblastoma multiforme, astrocytoma or melanoma. In some embodiments, the cancer is pancreatic cancer, ovarian cancer, breast cancer, prostate cancer, melanoma cancer, or lung cancer.

[0064] Controls and control levels Aspects of the present disclosure relate to the comparison of the CD36 level in a sample to a control level. In some embodiments, the control level is the level of CD36 in cells, tissues or body fluids obtained from a healthy subject or a population of healthy subjects. As used herein, a healthy subject is a subject who does not have an apparent disease such as cancer and does not have a medical history of a disease. In some embodiments, the control level is determined from a sample obtained from a subject having cancer. Thus, in some embodiments, the control level is obtained from the same subject from whom the sample is obtained. In some embodiments, the control level is the level of CD36 from non-cancerous cells or tissues obtained from a subject having cancer. In some embodiments, the control level is undetectable or a CD36 level below the background / noise level obtained using standard detection methods (e.g., Western blot or immunohistochemistry).

[0065] The present disclosure also includes comparing the level of CD36 in a sample from a subject to a predetermined level or value such that it is not necessary to measure the control level each time. The predetermined level or value can take various forms. This can be a single cut-off value such as a median or an average value. This can be established based on comparison groups, for example, where one defined group is known not to respond to treatment with the Psap peptide and another defined group is known to respond to treatment with the Psap peptide. This can also be a range, for example, dividing the test population evenly (or unevenly) into groups (non-responsive to treatment with the Psap peptide, somewhat responsive to treatment with the Psap peptide, and highly responsive to treatment with the Psap peptide), or dividing into four groups (the lowest group being subjects non-responsive to treatment with the Psap peptide and the highest group being subjects with maximum responsiveness to treatment with the Psap peptide). The predetermined value can depend on the particular population selected. For example, a clearly healthy group (without detectable cancer and no history of cancer) will have a different "normal" range of CD36 than a population of members known to have cancer and not respond to treatment with the Psap peptide. Thus, the selected predetermined value can take into account the category to which the subject belongs. Appropriate ranges and categories can be selected by those of ordinary skill in the art without undue experimentation.

[0066] Example Example 1 Method Cell Lines and Primary Cells The cell line PC3 has been previously described (Kang et al. PNAS. 2009; 106:12115-20). PC3 cells were cultured in RPMI containing 10% FBS. Human breast cancer cell lines MDA-MB-231 and MCF-7 have been previously described (Ryu et al. PLoS one, 6, 2011). The mouse Lewis lung carcinoma cell line LLC, which stably expresses RFP and firefly luciferase, was provided by Lea Eisenbach, Wiesmann Institute of Science, Rehovot, Israel (Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127:679-95; Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science. 2008;319:195-8; and Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2009;9:239-52) and cultured in DMEM supplemented with 10% fetal bovine serum.B16 melanoma cells, LNCaP prostate cancer cells, AsPc1 pancreatic cancer cells, and ID8 ovarian cancer cells have been described previously (Overwijk WW et al. B16 as a mouse model for human melanoma. Curr Protoc Immunol. 2001, May;Chapter 20:Unit 20.1;Horoszewicz JS, Leong SS, Kawinski E et al. LNCaP model of human prostatic carcinoma. Cancer Res. 1983, Apr;43(4):1809-18.;Chen WH, et al. Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites. In Vitro 18: 24-34, 1982; and Roby KF, et al. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis. 2000, 21:585-591). Primary ovarian cancer cells were obtained from the ascites of ovarian cancer patients.

[0067] Western blot analysis Cells were homogenized in lysis buffer (BioRad) containing protease inhibitor (Roche Applied Science). Samples were boiled in 1× SDS sampling buffer and loaded onto a 4-20% gradient Bis-Tris NuPAGE gel (Invitrogen). Western blotting was performed using an antibody specific for CD36 (AbCam, ab78054) or β-actin (Sigma-Aldrich).

[0068] In vitro cell proliferation assay Cell proliferation was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) assay. Cells were seeded into 50 μL of growth medium in a 96-well culture plate and allowed to adhere overnight. Next, 50 μL of growth medium + 2-fold concentrated treatment reagent was added. After each treatment time point, 10 μL of 5% MTT solution (buffered in PBS) was added to each well. The plate was incubated at 37 °C for an additional 4 hours to metabolically convert the MTT to formazan crystals in the mitochondria of the cells. The formazan crystals were finally solubilized by adding 100 μL of 10% sodium dodecyl sulfate in 50% N-N-dimethylformamide to each microplate well. Absorbance at 550 and 680 nm (corresponding to the formazan salt and reference wavelength, respectively) was measured using a colorimetric microplate reader. Wells containing only complete medium were used as controls. Each experiment was performed twice using six replicates for each drug concentration.

[0069] Results It was hypothesized that Tsp-1, upregulated by the Psap peptide, can act directly on cancer cells not only indirectly via an anti-angiogenic mechanism. To test this, LLC cells were treated with either recombinant Tsp-1 or the DWLPK (SEQ ID NO: 2) Psap peptide, and cell proliferation was measured using the MTT assay. It was found that Tsp-1 was able to decrease cell proliferation, while the Psap peptide had no effect on cell proliferation (Figure 1A). This supports the hypothesis that Tsp-1 can act directly on cancer cells, since this assay was performed in vitro in the absence of any blood vessels. These results also indicate that the Psap peptide alone does not appear to affect the proliferation of cancer cells, supporting the hypothesis that the Psap peptide can indirectly treat cancer via upregulation of Tsp-1. LLC cells have been shown to express CD36, a receptor for Tsp-1, indicating that Tsp-1 can act directly on cancer cells via CD36 (Figure 1B).

[0070] CD36 levels were measured in other cell lines to confirm whether CD36 was expressed in other cancer types. CD36 levels were measured by Western blot analysis in breast cancer (MDA-231, MCF-7), ovarian cancer (ID8), melanoma (B16), prostate cancer (PC3 and LNCaP), and lung cancer (LLC) cell lines. CD36 protein was detected in all cell lines tested, and particularly high levels of CD36 were found to be detected in the MDA-231, MCF-7, PC3, and LLC cell lines (Figure 2). MDA-231, ID8, B16, PC3, and LLC cells have previously been shown to respond to treatment with the Psap peptide in vivo. The pancreatic cell line AsPc1 was also examined and found to express CD36. CD36 levels were also measured in primary ovarian cancer cells derived from patients with ascites. CD36 protein was detectable in all primary ovarian cancer cells tested (Figure 3).

[0071] Example 2 Methods Mice and cell lines All animal work was carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee. Wild-type C57BL / 6J, and GFP transgenic C57BL / 6-Tg(ACTB-EGFP)1Osb / J are obtained from The Jackson Laboratory (Bar Harbor, Maine). CB-17 SCID mice are obtained from Charles River (Wilmington, MA). The cell lines PC3 and PC3M-LN4 have been described previously (14). The human breast cancer cell lines MDA-MB-231 and MDA-MB-LM2 have been described previously (Ryu et al. PLoS one, 6, 2011). The mouse Lewis lung carcinoma cell line LLC / D122, which stably expresses RFP and firefly luciferase, provided by Lea Eisenbach, Wiesmann Institute of Science, Rehovot, Israel (Gupta GP, Massague J. Cancer metastasis: building a framework. Cell. 2006;127:679-95; Gao D, Nolan DJ, Mellick AS, Bambino K, McDonnell K, Mittal V. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science. 2008;319:195-8; and Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev Cancer. 2009;9:239-52), is cultured in DMEM supplemented with 10% fetal bovine serum.

[0072] Tissue microarray and immunohistochemistry Archival specimens (radical prostatectomy specimens or biopsy materials of metastases) are obtained from the files of the Department of Pathology, The Gade Institute, Haukeland University Hospital. Formalin-fixed prostatectomy specimens are embedded in paraffin and examined with whole-mount step sections at 5-mm intervals. Tissue microarrays (TMAs) are constructed by selecting three tissue cores (0.6 mm in diameter) from the area of the highest tumor grade in each case. Thin paraffin sections (5 μm) from the TMA paraffin block were defatted with xylene / ethanol and then subjected to heat-induced microwave epitope retrieval in citrate buffer (pH 6.0) for 20 minutes and incubated with the CD36 antibody at room temperature for 60 minutes. Immunostaining was performed on a DAKO Autostainer (Dako Cytomation, Copenhagen, Denmark) using the EnVision linked polymer method as the detection system. Antigen localization was achieved using the DAB diaminobenzidine peroxidase reaction and counterstained with hematoxylin. Immunostaining was estimated semi-quantitatively, and the staining index (SI) was calculated as the product of the staining intensity (0 - 3) and the percentage of immunoreactive tumor cells (<10% = 1, 10 - 50% = 2, >50% = 3). The staining index (range 0 - 9) is a categorical scale with some variation expected within each category.

[0073] Knockdown of CD36 in tumor cells CD36 levels are reduced in cancer cell lines using retroviral or lentiviral vectors encoding miRNA or shRNA targeting CD36. The knockdown efficiency is tested using qPCR analysis. Total RNA is extracted using the PicoPure RNA extraction kit (Arcturus) according to the manufacturer's protocol. RNA is converted to cDNA using the qScript® cDNA SuperMix (Quanta biosciences). qPCR is performed using primers and the iQTM SYBR Green Master Mix (Biorad, Hercule, CA). The standard protocol of initial denaturation at 95°C for 10 minutes, then 40 cycles of 95°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes, and melting curve analysis, was performed on a BioRad CFX96 Real Time System (BioRad) combined with Bio-Rad-CFX Manager software. The relative abundance of each transcript was calculated using the delta Ct method compared to a control.

[0074] In vitro cell proliferation assay Cell proliferation was measured using the MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) assay. Cells were seeded into 50 μL of growth medium in 96-well culture plates and allowed to adhere overnight. Next, 50 μL of growth medium + 2-fold concentrated treatment reagent was added. After each treatment time point, 10 μL of 5% MTT solution (buffered in PBS) was added to each well. The plates were incubated at 37 °C for an additional 4 hours to metabolically convert the MTT to formazan crystals in the mitochondria of the cells. The formazan crystals were finally solubilized by adding 100 μl of 10% sodium dodecyl sulfate in 50% N-N-dimethylformamide to each microplate well. Absorbance at 550 and 680 nm (corresponding to the formazan salt and reference wavelength, respectively) was measured using a colorimetric microplate reader. Wells containing only complete medium were used as controls. Each experiment was performed twice using six replicates for each drug concentration.

[0075] Metastasis assay, bioluminescence imaging and analysis For experimental metastasis, 7-week-old C57BL / 6 mice were injected via the tail vein with 1×10 5 luciferase-labeled LLC cells. For the injection of syngeneic breast cancer cells, 5×10 6 MDA-MB-231 or its metastatic variant MDA-MB-LM2 cells were injected into the fat pad of CB-17 SCID mice in a volume of 0.1 ml. Tumor growth and lung metastasis (after resection of the primary tumor) were monitored once a week by bioluminescence imaging (Xenogen) of the live animals. For the injection of syngeneic prostate cancer cells, 2×10 6 live LN4 or cells were injected into the prostate of the mice. To determine the burden of metastasis in vivo, mice were anesthetized and injected intraperitoneally with 75 mg / kg of D-luciferin (100 μL of 30 mg / mL in PBS). Metastatic growth was monitored over time using bioluminescence imaging, which was performed on a Xenogen IVIS system incorporating Living Image acquisition and analysis software (Xenogen) using mice in the supine position for 5 minutes after D-luciferin injection. For BLI plots, the photon flux for each mouse was calculated using the same circular region of interest surrounding the chest of the mouse.

[0076] Psap peptide administration 8-week-old mice are treated by intraperitoneal injection at a dose of 30 mg / kg / day for up to 2 weeks with Psap peptide (e.g., DWLPK (SEQ ID NO: 2), DWLP (SEQ ID NO: 3), or a modification thereof) diluted in PBS.

[0077] Results CD36 levels are measured in tissue samples from human subjects with cancer. CD36 levels are knocked down in cancer cell lines, and these cell lines with reduced CD36 are injected into mice. The mice are then administered Psap peptide. The burden of tumor growth and metastasis is monitored. Knockdown of CD36 in cancer cells is expected to reduce the anti-cancer activity of Psap peptide in vivo.

[0078] Example 3 Methods Unless otherwise specified, the methods used in Example 3 are the same as those used in Examples 1 and 2. The cell lines tested for CD36 expression were cell lines of pancreatic cancer (AsPC1), ovarian cancer (DF-14 and ID-8), breast cancer (MDA-MB231 and LM2), prostate cancer (PC3, PC3-M-LN4, LN-CAP, and LN-CAP-LN3), melanoma (B16-BL6), and lung cancer (LLC). All of these cell lines are known in the art and / or are commercially available. Ovarian cancer cells expressing CD36 were treated with either control or thrombospondin (Tsp-1, 100 ng, 500 ng, or 1000 ng), and the percentage of viable cells was measured at 0 hours or 48 hours.

[0079] In a mouse model of ovarian cancer, 1 million ovarian cancer cells expressing luciferase were injected intraperitoneally. Treatment was started 17 days later, and cisplatin (4 mg / kg QOD), Psap peptide dWlP (SEQ ID NO: 47, 40 mg / kg QD), a combination of cisplatin and Psap peptide, or PBS QD was used. Luciferase intensity was measured starting around day 17 and over several days and measured over time. In a mouse model of pancreatic cancer, 1 × 10 6 AsPc1 human pancreatic cells were injected into the pancreas of SCID mice. The mice were treated with either control or Psap peptide dWlP (SEQ ID NO: 47, 20 mg / kg / day or 40 mg / kg / day). Treatment was started on day 25 and continued daily for 21 days. The mice were then euthanized, and the primary tumor mass was measured. The presence or absence of ascites was also measured. For the melanoma mouse model, B16-BL6 cells were injected into the mice. The mice were treated with either Psap peptide dWlP (SEQ ID NO: 47, 10 or 40 mg / kg) or control. Tumor volume was measured over time until approximately 20 - 25 days after cell injection.

[0080] Results Multiple cancer cell lines were tested for CD36 expression. CD36 protein was detected in all cell lines tested and was found to be at high levels particularly in the AsPC1, DF-14, MDA-MB231, and PC3 cell lines (Figure 5). Ovarian cells expressing CD36 were shown to be sensitive to Tsp-1-mediated cell killing in a dose-dependent manner (Figure 6). Two "high" CD36 cell lines (ovarian cancer cells and AsPC pancreatic cancer cells) and one "low" CD36 cell line (B16-B6 cancer cells) were injected into mice to test the effect of the Psap peptide on tumor growth and metastasis. The "high" CD36 cancer models were found to regress in response to treatment with the Psap peptide (Figs. 5 and 7). In the ovarian cancer model, regression of metastatic disease was also shown (Fig. 5). The pancreatic cancer model also showed inhibition of metastasis, as only 1 out of 19 mice treated with the Psap peptide developed ascites, while 4 out of 10 mice treated with the control developed ascites. In the "low" CD36 melanoma model, treatment with the Psap peptide was found to inhibit primary tumor growth but did not cause tumor regression (Fig. 8). These results indicate that "high" CD36 cancers may respond more strongly to Psap peptide treatment (e.g., regression of primary tumors and / or metastases), while "low" CD36 cancers may respond more weakly (e.g., more inhibition rather than regression of primary tumors).

[0081] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the above description, utilize the present disclosure to its fullest extent. Accordingly, the specific embodiments are to be construed as merely illustrative and in no way limiting of the remainder of the disclosure. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to therein. As used herein in the specification and claims, the indefinite articles "a", "an" are to be understood to mean "at least one" unless specifically stated otherwise. From the above description, one of ordinary skill in the art can readily ascertain the essential characteristics of the present disclosure and, without departing from the spirit and scope thereof, can make various changes and modifications to the present disclosure to adapt it to various uses and conditions. Accordingly, other embodiments are also within the scope of the claims.

Claims

**Claim 1** A composition for use in the treatment of a subject having cancer and having an elevated level of CD36 in a sample as compared to a control level, wherein the composition comprises cisplatin and a Psap peptide, and the Psap peptide consists of the amino acid sequence dWlP (SEQ ID NO: 47), said composition. **Claim 2** The composition according to claim 1, wherein the control level is the level of CD36 from non-cancerous cells or tissues obtained from a subject having cancer. **Claim 3** The composition according to claim 1, wherein the control level is the level of CD36 in cells or tissues obtained from a healthy subject or a population of healthy subjects. **Claim 4** The composition according to claim 1, wherein the control level is a predetermined level. **Claim 5** The composition according to claim 1, wherein the level of CD36 is the CD36 protein level. **Claim 6** The composition according to claim 1, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma. **Claim 7** Use of a composition in the manufacture of a medicament for treating a subject having cancer and having an elevated level of CD36 in a sample as compared to a control level, wherein the composition comprises cisplatin and a Psap peptide, and the Psap peptide consists of the amino acid sequence dWlP (SEQ ID NO: 47), said use. **Claim 8** The use according to claim 7, wherein the control level is the level of CD36 from non-cancerous cells or tissues obtained from a subject having cancer. **Claim 9** The use according to claim 7, wherein the control level is the level of CD36 in cells or tissues obtained from a healthy subject or a population of healthy subjects. **Claim 10** The use according to claim 7, wherein the control level is a predetermined level. **Claim 11** The use according to claim 7, wherein the level of CD36 is the CD36 protein level. **Claim 12** The use according to claim 7, wherein the cancer is prostate cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, glioblastoma multiforme, astrocytoma, or melanoma.

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