Methods and compositions relating to therapeutic peptides for cancer therapy

Abstract

Methods and compositions for the treatment of cancer are provided.SOLUTION: Aspects of the present invention provide solutions to problems associated with balancing patient toxicity and broad efficacy of cancer treatments. In particular, embodiments are directed to anti-cancer peptides that exhibit broad anti-cancer effects with limited toxicity to normal or non-cancer cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 782,690, filed December 20, 2018, which is incorporated herein by reference in its entirety.

[0002] I. FIELD OF THE INVENTION Embodiments of the compositions and methods described herein are generally directed to the fields of molecular biology, medicine, and cancer therapy. In particular, embodiments are directed to cancer therapy and anti-cancer peptide compositions. [Background technology]

[0003] II. Description of Related Art Cancer is a mutational, expansive disease that exhibits high genetic heterogeneity across space and time (Stratton et al., Nature, 2009; Vogelstein et al., Science, 2013). Overcoming this heterogeneity, as well as eradicating tumor cells while sparing non-cancerous cells, remains a formidable challenge. For these reasons, it has been difficult to identify agents with broad efficacy across cancer types while maintaining specificity and limiting host toxicity.

[0004] Broad efficacy and specificity are essential properties of innate immunity. The innate immune system evolved to protect against a wide range of infectious pathogens, including bacteria, fungi, and protozoa, whose genetic diversity far exceeds that of cancer. Neutrophils, key effectors of innate immunity, eliminate genetically diverse pathogens and therefore ideally should perform a comparable function in cancer. Indeed, human blood polymorphonuclear neutrophils (PMNs) can kill cancer cells (Sagiv, Cell Rep., 2015; Yan et al., Oncoimmunology, 2014), and their therapeutic potential is being explored in clinical trials aimed at delivering them as cell therapies. Despite the growing interest in human PMNs, the mechanisms by which they kill cancer cells remain incompletely understood.

[0005] In contrast to the anti-cancer function of PMNs, numerous studies have demonstrated that tumor-associated neutrophils (TANs) promote tumorigenesis. This discrepancy may be due to the source and activation state of neutrophils, which may result in significant functional differences (Coffelt et al., Nat Rev Cancer, 2016; Eruslanov et al., Trends Cancer, 2017; Kruger et al., PLoS Pathog., 2015). For example, mouse studies suggest that tumor cells hijack neutrophils to release molecules that promote metastatic spread (Coffelt et al., Nature, 2015; Finisguerra et al., Nature, 2015). Furthermore, increased TAN accumulation is a poor prognostic marker in many cancer types (Coffelt et al., Nat Rev Cancer, 2016; Shen et al., PLoS One, 2014; Powell et al., Immunol., 2016).

[0006] There remains a need for additional anti-cancer compositions and additional therapies for treating cancer that have broad anti-cancer efficacy and limited patient toxicity. Summary of the Invention

[0007] Embodiments of the present invention provide solutions to problems associated with balancing patient toxicity and broad efficacy in cancer treatments. In particular, embodiments are directed to anti-cancer peptides that exhibit broad anti-cancer efficacy with limited toxicity to normal or non-cancerous cells.

[0008] Given that human PMNs release extracellular factors that kill a wide range of pathogens, we sought to explore whether these factors have a similar ability to kill cancer cells. Using this strategy, we used human CD95 (hCD95) as a target for V 220 / A 221 and I 331 / Q 332 ELANE has been identified as a major anticancer protein released by human PMNs, with a mechanism of action involving cleavage of IL-1 at 200 kJ / kcal, liberating a death domain (DD)-containing proteolytic fragment that selectively kills cancer cells. ELANE has been shown to have broad anticancer efficacy and selectivity in multiple models.

[0009] ELANE has several attractive properties in preclinical models. First, its ability to kill a broad range of cancer cells may allow its implementation without knowledge of the genetic characteristics of the cancer cells. Second, its specificity for cancer and non-cancer cells may limit potential toxicity. Third, initial studies suggest that cancer cells have difficulty developing resistance to ELANE. Mechanistically, these properties are proposed to result from ELANE's ability to target CD95. Indeed, the killing program, broad efficacy, specificity, and resistance profile of ELANE were similar to those reported in cancer cells treated with shRNA to reduce CD95 levels (L. Chen et al., Nature, 2010), with one important difference: the proposed mechanism involves a gain-of-function of CD95 (i.e., release of DD-containing fragments) rather than a loss-of-function (i.e., knockdown of CD95). CD95 is known to exert both pro- and anti-apoptotic effects depending on the physiological context (Martin-Villalba, Liorens-Bobadilla, Wollny. Trends Mol Med, 2013), and research has identified ELANE as a potential therapeutic agent utilizing its pro-apoptotic function.

[0010] Our studies demonstrate that ELANE proteolytically liberates the CD95 DD, selectively killing a broad range of cancer cells. Indeed, transient overexpression of full-length CD95 (aa 1-335) or a C-terminal CD95 peptide (aa 212-335) containing both cleavage sites enhanced ELANE-mediated killing in human cancer cells, whereas transient overexpression of the N-terminal domain (aa 1-209) had no effect. However, transient overexpression of these hCD95 proteins / peptides was insufficient to kill cancer cells in the absence of ELANE. To induce apoptosis in the absence of ELANE, expression of C-terminal hCD95 peptides mimicking ELANE cleavage at one or both sites (site 1: aa 221-335, site 2: aa 212-331, or both sites: aa 221-331) was required. These hCD95 peptides were demonstrated to kill many types of human cancer cells without harming non-cancerous cells, supporting the approach of delivering specific CD95 peptides or DNA encoding CD95 peptides for the treatment of many cancers.

[0011] Our data indicate that some human CD95 peptides (containing the ELANE cleavage site) are not toxic to cancer cells but merely enhance the effects of ELANE. Meanwhile, transient expression of human CD95 peptides mimicking ELANE cleavage at one or both sites kills cancer cells in the absence of ELANE. While the C peptide is nontoxic, C1 (mimicking cleavage at site 1), C2 (mimicking cleavage at site 2), and C1-2 (mimicking cleavage at both sites) are all toxic. Furthermore, CD95 N-terminal domain peptides and full-length CD95 are not toxic to cancer cells. Finally, our results demonstrate that expression of the C1-2 peptide in MDA-MB-231 cells induces the same killing mechanism as treatment of these cells with ELANE (i.e., suppression of survival pathways and induction of DNA damage, mitochondrial ROS, and apoptotic effectors).

[0012] To investigate whether expression of the CD95 C1-2 peptide could attenuate tumor growth in vivo, we engineered MDA-MB-231 cancer cells (TNBC) stably expressing either the CD95 C peptide or the C1-2 peptide under the control of a doxycycline-inducible promoter. In vitro, expression of both proteins was induced by doxycycline treatment. Similar to the transient expression system, cells induced to express the C1-2 peptide underwent apoptosis, whereas cells induced to express the C peptide did not. Furthermore, doxycycline-induced expression of the C1-2 peptide in MCF10a cells (non-cancer cells) did not induce apoptosis. Analysis of distinct C1-2 peptide-expressing colonies of MDA-MB-231 cells revealed that the degree of apoptosis was significantly and positively correlated with the level of C1-2 expression. MDA-MB-231 cells expressing doxycycline-inducible C1-2 peptide were injected into the mammary fat pad of mice, causing tumors to grow to approximately 100 mm 3 The mice were allowed to grow to a size of 100 μg / cm. At this point, doxycycline was administered to the mice by IP injection or through the diet. Results showed that induction of C1-2 expression in MDA-MB-231 cells reduced tumor growth and increased intratumoral CD45 expression. + It was shown to increase the number of immune cells.

[0013] Certain embodiments are directed to therapeutic or anti-cancer compositions comprising various combinations of anti-cancer peptides or variants thereof, expression vectors or expression cassettes encoding same. In certain aspects, the peptide compositions comprise the amino acid sequence Certain embodiments may include one or more peptides comprising, consisting essentially of, or consisting of SEQ ID NO:2, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124 contiguous amino acids (including all values ​​and ranges therebetween), or variants thereof having an amino acid sequence that is 90, 92, 94, 96, 98, 99 to 100% identical (including all values ​​and ranges therebetween) to the amino acid sequence of the peptide or peptides.The functional segment of the anti-cancer peptide is amino acids 1, 2, 3, 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 of SEQ ID NO:2. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, Starting at 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 and amino acid 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, It may end in 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124. In certain aspects, the anti-cancer peptides described herein may be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by inclusion of heterologous peptide sequences at the amino or carboxy termini of the peptide.

[0014] The anti-cancer peptides may be individually present in the composition at a concentration of 1, 50, 100, 150, 200, 250, 300, 350, 400, 450 μg / mL to 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 μg / mL; or 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 mg / mL to 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 mg / mL (including all ranges and values ​​therebetween).

[0015] Certain embodiments are directed to one or more anti-cancer peptides and / or compositions comprising same. In certain aspects, one or more peptide components have 90-100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a functional segment thereof. In certain aspects, the peptide has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. The peptide component can be attached to a substrate. In certain aspects, the substrate is a delivery vehicle. The delivery vehicle can be a nanoparticle (e.g., a liposome).

[0016] Certain embodiments are directed to methods for treating cancer, including administering to a subject with cancer an effective amount of a therapeutic composition comprising one or more peptides 90-100% identical to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof. The therapeutic composition can be administered by injection. In certain aspects, the therapeutic composition can be administered intratumorally (e.g., by intratumoral injection). The cancer can be bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. In certain aspects, the peptide is present at a dose between 0.001 mg / kg body weight and 10 mg / kg body weight, preferably at least 0.1-5 mg / kg body weight, up to 0.1-5 mg / kg body weight, or about 0.1-5 mg / kg body weight, most preferably 0.5-1 mg / kg body weight. In other aspects, the method can include administering a second anti-cancer therapy. The second anti-cancer therapy can be chemotherapy, radiation therapy, immunotherapy (e.g., checkpoint inhibitors), or anti-hormonal therapy.

[0017] Certain embodiments are directed to methods for inducing apoptosis in cancer cells, comprising contacting the cancer cells with an effective amount of one or more peptides having 90-100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof. The cancer cells may be from a bladder, blood, bone, bone marrow, brain / nervous system, breast, colorectal, esophageal, gastrointestinal, head, kidney, liver, lung, nasopharyngeal, cervical, ovarian, pancreatic, prostate, skin, stomach, testis, tongue, or uterine tumor.

[0018] The anti-cancer peptides described herein are It was discovered as a fragment of human CD95, isoform 1, having the amino acid sequence of TIFF2026021440000002.tif41159 (SEQ ID NO:1, Accession No. NP_000034.1, which is incorporated by reference as of the filing date of this application). Amino acids 1-25 are the signal peptide, and the mature form contains amino acids 26-335. Certain embodiments are directed to one or more peptides or variants thereof having an amino acid sequence that is 90, 92, 94, 96, 98, 99 to 100% identical (including all values ​​and ranges therebetween) over 10, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or 255 consecutive amino acids (including all values ​​and ranges therebetween) to SEQ ID NO: 1. In certain aspects, the anti-cancer peptides described herein can be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by inclusion of heterologous peptide sequences at the amino or carboxy termini of the peptide. The N-terminus referred to herein is amino acids 1 to 209 of (SEQ ID NO:1), and the C-terminus referred to herein is amino acids 212 to 335 of (SEQ ID NO:1).

[0019] The compositions described herein can kill a wide variety of cancer cells, regardless of the genetic characteristics of the cancer cells. Thus, the compositions described herein can treat various types of cancer. In certain aspects, the cancer is bladder cancer, blood cancer, bone cancer (e.g., osteosarcoma), bone marrow cancer (e.g., leukemia), brain / nervous system cancer (e.g., neuroblastoma, glioblastoma), breast cancer, colorectal cancer (e.g., colon cancer), esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer), nasopharyngeal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (e.g., melanoma), stomach cancer, testicular cancer, tongue cancer, or uterine cancer. The compositions described herein are toxic to cancer cells, but have no or limited toxicity to non-cancer cells.

[0020] Certain embodiments are directed to methods for killing cancer cells by contacting cancer cells or tumors with an effective amount of a therapeutic anti-cancer peptide composition. In certain aspects, the anti-cancer peptide composition is administered to a patient with cancer. In certain aspects, the cancer is bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. In certain aspects, the anti-cancer peptide composition may further comprise or be administered with an additional anti-cancer agent to enhance the effectiveness of the peptide composition. In certain aspects, these additional anti-cancer agents may be administered before; during; after; before and between; before and after; between and after; or before, during, and after administration of the polypeptide composition. In certain aspects, the compositions described herein can be administered before, during, after, before and during, before and after, during and after, or before, during and after immunotherapy, chemotherapy, antihormonal therapy or radiation therapy.In certain aspects, the polypeptide compositions described herein are administered in combination with chemotherapy, such as doxorubicin and / or paclitaxel.

[0021] Certain embodiments are directed to expression vectors or expression cassettes encoding one or more anti-cancer peptides. A subject can be administered such a vector or cassette for the purpose of expressing the one or more anti-cancer peptides at or near the target cancer or tumor.

[0022] The term "effective amount" means an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0023] An "effective amount" of an anti-cancer agent (e.g., a peptide composition described herein) with respect to reducing cancer cell growth means an amount that is capable of reducing the growth of certain cancer or tumor cells to some extent. This term includes an amount that is capable of causing growth inhibition, cytostatic and / or cytotoxic effects, and / or apoptosis of cancer or tumor cells.

[0024] A "therapeutically effective amount" with respect to cancer treatment means an amount capable of producing one or more of the following effects: (1) some inhibition of cancer or tumor growth, including slowing or complete cessation of growth; (2) a reduction in the number of cancer or tumor cells; (3) a reduction in tumor size; (4) inhibition (i.e., reduction, slowing, or complete cessation) of cancer or tumor cell invasion into peripheral organs; (5) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (6) an enhancement of an antitumor immune response, which may, but need not, result in tumor regression or rejection; or (7) some alleviation of one or more symptoms associated with cancer or tumor. A therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of one or more anticancer agents to induce a desired response in the individual. A "therapeutically effective amount" is also an amount in which any toxic or adverse effects are outweighed by the therapeutically beneficial effects.

[0025] The phrases "treating cancer" and "treatment of cancer" mean reducing, decreasing, or inhibiting the replication of cancer cells; reducing, decreasing, or inhibiting the spread of cancer (formation of metastases); reducing tumor size; reducing the number of tumors (i.e., reducing tumor burden); reducing or decreasing the number of cancerous cells in the body; preventing the recurrence of cancer after surgical resection or other anti-cancer therapy; or ameliorating or alleviating the symptoms of disease caused by cancer.

[0026] The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that (in conjunction with the host cell) direct and / or control the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control the transcription, translation of the coding regions operably linked thereto, and affect RNA splicing of introns, if present.

[0027] The term "expression cassette" refers to a nucleotide sequence containing at least one coding sequence along with sequence elements that direct the initiation and termination of transcription. Expression cassettes can contain additional sequences, including, but not limited to, promoters, enhancers, and sequences involved in post-transcriptional or post-translational processes.

[0028] The terms "inhibit," "reduce," or "prevent," or any variation of these terms, as used in the claims and / or this specification, include any measurable decrease or complete inhibition to achieve a desired result.

[0029] The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0030] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists among test subjects.

[0031] Use of the term "or" in the claims is used to mean "and / or," unless expressly stated to refer to alternatives only or where the alternatives are mutually exclusive, but the present disclosure supports a definition that refers to alternatives only and "and / or." It is also contemplated that anything listed with the term "or" may also be expressly excluded.

[0032] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unspecified elements or method steps.

[0033] The terms "consisting of" or "consisting essentially of" may be used in place of the term "comprising" in any embodiment discussed herein.

[0034] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition, and vice versa. Additionally, compositions and kits can be used to achieve methods.

[0035] Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect applies to other aspects as well, and vice versa. An embodiment in the Examples section is understood to be an embodiment applicable to all aspects. Further objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [The present invention 1001] A method for treating cancer, comprising administering to a subject having cancer an effective amount of a therapeutic composition comprising one or more peptides that are 90 to 100% identical to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof. [The present invention 1002] 1001. The method of claim 1001, wherein said therapeutic composition is administered by injection. [The present invention 1003] 1001. The method of claim 1001, wherein said therapeutic composition is administered intratumorally. [The present invention 1004] 1001. The method of claim 1001, wherein the cancer is bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer. [The present invention 1005] The method of the present invention 1001, wherein the peptide is present in a dose between 0.001 mg / kg body weight and 10 mg / kg body weight, preferably at least 0.1-5 mg / kg body weight, at most 0.1-5 mg / kg body weight, or about 0.1-5 mg / kg body weight, most preferably 0.5-1 mg / kg body weight. [The present invention 1006] 1001. The method of claim 1001, further comprising administering a second anti-cancer treatment. [The present invention 1007] 1006. The method of claim 10, wherein said second anti-cancer treatment is chemotherapy, radiation therapy, immunotherapy, or anti-hormonal therapy. [The present invention 1008] 1006. The method of claim 10, wherein said second anti-cancer treatment is ELANE protease. [The present invention 1009] A method for inducing apoptosis in cancer cells, comprising contacting the cancer cells with an effective amount of one or more peptides having 90 to 100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof. [The present invention 1010] The method of claim 1009, wherein the cancer cells are present in a tumor of the bladder, blood, bone, bone marrow, brain / nervous system, breast, colorectal, esophageal, gastrointestinal, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. [The present invention 1011] An anti-cancer peptide composition comprising one or more peptide components having 90-100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof. [The present invention 1012] 1011. The composition of claim 1011, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:2. [The present invention 1013] 1011. The composition of claim 1011, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:3. [The present invention 1014] 1011. The composition of claim 1011, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:4. [The present invention 1015] The composition of claim 1011, wherein the peptide component is bound to a substrate. [The present invention 1016] The composition of claim 1015, wherein said substrate is a delivery vehicle. [The present invention 1017] The composition of claim 1016, wherein said delivery vehicle is a nanoparticle. [Brief explanation of the drawings]

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. [Figure 1A] ELANE cleaves CD95 to selectively kill cancer cells. (A): Heatmap summary of the effects of ELANE on survival, stress, and apoptosis pathways in cancer and healthy cells. See Figure 2 for quantification. [Figure 1B] ELANE cleaves CD95 and selectively kills cancer cells. (B): Cancer cells were treated with ELANE (100 nM), and CD95 cleavage was assessed by Western blotting using an anti-N-terminal antibody. *, low molecular weight CD95 fragment. [Figure 1C] ELANE cleaves CD95 and selectively kills cancer cells. (C): Cancer cells were treated with ELANE (100 nM), and CD95 cleavage was assessed by Western blotting using an anti-C-terminal antibody. *, low molecular weight CD95 fragment. [Figure 1D]ELANE cleaves CD95 and selectively kills cancer cells. (D): Cancer or non-cancer cells were transduced to overexpress CD95 protein, and the effect on ELANE-mediated apoptosis was quantified by ANXA5 at 30 minutes. n=2 / group. See Figure 1I for transduction and CD95 overexpression. [Figure 1E] ELANE cleaves CD95 and selectively kills cancer cells. (E): Cleavage of recombinant human CD95 N-terminal (aa 1-173) or C-terminal (aa 212-335) proteins by ELANE was assessed by SDS-PAGE and Coomassie blue staining. [Figure 1F] ELANE cleaves CD95 and selectively kills cancer cells. (F): The band from (E) was digested with trypsin and analyzed by mass spectrometry to identify putative ELANE cleavage sites (i.e., peptides with nontryptic ends). Major nontryptic peptides were quantified by ion chromatogram. [Figure 1G] ELANE cleaves CD95 to selectively kill cancer cells. (G): Schematic of ELANE cleavage sites in CD95. The heatmap shows the overlap between the two CD95 cleavage sites and the sequence specificity of ELANE (at URL web.expasy.org). [Figure 1H] ELANE cleaves CD95 and selectively kills cancer cells. (H): Cleavage of recombinant peptides corresponding to aa 214-231 and aa 317-335 of CD95 by ELANE was monitored by mass spectrometry. [Figure 1I] ELANE cleaves CD95 and selectively kills cancer cells. (I) Cancer or non-cancer cells were treated with fluorescein-labeled ELANE for 10 minutes, and uptake was visualized by immunofluorescence. [Figure 1J]ELANE cleaves CD95 and selectively kills cancer cells. (J): ELANE catalytic activity in cancer cell lysates after 30 minutes of exposure to ELANE in the presence and absence of Dynasore (60 μM, an endocytosis inhibitor). Effect of Dynasore on the ability of ELANE to kill cancer cells (measured by calcein AM). n=6 / group. [Figure 1K] ELANE cleaves CD95 and selectively kills cancer cells. (K): Cancer or non-cancer cells were transduced to overexpress various CD95 proteins, and cell viability in the absence of ELANE was determined using calcein AM. n=10 / group. *, p<0.05 Student's t-test. [Figure 2A] Overexpression of CD95 protein enhances cancer cell killing by ELANE. Polycistronic adenoviral vectors were prepared to express human and mouse CD95 sequences, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRS) and dTomato sequences, under the control of a cytomegalovirus (CMV) promoter. Human and mouse cancer or non-cancer cells were transduced to overexpress full-length CD95, N-terminal CD95 (human: aa 1-209; mouse: aa 1-204), or C-terminal CD95 (human: aa 212-335; mouse: aa 204-327). (A): Transduction efficiency quantified by dTomato (left). CD95 levels in dTomato- and dTomato+ cells quantified by geometric mean index (MFI) (center). ANXA5 levels in dTomato- and dTomato+ cells after 30 min of treatment with ELANE (40 nM) (right). Representative data from A549 cells are shown. [Figure 2B]Overexpression of CD95 protein enhances cancer cell killing by ELANE. Polycistronic adenoviral vectors were prepared to express human and mouse CD95 sequences, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (RIS) and dTomato sequence, under the control of the cytomegalovirus (CMV) promoter. Human and mouse cancer or non-cancer cells were transduced to overexpress full-length CD95, N-terminal CD95 (human: aa 1-209; mouse: aa 1-204), or C-terminal CD95 (human: aa 212-335; mouse: aa 204-327). (B): Quantification of transduction efficiency of cancer and non-cancer cells. [Figure 2C] Overexpression of CD95 protein enhances cancer cell killing by ELANE. Polycistronic adenoviral vectors were prepared to express human and mouse CD95 sequences, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRS) and dTomato sequence, under the control of the cytomegalovirus (CMV) promoter. Human and mouse cancer or non-cancer cells were transduced to overexpress full-length CD95, N-terminal CD95 (human: aa 1-209; mouse: aa 1-204), or C-terminal CD95 (human: aa 212-335; mouse: aa 204-327). (C): Quantification of relative CD95 overexpression in cancer and non-cancer cells. [Figure 2D]Overexpression of CD95 protein enhances cancer cell killing by ELANE. Polycistronic adenoviral vectors were prepared to express human and mouse CD95 sequences, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRS) and dTomato sequence, under the control of the cytomegalovirus (CMV) promoter. Human and mouse cancer or non-cancer cells were transduced to overexpress full-length CD95, N-terminal CD95 (human: aa 1-209; mouse: aa 1-204), or C-terminal CD95 (human: aa 212-335; mouse: aa 204-327). (D): Quantification of ANXA5 levels after ELANE treatment (40 nM, 30 min) in mouse cancer cell lines (E0771, B16F10). *, p<0.05 Student's t-test. [Figure 3] Efficacy of C1-2 peptide. Heat map of the effect of C1-2 expression on survival, stress, and apoptosis pathways in cancer and non-cancer cells. [Figure 4] Inducible expression system. Scheme of the doxycycline-inducible Tet-on system for expression of either C (aa. 157-335) or C1-2 (aa. 221-331). C1-2 (DD-containing fragment) upon addition of doxycycline (both in vitro and in vivo). [Figure 5]Figures 5A-5C. Confirmation of C1-2 expression and selective cancer cell death. (A) MDA-MB-231 cells transduced with Tet-on C or C1-2 were treated with 2 μg / mL doxycycline (2 μg / mL) for 24 hours. Cell lysates were collected and Western blotted. Expression of C or C1-2 was confirmed. (B) MDA-MB-231 cells transduced with Tet-on C or C1-2 were treated with 0.2 or 2 μg / mL doxycycline for 72 hours. Cell viability was measured using calcein AM. Expression of C1-2, but not C, led to MDA-MB-231 cell death. (C) MCF10A cells transduced with Tet-on C1-2 were treated with 2 μg / mL doxycycline. Cell viability was measured at various time points using calcein AM. Expression of C1-2 in MCF10A cells did not result in cell death. [Figure 6] Figures 6A-6C. In vitro study of MDA-MB-231 cell death after C1-2 induction. (A) Various single colonies of MDA-MB-231 cells transduced with Tet-on C1-2 were treated with 2 μg / mL doxycycline (2 μg / mL) for 72 hours. Cell viability was measured using calcein AM. Different colonies exhibited different susceptibility to death after C1-2 induction. (B) C1-2 expression was measured by Western blot after doxycycline treatment in various single colonies of MDA-MB-231 cells transduced with Tet-on C1-2 (C), and levels at 24 hours were correlated with % death (normalized by full-length). C1-2 expression correlates with MDA-MB-231 cell death. (C) Immunoblot of C1-2 and full-length (FL) CD95 expression in various colonies. [Figure 7] Figures 7A-7C. In vivo study of MDA-MB-231 tumor regression after C1-2 induction. (A) Tumor weight. (B) Tumor cell count (million) (after ficoll gradient). (C) Percent immune cells in the tumor (mouse CD45+) measured by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention ELANE cleaves CD95 to release proteolytic fragments that selectively kill a wide range of cancer cells. These results, combined with previous studies (Chen et al., Nature, 2010; Hadji et al., Cell Rep., 2014; Peter et al., Cell Death Differ., 2015), clearly demonstrate the critical and selective importance of CD95 for cancer cell viability. From a therapeutic perspective, delivering CD95 fragments to tumors can overcome defense mechanisms and kill cancer cells through a genotype-independent mechanism with a broad therapeutic window. CD95 degradation (generation of CD9 fragments) has been identified as the mechanism of action by ELANE for cancer cell killing, further demonstrating that a wide range of proteases can mimic ELANE's CD95-degrading and cancer cell-killing properties. Aspects of the present invention are based, in part, on the following findings. Treatment of ELANE or neutrophil-conditioned medium with α-1 antitrypsin or PMSF, two irreversible, noncompetitive ELANE inhibitors, protects cancer cells from apoptosis. ELANE cleaves the C-terminal domain of purified CD95. Importantly, this cleavage pattern differs from that produced by MMP7, which has previously been shown to cleave the extracellular N-terminal domain of CD95 and protect cancer cells from FASL-mediated apoptosis (Strand et al., Oncogene, 2004).

[0038] Certain embodiments are directed to therapeutic or anti-cancer compositions comprising various combinations of anti-cancer peptides or variants thereof, expression vectors or expression cassettes encoding same. In certain aspects, the peptide compositions comprise the amino acid sequence Certain embodiments may include one or more peptides comprising, consisting essentially of, or consisting of SEQ ID NO: 2, 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124 contiguous amino acids (including all values ​​and ranges therebetween), or variants thereof.The functional segment of the anti-cancer peptide is amino acids 1, 2, 3, 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 of SEQ ID NO:2. 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, Starting at 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 and amino acid 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, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, It may end in 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124. In certain aspects, the anti-cancer peptides described herein may be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by inclusion of heterologous peptide sequences at the amino or carboxy termini of the peptide.

[0039] I. Polypeptide Compositions and Formulations In certain embodiments, polypeptides and peptides include polypeptides having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and / or SEQ ID NO:5, as well as functional segments thereof. A "polypeptide" refers to any peptide or protein comprising amino acids linked by peptide bonds or modified peptide bonds. A "polypeptide" can include short polypeptides, including peptides, oligopeptides, or oligomers, and longer polypeptides, including proteins. A polypeptide can contain amino acids other than the 20 genetically encoded amino acids. A "polypeptide" includes amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification or other synthetic techniques well known in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxy termini. It will be understood that the same type of modification may be present in the same or varying degrees at several sites within a given peptide. A given polypeptide may also contain many types of modifications. Modifications include terminal fusions (N- and / or C-terminus), acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids to proteins via transfer RNA such as arginylation, and ubiquitination.

[0040] There are a wide variety of detectable labels that can be attached to polypeptides and their variants. For flow cytometry applications for both extracellular and intracellular detection, common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, fluorescence resonance energy tandem fluorophores such as PerCPCy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7. Other fluorophores include, among others, Alexa Fluor® 350, Alexa Fluor® 488, Alexa 25 Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, OR, USA), BODIPY dyes, such as BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY Examples of suitable dyes include 630 / 650, BODIPY 650 / 665, etc., Cascade Blue, Cascade Yellow, dansyl, Lissamine Rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR, USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7 can be used to label the polypeptide with biotin for secondary detection using labeled avidin, streptavidin, captavidin, or neutravidin. The polypeptide can be 33P, 32 P, 35 S, 3 H, and 125 The polypeptide may be labeled with a radioisotope such as I. As another example, if the polypeptide is to be used for targeted radiotherapy, the label may be 3 H, 228 Th, 227 Ac, 225 Ac, 223 Ra, 213 Bi, 212 Pb, 212 Bi, 211 At, 203 Pb, 194 Os, 188 Re, 186 Re, 153 Sm, 149 Tb, 131 I, 125 I, 111 In, 105 Rh, 99 mTc, 97 Ru, 90 Y, 90 Sr, 88 Y, 72 Se, 67 Cu, or 47 It may be Sc.

[0041] The term "isolated" can refer to a nucleic acid or polypeptide that is substantially free of its original source cellular material, bacterial material, viral material, or culture medium (if produced by recombinant DNA technology), or chemical precursors or other chemicals (if chemically synthesized). Furthermore, an isolated polypeptide refers to one that can be administered to a subject as an isolated polypeptide; in other words, a polypeptide cannot simply be considered "isolated" if it is attached to a column or embedded in a gel. Furthermore, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that is not naturally occurring as a fragment and / or is not typically in a functional state.

[0042] The term "amino acid" or "residue" should be understood to mean a compound containing an amino group (NH), a carboxylic acid group (COOH), and any of a variety of side groups, having the basic formula NHCHRCOOH, and linked together by peptide bonds to form proteins. Amino acids can be, for example, acidic, basic, aromatic, polar, or derivative. Non-standard amino acids are sometimes referred to as "non-canonical amino acids." Amino acids are naturally found in α- and L-forms, although β- and D-forms of amino acids can also be prepared.

[0043] A one-letter abbreviation system is often applied to designate the identities of the 20 "canonical" amino acid residues commonly incorporated into naturally occurring peptides and proteins, and these designations are well known in the art. Such one-letter abbreviations are fully interchangeable in meaning with the three-letter abbreviations or unabbreviated amino acid names. Canonical amino acids and their three-letter and one-letter codes include alanine (Ala) A, glutamine (Gln) Q, leucine (Leu) L, serine (Ser) S, arginine (Arg) R, glutamic acid (Glu) E, lysine (Lys) K, threonine (Thr) T, asparagine (Asn) N, glycine (Gly) G, methionine (Met) M, tryptophan (Trp) W, aspartic acid (Asp) D, histidine (His) H, phenylalanine (Phe) F, tyrosine (Tyr) Y, cysteine ​​(Cys) C, isoleucine (Ile) I, proline (Pro) P, and valine (Val) V.

[0044] Certain embodiments also include variants of the polypeptides described herein. Variants of the disclosed polypeptides can be made by adding or inserting amino acids, deleting or substituting amino acids, and / or chemically derivatizing amino acid residues within the polypeptide sequence. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art following the guidelines provided herein to improve stability while maintaining or enhancing the efficacy of the polypeptide. In certain embodiments, conservative amino acid substitutions can include unnatural amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in a biological system.

[0045] Conservative modifications may result in peptides with similar functional, physical, and chemical characteristics to the peptides in which such modifications are made. In contrast, substantial modifications of the functional and / or chemical characteristics of a peptide may be achieved by selecting substitutions within the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the region of the substitution, e.g., as an α-helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the size of the molecule. For example, a "conservative amino acid substitution" may involve substituting a non-natural amino acid residue for a natural amino acid residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.

[0046] Protein expression via recombinant DNA and / or RNA, as well as protein engineering techniques, or any other method of peptide preparation, are applicable to the production of the polypeptides disclosed herein or their expression in target cells or tissues. The term "recombinant" should be understood to mean that a substance (e.g., a nucleic acid or polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. Modifications can be made to a substance in or removed from its natural environment or state. For example, a "recombinant nucleic acid" is one produced by recombining nucleic acids, e.g., during cloning, DNA shuffling, or other well-known molecular biology techniques. Examples of such molecular biology techniques can be found in Maniatis et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. A "recombinant DNA molecule" is composed of segments of DNA linked together using such molecular biology techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and / or expresses a recombinant nucleic acid.

[0047] The polypeptides can be produced in transformed host cells according to methods known to those skilled in the art. Briefly, a recombinant DNA molecule or construct encoding the peptide is prepared. Methods for preparing such DNA molecules are well known in the art. For example, the peptide-encoding sequence can be excised from the DNA using appropriate restriction enzymes. Any of a large number of available and well-known host cells can be used in implementing various embodiments. The selection of a particular host depends on many factors, including, for example, compatibility with the selected expression vector, toxicity of the polypeptide encoded by the DNA molecule, rate of transformation, ease of polypeptide recovery, expression characteristics, biosafety, and cost. These factors should be balanced with the understanding that not all hosts are equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial host cells in culture include bacteria (e.g., Escherichia coli species), yeast (e.g., Saccharomyces species), as well as other fungal cells, insect cells, plant cells, and mammalian (including human) cells, such as CHO cells and HEK293 cells. Modifications can also be made at the DNA level. The DNA sequence encoding the peptide may be altered to use codons more compatible with the selected host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to remove restriction sites or include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The transformed host is then cultured and purified. The host cell can be cultured under conventional fermentation conditions to express the desired polypeptide. In addition, the DNA optionally further encodes a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed polypeptide 5' of the coding region of the fusion protein.

[0048] The polypeptides can also be produced by synthetic methods. Solid-phase synthesis is the most cost-effective method for producing small peptides, and can therefore be used as a technique for producing individual polypeptides. For example, well-known solid-phase synthesis techniques involve the use of protecting groups, linkers, and solid-phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, the use of scavengers, and other aspects of solid-phase peptide synthesis. Suitable techniques are well known in the art. See, e.g., Merrifield, Chem. Polypeptides, Katsoyannis and Panayotis eds., pp. 335-361, 1973; Merrifield, J. Am. Chem. Soc. 85: 2149, 1963; Davis et al., Biochem. Intl. 10:394-414, 1985; Stewart and Young, Solid Phase Peptide Synthesis, 1969; U.S. Pat. No. 3,941,763; Finn et al., The Proteins, 3rd ed., 2:105-253, 1976; and Erickson et al., The Proteins, 3rd ed., 2: 257-527, 1976; "Protecting Groups in Organic Synthesis," 3rd ed., T.W. Greene and P.G.M. Wuts, Eds., John Wiley & Sons, Inc., 1999;NovaBiochem Catalog, 2000;“Synthetic Peptides, A User's Guide,” GA Grant, Ed., WH Freeman & Company, New York, NY, 1992;“Advanced Chemtech Handbook of Combinatorial & Solid Phase Organic Chemistry,” WD Bennet, JW Christensen, LK Hamaker, ML Peterson, MR Rhodes, and H.H. Saneii, Eds.See, e.g., Advanced Chemtech, 1998; “Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds., Oxford Press, 2000; G. B. Fields et al., Synthetic Peptides: A User's Guide, 77-183, 1990.

[0049] A composition comprising a polypeptide covalently linked, attached, or bound to another peptide, vehicle (e.g., carrier), or half-life extending moiety directly or indirectly via a linker moiety is a "conjugate" or "conjugated" molecule, regardless of whether the conjugation is by chemical means (e.g., post-translationally or post-synthetically) or by recombinant fusion. Conjugation of the polypeptide may be via the N-terminus and / or C-terminus of the polypeptide, or may be inserted into the primary amino acid sequence of the peptide. Due to the specificity of the polypeptide for cancer cells, the polypeptide can be linked to other cytotoxic moieties to facilitate specific delivery to cancer cells and enhance the cytotoxicity of the polypeptides described herein. Linkers can be used to create fusion proteins that allow the introduction of additional moieties to enhance the killing or localization of the polypeptide. Particular moieties of interest may include chemotherapeutic agents, proapoptotic factors, targeted therapeutic agents (e.g., kinase inhibitors, etc.), or other agents that promote killing.

[0050] In some embodiments, one, two, three, or four polypeptides are bound to or encapsulated in the same or different delivery vehicles, such as carriers (e.g., particles) or liposomes. In some embodiments, the binding of the polypeptides to the carriers involves one or more covalent and / or non-covalent interactions. In one embodiment, the carriers are metallic or polymeric particles. In one embodiment, the carriers are liposomes. The size of the particles can be microscopic or nanoscale. In certain aspects, the particles have a diameter of at least 0.1 μm, at most 0.1 μm, or about 0.1 μm to at least 10 μm, at most 10 μm, or about 10 μm. In another aspect, the particles have an average diameter of at least 0.3 μm, at most 0.3 μm, or from about 0.3 μm to at least 5 μm, at most 5 μm, or about 5 μm, from 0.5 μm to at least 3 μm, at most 3 μm, or about 3 μm, or from 0.2 μm to at least 2 μm, at most 2 μm, or about 2 μm.In certain aspects, the particles are at least 0.1 μm, at most 0.1 μm, or about 0.1 μm, or at least 0.2 μm, at most 0.2 μm, or about 0.2 μm, or at least 0.3 μm, at most 0.3 μm, or about 0.3 μm, or at least 0.4 μm, at most 0.4 μm, or about 0.4 μm, or at least 0.5 μm, at most 0.5 μm, or about 0.5 μm, or at least 1.0 μm, at most 1.0 μm, or about 1.0 μm, or at least 1.5 μm, at most 1.5 μm, or about 1.5 μm, or at least They may have an average diameter of 2.0 μm, up to 2.0 μm, or about 2.0 μm, or at least 2.5 μm, up to 2.5 μm, or about 2.5 μm, or at least 3.0 μm, up to 3.0 μm, or about 3.0 μm, or at least 3.5 μm, up to 3.5 μm, or about 3.5 μm, or at least 4.0 μm, up to 4.0 μm, or about 4.0 μm, or at least 4.5 μm, up to 4.5 μm, or about 4.5 μm, or at least 5.0 μm, up to 5.0 μm, or about 5.0 μm (including all values ​​and ranges therebetween).

[0051] In some embodiments, the charge of the carrier (e.g., positive, negative, neutral) is selected to confer application-specific benefits (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier has a net neutral or negative charge (e.g., to reduce non-specific binding to cell surfaces, which generally carry a net negative charge). In some cases, the carrier is coupled to multiple polypeptides, and the carrier can have 2, 3, 4, 5, 6, 7, 8, 9, 10... 20... 50... 100, or more copies of a particular polypeptide or combination of polypeptides exposed on its surface. In some embodiments, the carrier displays a single type of polypeptide. In some embodiments, the carrier displays multiple different polypeptides on its surface.

[0052] As used herein, the terms "packaged," "encapsulated," and "entrapped" refer to the incorporation of or association of a polypeptide within a liposome or similar vesicle. The polypeptide may be associated with the lipid bilayer or may reside in the aqueous interior of the liposome, or both.

[0053] The liposomes can be formed from standard vesicle-forming lipids, typically containing neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipid is generally guided by considerations such as liposome size and stability in the bloodstream. Various types of lipids can be used to form liposomes. For example, the amphipathic lipids used can be zwitterionic, acidic, or cationic. Examples of zwitterionic amphipathic lipids include phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, etc. Examples of acidic amphipathic lipids include phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, etc. Examples of cationic amphipathic lipids include diacyltrimethylammoniumpropane, diacyldimethylammoniumpropane, stearylamine, etc. Examples of neutral lipids include diglycerides, such as diolein, dipalmitolein, and mixed caprylin-capric acid; triglycerides, such as triolein, tripalmitolein, trilinolein, tricaprylin, and trilaurin; and combinations thereof. In addition, cholesterol or plant sterols are used, for example, to prepare multivesicular liposomes.

[0054] Various methods are available for preparing liposomes, such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Patent Nos. 4,235,871; 4,501,728; and 4,837,028, all of which are incorporated herein by reference. One method produces multilamellar vesicles of heterogeneous sizes. In this method, vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or inert gas to form a lipid film. Alternatively, lipids may be dissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture in a powder form that is more easily hydrated. This film or powder is then coated with an aqueous buffer and allowed to hydrate, typically with stirring, for 15 to 60 minutes. The size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous stirring conditions or by adding a surfactant, such as deoxycholic acid.

[0055] Multilamellar liposomes are formed by agitating the dispersion, for example, preferably by using a thin film evaporator or by shaking or vortex mixing.Unilamellar vesicles are formed by applying shear force to the aqueous dispersion of lipid solid phase, for example, by using ultrasonic treatment or a microfluidizer such as a homogenizer or French press.Shear force can also be applied by injection, freezing and thawing, dialysis of surfactant solution from lipids, or other known methods used to prepare liposomes.Liposome size can be controlled using various known techniques, including controlling the duration of shear force.

[0056] "Unilamellar liposomes," also known as "unilamellar vesicles," are spherical vesicles containing a lipid bilayer membrane that defines a single, closed aqueous compartment. The bilayer membrane comprises two lipid layers (or "leaflets"): an inner layer and an outer layer. The outer layer of lipid molecules is oriented with their hydrophilic heads facing the external aqueous environment and their hydrophobic tails pointing downward toward the interior of the liposome. The inner layer of lipids is located directly below the outer layer, with the lipids oriented with their heads facing the aqueous interior of the liposome and their tails pointing toward the tails of the outer layer of lipids.

[0057] "Multilamellar liposomes," also called "multilamellar vesicles" or "multilamellar vesicles," contain two or more lipid bilayer membranes that define two or more enclosed aqueous compartments. The membranes are arranged concentrically, like an onion, with the different membranes separated by aqueous compartments.

[0058] II. Pharmaceutical Formulations and Administration Some embodiments relate to compositions comprising one, two, three, four or more anti-cancer peptides or their variants or functional segments, together with one or more of pharmaceutically acceptable diluents; carriers; solubilizers; emulsifiers; and / or preservatives.Such compositions can contain an effective amount of at least one anti-cancer drug or complex.Therefore, also included is the use of one or more anti-cancer drugs as described herein in the preparation of pharmaceutical compositions of medicines.Such compositions can be used in the treatment of various cancers.

[0059] The anticancer agent can be formulated into a therapeutic composition in various dosage forms, including, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred dosage form depends on the mode of administration and the specific disease to be targeted. The composition also preferably includes a pharmaceutically acceptable vehicle, carrier, or adjuvant well known in the art.

[0060] Acceptable formulation ingredients for pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the anticancer agent provided, the composition may contain ingredients to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissociation or release, adsorption, or permeability of the composition. Suitable materials for formulating pharmaceutical compositions include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as acetic acid, boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (ethylenediaminetetraacetic acid (EDTA) etc.); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, etc.) solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapar); stability enhancers (such as sucrose or sorbitol); isotonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants.(See Remington's Pharmaceutical Sciences, 18th Ed., (AR Gennaro, ed.), 1990, Mack Publishing Company, incorporated herein by reference).

[0061] The formulation components are present in concentrations acceptable to the site of administration. A buffer is advantageously used to maintain the composition at physiological pH or slightly lower, typically within a pH range of at least 4.0, up to 4.0, or about 4.0 to at least 8.5, up to 8.5, or about 8.5, or alternatively within a pH range of at least 5.0 to 8.0, up to 5.0 to 8.0, or about 5.0 to 8.0 (including all values ​​and ranges therebetween). The pharmaceutical composition may comprise a TRIS buffer of at least pH 6.5-8.5, up to pH 6.5-8.5, or about pH 6.5-8.5 (including all values ​​and ranges therebetween), or an acetate buffer of at least pH 4.0-5.5, up to pH 4.0-5.5, about pH 4.0-5.5 (including all values ​​and ranges therebetween), and may further comprise sorbitol or a suitable substitute therefor.

[0062] The pharmaceutical composition to be used for in vivo administration is typically sterile.Sterilization can be achieved by filtration through sterile filtration membrane.When composition is lyophilized, sterilization can be carried out either before or after lyophilization and reconstitution.The composition for parenteral administration can be stored in lyophilized form or in solution.In certain embodiments, parenteral composition is placed in a container with sterile access port, for example, the bag or vial of intravenous solution with the stopper that can be pierced by hypodermic injection needle, or the sterile pre-filled syringe that can be used immediately for injection.

[0063] The above compositions can be administered using conventional delivery methods, including, but not limited to, intravenous, intraperitoneal, oral, intralymphatic, subcutaneous, intraarterial, intramuscular, intrapleural, intrathecal, and by perfusion through a regional catheter. Local administration to the tumor in question (e.g., intratumoral) is also contemplated. When the composition is administered by injection, administration can be by continuous infusion or by single or multiple boluses. For parenteral administration, the anti-metastatic agent can be administered in a pyrogen-free, parenterally acceptable aqueous solution containing the desired anti-cancer agent in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which one or more anti-cancer agents are formulated as a properly preserved, sterile, isotonic solution.

[0064] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

[0065] If necessary, stabilizers conventionally used in pharmaceutical compositions, such as sucrose, trehalose, or glycine, may be used. Typically, such stabilizers are added in small amounts, for example, from at least 0.1%, at most 0.1%, or about 0.1% to at least 0.5%, at most 0.5%, or about 0.5% (w / v). Surfactant stabilizers, such as TWEEN®-20 or TWEEN®-80 (ICI Americas, Inc., Bridgewater, NJ, USA), may also be added in conventional amounts.

[0066] Components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic materials. Compositions for parenteral administration are also sterile, substantially isotonic, and manufactured under GMP conditions.

[0067] For a compound described herein, alone or as part of a pharmaceutical composition, such a dose is at least 0.001 mg / kg body weight to 10 mg / kg body weight, up to 0.001 mg / kg body weight to 10 mg / kg body weight, or about 0.001 mg / kg body weight to 10 mg / kg body weight, preferably at least 1 to 5 mg / kg body weight, up to 1 to 5 mg / kg body weight, or about 1 to 5 mg / kg body weight, and most preferably 0.5 to 1 mg / kg body weight (including all values ​​and ranges therebetween).

[0068] A therapeutically effective dose may be readily determined by one of skill in the art and will depend on the severity and course of the disease, the patient's health and response to treatment, the patient's age, weight, height, sex, medical history, and the judgment of the treating physician.

[0069] In some methods, the cancer cells are tumor cells. The cancer cells may be present in a patient. The patient may have a solid tumor. In such cases, embodiments may further involve performing surgery on the patient, such as removing all or part of the tumor. The composition may be administered to the patient before, after, or simultaneously with the surgery. In further embodiments, the patient may also be administered directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, locally, percutaneously, topically, intraarterially, intravesically, or subcutaneously. Therapeutic compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times, and they can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or every 1, 2, 3, 4, 5, 6, 7 days, or every 1, 2, 3, 4, 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

[0070] The method can further comprise administering to the subject a second cancer treatment selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy or gene therapy.The method can further comprise administering to the subject one, two, three, four or all five polypeptides or their variants two or more times.In certain aspects, the second cancer treatment can be the administration of ELANE or similar protease in combination with the anti-cancer peptide described herein.

[0071] The method for treating cancer may further include administering chemotherapy or radiation therapy to the patient, which may be administered two or more times. Chemotherapy includes, but is not limited to, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, transplatinum, 5-fluorouracil, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof. Radiation therapy includes, but is not limited to, X-ray irradiation, UV irradiation, gamma irradiation, electron beam irradiation, or microwave. Additionally, the cells or patient may be administered a microtubule-stabilizing agent, including but not limited to a taxane, as part of the method. It is specifically contemplated that any of the compounds or derivatives or analogs may be used with these combination therapies.

[0072] In certain aspects, other therapeutic agents useful for cancer therapy in combination with the polypeptide described herein include anti-angiogenic agents.Many anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitor of metalloproteinases (TIMP1 and TIMP2), prolactin (16 Kd fragment), angiostatin (38 Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptor, placental proliferin-related protein, and those listed by Carmeliet and Jain (2000). In one embodiment, the inhibitor may be used in combination with a VEGF antagonist or VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0073] Immunotherapy or biological response modifier therapy can be used in combination with the therapies described herein. These treatments use the immune system to fight disease. Immunotherapy can help the immune system recognize cancer cells or enhance the response to cancer cells. Immunotherapy includes active immunotherapy and passive immunotherapy. Active immunotherapy stimulates the body's own immune system, while passive immunotherapy generally uses immune system components produced outside the body.

[0074] Examples of active immunotherapies include, but are not limited to, cancer vaccines, tumor cell vaccines (autologous or allogeneic), viral vaccines, dendritic cell vaccines, antigen vaccines, anti-idiotype vaccines, DNA vaccines, or vaccines including tumor infiltrating lymphocyte (TIL) vaccines with interleukin-2 (IL-2), or lymphokine-activated killer (LAK) cell therapy.

[0075] Examples of passive immunotherapy include, but are not limited to, targeted therapy involving monoclonal antibodies and toxins. Monoclonal antibodies include naked antibodies and conjugated antibodies (also called tagged antibodies, labeled antibodies, or loaded antibodies). Naked monoclonal antibodies do not have any drugs or radioactive substances bound to them, while conjugated monoclonal antibodies are bound to, for example, chemotherapeutic drugs (chemically labeled), radioactive particles (radiolabeled), or toxins (immunotoxins).

[0076] In certain embodiments, passive immunotherapy, such as naked monoclonal antibody drugs, can be used in combination with the polypeptide compositions described herein to treat cancer.Examples of these naked monoclonal antibody drugs include, but are not limited to, rituximab (Rittman), an antibody against CD20 antigen, for example, used to treat B-cell non-Hodgkin's lymphoma; trastuzumab (Herceptin), an antibody against HER2 protein, for example, used to treat advanced breast cancer; alemtuzumab (Campath), an antibody against CD52 antigen, for example, used to treat B-cell chronic lymphocytic leukemia (B-CLL); cetuximab (Erbitux), an antibody against EGFR protein, for example, used in combination with irinotecan to treat advanced colorectal cancer and head and neck cancer; and bevacizumab (Avastin), an antiangiogenic therapy that acts on VEGF protein, for example, used in combination with chemotherapy to treat metastatic colorectal cancer. Further examples of therapeutic antibodies that can be used include HERCEPTIN® (trastuzumab), a humanized anti-HER2 monoclonal antibody for treating metastatic breast cancer patients (Genentech, CA); REOPRO® (abciximab), an anti-glycoprotein IIb / IIIa receptor on platelets (Centocor) for preventing blood clot formation; ZENAPAX® (daclizumab), an immunosuppressive humanized anti-CD25 monoclonal antibody for preventing acute kidney allograft rejection (Roche Pharmaceuticals, Switzerland); PANOREX™, a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome / Centocor); BEC2 (ImClone Systems), a murine anti-idiotypic (GD3 epitope) IgG antibody; IMC-C225 (ImClone Systems), a chimeric anti-EGFR IgG antibody; VITAXIN™, a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution / MedImmune);Campath 1H / LDP-03 (Leukosite), a humanized anti-CD52 IgG1 antibody; Smart M195 (Protein Design Lab / Kanebo), a humanized anti-CD33 IgG1 antibody; RITUXAN™ (IDEC Pharm / Genentech, Roche / Zettyaku), a chimeric anti-CD20 IgG1 antibody; LYMPHOCIDE™ (Immunomedics), a humanized anti-CD22 IgG antibody; LYMPHOCIDE™ Y-90 (Immunomedics); Lymphoscan (Tc-99m labeled; radioimaging; Immunomedics); Nuvion (anti-CD3; Protein Design Labs); CM3, a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114, a primatied anti-CD80 antibody (IDEC Pharm / Mitsubishi); and ZEVALIN™, a radiolabeled murine anti-CD20 antibody. (IDEC / Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC / Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody (Mexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT / BASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm / SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody. (Medarex / Eisai / Genmab); CD20-streptavidin (+biotin-yttrium 90; NeoRx); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7 antibody (LeukoSite / Genentech);Examples of antibodies may include, but are not limited to, Orthodone OKT4A, a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™, a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™, a humanized anti-VLA-4 IgG antibody (Elan); and CAT-152, a human anti-TGF-β2 antibody (Cambridge Ab Tech).

[0077] In certain embodiments, passive immunotherapy, such as conjugated monoclonal antibodies, can be used in combination with the polypeptide compositions described herein to treat cancer. Examples of conjugated monoclonal antibodies include, but are not limited to, the radiolabeled antibody ibritumomab tiuxetan (Zevalin), which delivers radioactivity directly to cancerous B lymphocytes and is used, for example, to treat B-cell non-Hodgkin's lymphoma; the radiolabeled antibody tositumomab (Bexxar), which is used, for example, to treat certain types of non-Hodgkin's lymphoma; and the immunotoxin gemtuzumab ozogamicin (Mylotarg), which contains calicheamicin and is used, for example, to treat acute myeloid leukemia (AML). BL22 is a conjugated monoclonal antibody, for example, for treating hairy cell leukemia; immunotoxins for treating leukemia, lymphoma, and brain tumors; and radiolabeled antibodies such as OncoScint, for example, for colorectal and ovarian cancer, and ProstaScint, for example, for prostate cancer.

[0078] In certain embodiments, targeted therapies comprising toxins can be used in combination with the polypeptide compositions described herein to treat cancer. The targeted therapies comprising toxins are growth factors or, in certain embodiments, toxins linked to the polypeptides described herein, and do not include antibodies.

[0079] Some embodiments also include the use of adjuvant immunotherapies in combination with the polypeptide compositions described herein, including cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1α, interleukins (including IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27), tumor necrosis factors (including TNF-α), and interferons (including IFN-α, IFN-β, and IFN-γ); aluminum hydroxide (alum); attenuated bovine tuberculosis vaccine (BCG); keyhole limpet hemocyanin (KLH); incomplete Freund's adjuvant (IFA); QS-21; DETOX; levamisole; and dinitrophenyl (DNP), as well as combinations thereof, such as combinations of interleukins, such as IL-2, with other cytokines, such as IFN-α, and the like.

[0080] Some embodiments also include the use of hormone therapy (antihormonal agents) in combination with the peptide compositions described herein. Antihormonal agents include agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), such as tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, and formestany. (formestanie), fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in cell proliferation, such as PKC-α, Ralf, and H-Ras; Ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; vaccines, such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0081] In some embodiments, the cancer to which the compositions described herein are administered may be a bladder, blood, bone, bone marrow, brain, breast, colorectal, esophageal, gastrointestinal, head, kidney, liver, lung, nasopharynx, cervical, ovarian, pancreatic, prostate, skin, stomach, testis, tongue, or uterine cell.

[0082] III. Expression and Expression Vectors Nucleic acids encoding any of the polypeptides (anti-cancer peptides) described herein can be inserted into or used with any suitable expression system. Recombinant expression can be achieved using vectors, such as plasmids and viruses. A vector can contain a promoter operably linked to one or more polypeptide-encoding nucleic acids. A vector can also contain other elements necessary for transcription and translation. As used herein, a vector refers to any carrier containing exogenous DNA. Thus, a vector is an agent that transports an exogenous nucleic acid into a cell without degradation and contains a promoter that confers expression of the nucleic acid in the cell to which it is delivered. Vectors include, but are not limited to, plasmids, viral nucleic acids, viruses, phage nucleic acids, phages, cosmids, and artificial chromosomes. Various prokaryotic and eukaryotic expression vectors suitable for transporting, encoding, and / or expressing protease-encoding nucleic acids can be generated. Such expression vectors include, for example, pET, pET3d, pCR2.1, pBAD, pUC, and yeast vectors. Vectors can be used, for example, in a variety of in vivo and in vitro contexts. The vector may be a gene therapy vector, for example, an adenoviral vector, a lentiviral vector, or a CRISP-R vector.

[0083] Expression cassettes, expression vectors, and sequences in the cassettes or vectors may be heterologous. As used herein, the term "heterologous" when used with respect to an expression cassette, expression vector, regulatory sequence, promoter, or nucleic acid refers to an expression cassette, expression vector, regulatory sequence, or nucleic acid that has been manipulated in some way. For example, a heterologous promoter may be a promoter that is not naturally linked to the nucleic acid to be expressed or that has been introduced into a cell by a cell transformation technique. A heterologous nucleic acid or promoter also includes a nucleic acid or promoter that is native to an organism but has been modified in some way (e.g., located at a different chromosomal location, mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). A heterologous nucleic acid may include sequences that include cDNA. A heterologous coding region can be distinguished from an endogenous coding region, for example, if it is linked to a nucleotide sequence containing regulatory elements, such as a promoter, that are not found associated with that coding region in nature, or if it is associated with a part of a chromosome where it is not found in nature (e.g., a gene that is expressed at a locus where the protein encoded by the coding region is not normally expressed). Similarly, a heterologous promoter can be a promoter that is linked to a coding region with which it is not naturally linked.

[0084] Viral vectors that can be used include those related to lentivirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, AIDS virus, neurotrophic virus, Sindbis virus, and other viruses. Any virus family that shares the characteristics of these viruses that make them suitable for use as vectors is also useful. Retroviral vectors that can be used include those described in Verma, IM, Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229-232, Washington, (1985). For example, such retroviral vectors can include murine Moloney leukemia virus, MMLV, and other retroviruses that express desired characteristics. Typically, viral vectors contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and a promoter for controlling transcription and replication of the viral genome. When engineered as a vector, a virus typically has one or more of the early genes removed and a gene or gene / promoter cassette inserted into the viral genome in place of the removed viral nucleic acid.

[0085] Various regulatory elements, including promoters, enhancers, translation initiation sequences, transcription termination sequences, and other elements, can be included in an expression cassette and / or expression vector. A "promoter" generally refers to one or more sequences of DNA that function when located at a relatively fixed position relative to the transcription start site. For example, a promoter may be located upstream of a nucleic acid segment encoding a protease. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors and may include upstream elements and response elements. An "enhancer" generally refers to a sequence of DNA that functions at an unfixed distance from the transcription start site and may be located either 5' or 3' of the transcription unit. Furthermore, enhancers can be located within introns and within the coding sequence itself. They are typically 10-300 nucleotides in length and function in cis. Enhancers function to increase transcription from nearby promoters. Like promoters, enhancers often also contain response elements that mediate transcriptional control. Enhancers often determine the regulation of expression.

[0086] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) may also contain sequences necessary for the termination of transcription, which can affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also contains a transcription termination site. It is preferred that the transcription unit also contain a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that the homologous polyadenylation signal be used in the expression construct.

[0087] Expression of one or more proteases from an expression cassette or expression vector can be controlled by any promoter capable of expression in prokaryotic or eukaryotic cells. Examples of prokaryotic promoters that can be used include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters. Examples of eukaryotic promoters that can be used include, but are not limited to, constitutive promoters, such as viral promoters, for example, CMV, SV40, and RSV promoters, and regulatable promoters, such as inducible or repressible promoters, for example, tet promoters, hsp70 promoters, and synthetic promoters regulated by CRE. Vectors for bacterial expression include pGEX-5X-3, and vectors for eukaryotic expression include pCIneo-CMV.

[0088] The expression cassette or vector may contain a nucleic acid sequence encoding a marker product. This marker product is used to determine whether the gene has been delivered to the cell and whether it is expressed once delivered. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein. In some embodiments, the marker may be a selectable marker. If such a selectable marker is successfully transferred into a host cell, the transformed host cell can survive when placed under selection pressure. There are two widely used distinct categories of selection schemes. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independent of supplemented media. The second category is dominant selection, which refers to a selection scheme that can be used in any cell type and does not require the use of mutant cell lines. These schemes typically use drugs to stop host cell growth. Cells harboring the novel gene express proteins that convey drug resistance and survive selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5: 410-413 (1985)).

[0089] Gene transfer can be achieved using, but is not limited to, direct transfer of genetic material in plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, and artificial chromosomes, or through transfer of genetic material in carriers such as cells or cationic liposomes or viruses. Such methods are well known in the art and can be easily adapted for use in the methods described herein. Transfer vectors can be any nucleotide constructs used to deliver genes into cells (e.g., plasmids), or any nucleotide constructs used as part of a general strategy for delivering genes, such as recombinant retroviruses or adenoviruses (Ram et al. Cancer Res. 53:83-88, (1993)). Suitable means for transfection, including viral vectors, chemical transfectants, or physical and mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, by Wolff et al., Science, 247, 1465-1468, (1990); and Wolff, Nature, 352, 815-818, (1991).

[0090] For example, a nucleic acid molecule, expression cassette, and / or vector encoding a protease can be introduced into cells by any method, including, but not limited to, calcium-mediated transformation, electroporation, microinjection, lipofection, biolistics, and the like. The cells can be expanded in culture and then administered to a subject, e.g., a mammal, such as a human. The amount or number of cells administered can vary, but typically ranges from about 10 cells to about 10 cells. 6 ~about 10 9 Amounts within the range of 1000 to 10000 can be used. Cells are generally delivered in a physiological solution such as saline or buffered saline. Cells can also be delivered in a vehicle such as a liposome, exosome, or population of microvesicles.

[0091] The protease can be produced by transgenic cells that produce exosomes or microvesicles that contain proteases.Exosomes and microvesicles can mediate the secretion of a wide variety of proteins, lipids, mRNAs, and microRNAs, and can interact with neighboring cells, thereby transmitting signals, proteins, lipids, and nucleic acids from cell to cell (see, for example, Shen et al., J Biol Chem. 286(16): 14383-14395 (2011); Hu et al., Frontiers in Genetics 3 (April 2012); Pegtel et al., Proc. Nat'l Acad Sci 107(14): 6328-6333 (2010); WO / 2013 / 084000; each of which is incorporated herein by reference in its entirety).

[0092] Thus, transgenic cells carrying a heterologous expression cassette or expression vector expressing one or more proteases can be administered to a subject, and exosomes produced by the transgenic cells deliver the proteases to tumor and / or cancer cells in the subject.

[0093] In accordance with the above, the present disclosure relates to methods for deriving vectors, particularly plasmids, cosmids, viruses, and bacteriophages, commonly used in genetic engineering and gene therapy, which contain nucleic acid molecules encoding the polypeptide sequences of the proteases defined herein. In certain cases, the vectors are expression vectors and / or gene transfer or gene targeting vectors. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papillomaviruses can be used to deliver the listed polynucleotides or vectors to targeted cell populations. Recombinant vectors can be constructed using methods well known to those skilled in the art. Alternatively, the listed nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the present disclosure can be introduced into host cells by well-known methods, which vary depending on the type of cellular host.

[0094] Another aspect of the present invention is directed to a gene therapy vector comprising an anti-cancer peptide construct.Gene therapy vectors are known in the art, including but not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids, and the like.The gene therapy vector of the present invention can be constructed by methods known in the art.In certain aspects, the gene therapy vector can be about 10, about 100, about 1000, about 1x10 4 , about 1×10 5 , about 1×10 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 pieces, up to 10, up to 100, up to 1000, up to 1×10 4 , up to 1 × 10 5 , up to 1 × 10 6 , up to 1 × 10 7, up to 1 × 10 8 , up to 1 × 10 9 , up to 1 × 10 10 , up to 1 × 10 11 , up to 1 × 10 12 pieces, or at least 10, at least 100, at least 1000, at least 1 x 10 4 , at least 1 x 10 5 , at least 1 x 10 6 , at least 1 x 10 7 , at least 1 x 10 8 , at least 1 x 10 9 , at least 1 x 10 10 , at least 1 x 10 11 , at least 1 x 10 12 The vaccine may be administered in amounts of virus particles (VP) or colony forming units (CFU), including all values ​​and ranges therebetween.

[0095] As an example of a gene therapy vector, the expression cassette can be contained in a lentiviral vector. The therapeutic vector can be transduced into cells ex vivo and the cells can be delivered to a patient. Similarly, the therapeutic vector of the present invention can be delivered directly to a patient. [Example]

[0096] IV. Working Examples The following examples and figures are included to demonstrate preferred embodiments of the invention. It should be recognized by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0097] Example 1 Neutrophil elastase cleaves CD95 to broadly and safely kill cancer cells A. Results Human neutrophils release factors that selectively kill cancer cells. Human peripheral polymorphonuclear neutrophils (PMNs) have a short in vivo half-life (approximately 8 hours) and rapidly undergo apoptosis, releasing factors with potent antibacterial properties (Dancey et al., J Clin Invest., 1976; Nathan et al., Nat Rev Immunol., 2006). To determine whether apoptotic neutrophils also release factors that kill cancer cells, 35 different human and mouse cancer cell lines were treated with serum-free conditioned medium (PMN medium) from purified PMNs that had been circulating until apoptosis. PMN medium effectively killed all cancer cell lines tested within 24 hours. In stark contrast, PMN medium did not kill any of the six human or mouse normal or non-cancerous cell types tested. Conditioned medium from omental neutrophils (ONs) isolated from healthy subjects also selectively killed cancer cells. However, murine neutrophils from various sources and activation states lacked this cancer cell-killing ability.

[0098] The cancer cell-killing ability of PMN medium was inhibited by culturing cancer cells in fetal bovine serum, mouse serum, or human serum. However, delivery of serum after 5 minutes of exposure to PMN medium under serum-free conditions failed to rescue the cancer cells, suggesting that serum contains inhibitors that directly antagonize the anticancer factors in the PMN medium (see below). The dependence of PMN medium killing on serum-free conditions may explain why previous studies conducted in the presence of serum showed that cell-cell contact is required for human neutrophils to kill cancer cells (Yan et al., Oncoimmunology, 2014).

[0099] To begin to understand the mechanism by which PMN medium kills cancer cells, a representative study focused on three cancer types with distinct mutational spectra: melanoma (MEL888, B16F10), lung cancer (A549, LLC1), and triple-negative breast cancer (MDA-MB-231, E0771). PMN medium was found to kill all of these cancer cells by inducing apoptosis.

[0100] To determine whether PMN media could induce apoptosis in cancer cells in vivo, we performed a study in which mice were injected with various cancer cells in syngeneic models (E0771, LLC1, B16F10) and triple-negative breast cancer PDX models (TNBC, 4195). 3 Tumors measuring 100x the size of a human were generated. Because serum antagonized the cancer-killing ability of PMN medium in vitro, PMN medium was delivered intratumorally (IT). Tumors were injected with either human serum albumin (HSA) or PMN medium once daily for 5 days, and apoptosis was examined by staining for TUNEL, cleaved PARP (cPARP), and cleaved CASP3 (cCASP3). PMN medium attenuated tumor growth and induced cancer cell apoptosis in all models tested. Because inactivation of bioactive factors in PMN medium blocked its antitumor activity, the effects in the syngeneic models were not due to the injection of human proteins into immunocompetent mice. In contrast, medium derived from murine bone marrow-derived neutrophils (BMDNs) failed to attenuate tumor formation in vivo, consistent with its inability to kill cancer cells in vitro. Consistent with its lack of toxicity to normal or non-cancerous cells in vitro, injection of PMN culture medium into the mammary fat pad of tumor-free C57BL / 6 mice (once daily for 5 days) did not induce apoptosis at the injection site or affect body weight, spleen weight, or liver function.

[0101] ELANE is a major anti-cancer protein released by human PMNs. Findings suggest that PMNs release a factor that selectively kills a wide range of cancer cells in vitro and in vivo. To identify the responsible factor, we developed a quantitative cancer cell killing assay to track the bioactive factor and used boiling, dialysis, and centricon experiments to validate the protein search. Next, we conducted studies to purify the responsible protein. PMN culture medium was prepared for fractionation by clarification through a 0.22 μm filter. Surprisingly, this step eliminated the cancer-killing activity of PMN culture medium from two independent donors without reducing total protein levels, suggesting selective depletion of the bioactive protein.

[0102] Shotgun proteomics analysis identified 890 proteins (≥2 peptides, FDR <1%) in PMN culture media, of which only two were significantly reduced by filtration in both donors (p < 0.05 by G test, Bonferroni correction): neutrophil elastase (ELANE) and eosinophil cationic protein (ECP). ELANE is a serine protease previously implicated in promoting tumorigenesis (Houghton et al., Nat Med., 2010), whereas ECP is a pore-forming protein toxic to both cancer and healthy cells (Young et al., Nature, 1986).

[0103] To determine whether ELANE and / or ECP are involved in the selective cancer-killing activity of PMN media, we used two approaches. First, we immunodepleted ELANE or ECP from PMN media and found that depletion of either protein attenuated the ability of PMN media to kill MDA-MB-231 cells. Second, we treated cancer cells or normal or non-cancerous cells with purified ELANE or ECP and monitored cell viability. ELANE killed MDA-MB-231 cells in a dose-dependent manner, and this effect was selective, as it did not kill human monocyte-derived macrophages (HMDMs). In contrast, ECP killed MDA-MB-231 cells only at doses that were also toxic to HMDMs.

[0104] Next, MDA-MB-231 cells were treated with ELANE (0.25 μg / mL) and ECP (0.05 μg / mL), concentrations present in PMN medium. PMN medium levels of ECP alone were unable to kill these cells, but significantly enhanced the selective killing ability of ELANE, suggesting a synergistic effect between the two proteins.

[0105] Because ELANE affects biological pathways through proteolysis (Pham, Nat Rev Immunol., 2006), it was hypothesized that its catalytic activity may be required for its anticancer function, and that ECP may enhance this activity to support synergistic killing. Treatment of ELANE with PMSF or alpha-1 antitrypsin (A1AT) and confirmation of inactivation using a chromogenic substrate activity assay revealed that catalytically inactive ELANE was no longer able to kill cancer cells. PMSF or A1AT treatment also eliminated the ability of PMN cultures to kill cancer cells in vitro and in vivo. These results suggest that ELANE is a major anticancer factor in PMN cultures. Indeed, ELANE catalytic activity in PMN cultures from nine healthy donors strongly correlated with its ability to kill MDA-MB-231 cells.

[0106] Understanding the anti-cancer function of ELANE in human neutrophils also helps explain why mouse neutrophils lack this ability: mouse ELANE kills cancer cells, and mouse neutrophils retain catalytically active ELANE intracellularly and release it during apoptosis, but this released ELANE is catalytically inactive.

[0107] To further determine whether ECP can enhance the catalytic activity of ELANE, studies were performed. ELANE was incubated with increasing concentrations of ECP, which showed that ECP acts as a type II allosteric activator of ELANE, binding with high affinity (K D =17nM), its catalytic turnover (k cat ) by approximately 12-fold. Coimmunoprecipitation experiments confirmed that ECP binds to ELANE in human PMN culture media. These results may explain why depleting ECP (and ELANE) from PMN culture media attenuates its killing activity, even though ECP is not toxic to cancer cells at the doses present in PMN culture media. Furthermore, because ECP has a high affinity for biological membranes (Young et al., Nature, 1986), these findings may help explain why filtration through a 0.22 μm filter selectively depletes ECP (and associated ELANE) from PMN culture media. Next, we focused on ELANE because it is effective and safe in vitro and because increasing its concentration can mimic ECP's ability to enhance its catalytic activity. Indeed, a high dose of ELANE (3 μg / ml) effectively induced apoptosis in all cancer cell lines tested, but was not toxic to any normal or non-cancerous cells tested.

[0108] ELANE selectively kills cancer cells by cleaving CD95. How does ELANE selectively kill cancer cells? We focused on the CD95 pathway because CD95 is essential for the survival of a wide range of cancer cells but is dispensable for the survival of normal or non-cancerous cells (Chen et al., Nature, 2010), a unique property that reflects ELANE's broad anticancer activity and low toxicity profile. Furthermore, CD95 function can be regulated by proteolysis (Strand et al., Oncogene, 2004). Previous studies have shown that reducing CD95 in cancer cells using shRNA activates a robust killing program characterized by the suppression of survival pathways and the induction of DNA damage, mitochondrial ROS (MT ROS), and apoptotic effectors (Chen et al., Nature, 2010; Hadji, et al., Cell Rep, 2014). In contrast, this killing program was not induced in Cd95- / - normal or non-cancer cells. Therefore, it was hypothesized that ELANE kills cancer cells through a mechanism involving CD95 cleavage. To begin testing this hypothesis, studies were conducted to determine whether ELANE could mimic the unique killing program triggered by CD95 knockdown in cancer cells (Chen et al., Nature, 2010). ELANE treatment (i) suppressed at least one survival pathway (phosphorylation of ERK, NFκB, or JNK), (ii) induced DNA damage (γH2AX), (iii) increased MT ROS, and (iv) activated pro-apoptotic pathways (cCASP3 and cPARP) in all six cancer cell lines tested (Figure 1a). Consistent with its lack of toxicity to normal or non-cancer cells, ELANE did not induce this program in any normal or non-cancer cell lines tested (Figure 1a).

[0109] Given that ELANE mimicked the killing program observed in CD95-null cancer cells, we determined whether ELANE could cleave CD95 in cancer cells and, if so, whether this cleavage resulted in the loss of CD95. When cancer cells were treated with ELANE, a low-molecular-weight CD95 band was observed immediately prior to cell death (Figures 1b-1c). Although ELANE cleaved CD95 in all cancer cells tested, this cleavage did not result in the loss of full-length CD95, suggesting that ELANE-mediated CD95 cleavage may kill cancer cells through a gain-of-function process rather than a loss-of-function process. Consistent with this interpretation, overexpression of human CD95 (hCD95) or mouse CD95 (mCD95) in cancer cells promoted, rather than prevented, ELANE-mediated killing (Figures 1d and 2).

[0110] To identify the ELANE cleavage site in hCD95, recombinant proteins corresponding to the N-terminal domain (aa. 1-173 of SEQ ID NO: 1) or C-terminal domain (aa. 212-335 of SEQ ID NO: 1) of hCD95 were incubated with ELANE. The results showed that ELANE preferentially cleaves the C-terminal domain (Fig. 1e), and mass spectrometry revealed two major cleavage sites—site 1:V 220 and A 221 Between, Part 2:I 321 and Q 332 These sites were identified as being involved in the proteolytic release of the CD95 death domain (DD) (Fig. 1f). Both of these sites closely match the sequence specificity of ELANE, and studies using synthetic peptides confirmed these findings (Fig. 1g-h).

[0111] Because CD95 is a plasma membrane protein and its C-terminal domain is present within cells, we hypothesized that its internalization must be required for the anticancer function of ELANE. Indeed, cancer cells rapidly internalized fluorescein-labeled ELANE, and the internalized ELANE retained its catalytic activity (Figures 1i–1j). Treatment with the endocytosis inhibitor Dynasore attenuated ELANE internalization and protected cancer cells from apoptosis (Figure 1j). Dynasore also protected cancer cells from PMN-mediated killing. Furthermore, overexpression of the CD95 C-terminal domain (aa. 212–335 of SEQ ID NO:1), which contains both cleavage sites, enhanced ELANE-mediated killing in both human and mouse cancer cells, whereas overexpression of the N-terminal domain (aa. 1–209 of SEQ ID NO:1) had no effect (Figures 1d and 2). These results suggest that the C-terminal CD95 proteolytic fragment released by ELANE cleavage may be sufficient to induce apoptosis in cancer cells.

[0112] To further test this possibility, we overexpressed C-terminal hCD95 proteins that mimic ELANE cleavage at site 1 (aa. 221–335 of SEQ ID NO:1), site 2 (aa. 212–331 of SEQ ID NO:1), or both sites (aa. 221–331 of SEQ ID NO:1). We found that expression of any of these proteins induced cancer cell death in the absence of ELANE (Figure 1k). In sharp contrast, overexpression of these same C-terminal hCD95 proteins in MCF10A cells or human omental adipose tissue fibroblasts derived from healthy subjects did not induce toxicity (Figure 1k). Thus, normal or non-cancer cells internalize ELANE, and while the internalized ELANE is catalytically active, the CD95 proteolytic fragments are not toxic to them (Figures 1a, 1d, 1i, and 1k). These findings reinforce the specificity of this killing mechanism for cancer cells.

[0113] IT-delivered ELANE attenuates tumor formation. Having demonstrated that ELANE selectively kills cancer cells in vitro, we investigated whether IT-delivered ELANE could attenuate tumor progression in vivo. To establish treatment conditions, various doses of ELANE were injected into the E0771 TNBC model, and 12 μg / day was found to produce reproducible and favorable therapeutic effects. Inactivation of ELANE with PMSF eliminated this therapeutic effect, and PMSF-ELANE did not produce any unexpected adverse effects. Finally, injection of ELANE (12 μg / day) into the mammary fat pad of non-tumor-bearing C57BL6 mice was shown to produce no apparent adverse effects.

[0114] We next explored the therapeutic potential of ELANE in models of TNBC, lung cancer, and melanoma. Athymic nude mice were injected with MDA-MB-231, A549, or MEL888 cells (xenograft models); SCID mice were injected with M1 or 4195 tumors (TNBC PDX models); and C57BL / 6 mice were injected with E0771, LLC1, or B16F10 cells (syngeneic models), resulting in tumor growth of approximately 100 mm. 3 At this point, PMSF-ELAN or ELANE (12 μg / day) was injected IT and the effect on tumor growth was monitored.

[0115] ELANE attenuated tumor growth in all models tested. Immunohistochemistry showed increased staining for TUNEL, cPARP, and cCASP3 in all ELANE-treated tumors, suggesting that ELANE induced apoptosis of cancer cells in vivo. In sharp contrast, no evidence of apoptosis was found when ELANE was injected into tumor-free C57BL / 6 mice. These findings are consistent with in vitro mechanistic studies, suggesting that the antitumor effects of ELANE are on-target and safe.

[0116] To explore whether cancer cells can acquire resistance to ELANE, we used a combination of in vitro and in vivo approaches. Repeated exposure of MDA-MB-231 cells to ELANE in vitro (resulting in nearly 90% death per exposure after seven exposures) did not confer resistance. Similarly, repeated treatment of E0771 or MEL888 tumors in vivo with ELANE (once daily for seven days) did not attenuate ELANE's ability to kill isolated cancer cells ex vivo. These findings suggest that resistance to ELANE may be difficult to develop, similar to what was previously reported in CD95-knockdown cancer cells (Murmann et al., Oncotarget, 2017).

[0117] IT-delivered ELANE induces CD8+ T cells to attack distant tumors. Because the anticancer function of ELANE relies on its catalytic activity and the abundance of serine protease inhibitors in the blood, its therapeutic delivery is limited to the IT pathway, hindering its access to metastatic sites. This obstacle could be overcome if ELANE's action on primary tumors could induce / activate immune cells to attack distant tumors, a property known as the abscopal effect (Ngwa et al. Rev Cancer, 2018).

[0118] To test this possibility, we conducted a study to determine whether ELANE could increase tumor immune cells. ELANE increased the number of dendritic cells (DCs), CD8 + T cells, and CD8 + T effector cells (CD8 + T eff) in the mammary fat pad of tumor-free mice. This increase in immune cells was not observed when ELANE was injected into the mammary fat pad of tumor-free mice, nor was it observed when ELANE was injected into the contralateral site (i.e., opposite the tumor) of tumor-bearing mice; thus, ELANE failed to attenuate tumor formation. Thus, ELANE increased innate and adaptive immune cells within tumors, and this effect was not due to an immune response to human ELANE.

[0119] We conducted a study to determine whether treatment of primary tumors with ELANE would generate an immune response and reduce tumor formation at distant sites. Genetically identical tumors were generated by injecting E0771 cells into the left (1° tumor) and right (2° tumor) mammary fat pads. Treatment of 1° tumors with ELANE (12 μg / day for 5 days) attenuated tumor formation at both sites. This effect was specific and not due to ELANE spillover, as treatment of 1° E0071 tumors with ELANE did not affect tumor formation in 2° B16F10 tumors. To test this in a different model, 1° B16F10 tumors were generated in the flank and 2° lung metastases were generated by injecting B16F10 cells via the tail vein. Treatment of 1° tumors with ELANE (12 μg / day for 5 days) reduced the number of lung metastases. Importantly, depletion of CD8+ T cells attenuated the abscopal effect of ELANE in both models.

[0120] Remarkably, ELANE therapy produced "crater" formation in nearly 40% of tumors in all immunocompetent models tested. Crater formation was not observed in tumor-free mice, but was observed in CD8 + This was not observed in any immunodeficient cancer model, including T cell-depleted C57BL / 6 mice. These data suggest that ELANE-mediated cancer cell killing, combined with the subsequent adaptive immune response, results in the formation of tumor "craters." However, the underlying mechanisms of crater formation and its significance for therapeutic efficacy require further investigation.

[0121] In vitro and in vivo expression of peptides. Figure 3 shows the effect of C1-2 expression on survival, stress, and apoptosis pathways in cancer cells (e.g., MDA-MB-231, MEL888, and A549) and non-cancer cells (MCF10A and fibroblasts). Figure 4 shows a scheme of the doxycycline-inducible Tet-on system for expressing either C (aa. 157-335) or C1-2 (aa. 221-331). C1-2 (DD-containing fragment) upon doxycycline addition (both in vitro and in vivo). C1-2 expression can selectively kill cancer cells (Figures 5A-5C). In vitro studies confirm the induction of cell death by C1-2 in MDA-MB-231 cells (Figures 6A-6C). In vivo studies demonstrate that C1-2 induced regression of MDA-MB-231 tumors (FIGS. 7A-7C).

[0122] B. Method Regulations: Human studies were approved by the University of Chicago Institutional Review Board (IRB 16-0321). Animal studies were approved by the University of Chicago Institutional Animal Care and Use Committee (ACUP72209, 72504). Cancer cell line and virus testing was approved by the Institutional Biosafety Committee (IBC1503).

[0123] Cell lines. The ovarian cancer cell lines CAOV3, OVCAR3, OVCAR4, OVCAR5, A2780, A2780 / CP70, HeyA8, TykNu, SKOV3, ID8, and ID8p53- / - were kindly provided by Dr. Ernst Lengyel at the University of Chicago. The breast cancer cell lines MDA-MB-231, MDA-MB-231.BM1, MCF-7, M6C, E0771, and E0771.LMB were kindly provided by Dr. Marsha Rosner at the University of Chicago. The colon cancer cell line RKO, glioblastoma cancer cell line T98G, osteosarcoma cell lines U-2OS and Saos-2, and hepatocellular carcinoma cell line HepG2 were kindly provided by Dr. Kay McLeod at the University of Chicago. The lung cancer cell line NCI-H552 was a gift from Dr. Stephanie Huang of the University of Chicago, and A549 and LLC1 cells were purchased from ATTC. The melanoma cell lines B16F10, Mel888, Mel1106, and SK-MEL-28 cells were a gift from Dr. Thomas Gajewski of the University of Chicago. The pancreatic cancer cell line PANC1 was a gift from Dr. Yamuna Krishnan of the University of Chicago. The leukemia cell line K562 was a gift from Dr. Amittha Wickrema of the University of Chicago. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone) supplemented with 10% heat-inactivated FBS (Gemini Bio Products) and 1% penicillin / streptomycin (Gibco).

[0124] The prostate cancer cell lines CWR22Rvl, LAPC4, and LNCaP were kindly provided by Dr. Donald Vander Griend of the University of Chicago. The neuroblastoma cell lines SK-N-BE(2) and NBL-WN were kindly provided by Dr. Lucy Godley of the University of Chicago. Cells were cultured in RPMI 1640 medium (Hyclone) containing 10% heat-inactivated FBS (Gemini Bio Products) and 1% penicillin / streptomycin (Gibco).

[0125] The mammary epithelial cell line MCF10A was a gift from Dr. Marsha Rosner at the University of Chicago. Cells were cultured in DMEM (HyClone) supplemented with 10% heat-inactivated FBS (Gemini Bio Products), 20 ng / mL EGF (Peprotech), 0.5 mg / mL hydrocortisone (Sigma), 100 ng / mL cholera toxin (Sigma), 10 μg / mL insulin (Sigma), and 1% penicillin / streptomycin (Gibco).

[0126] Primary human blood-derived cells. Following approval by the University of Chicago Institutional Review Board (IRB16-0321), human peripheral blood was provided by healthy volunteers with written consent. Blood was collected into EDTA-coated collection tubes (BD Vacutainer), and cells were separated using Ficoll-Paque Plus (GE Healthcare) to obtain the buffy coat (containing monocytes and lymphocytes) and bottom layer (containing neutrophils and red blood cells). Human peripheral blood neutrophils (PMNs) were purified from the bottom layer by repeated RBC lysis as previously described (Kuhns et al., Curr Protoc Immunol., 2015). Monocytes were purified from buffy coats using HMDM-CD14 microbeads (Miltenyi Biotec) and differentiated into HMDMs using human M-CSF (125 ng / mL, R&D Systems) as previously described (Kratz et al., Cel Metab., 2014). Human lymphocytes were isolated from buffy coats by collecting the flow-through from CD14 and CD16 microbeads (Miltenyi Biotec). The resulting cell population consisted of approximately 90% T cells and approximately 10% B cells.

[0127] Primary human omental adipose-derived cells. Human omental adipose tissue was obtained from healthy volunteers with written consent and approval from the University of Chicago Institutional Review Board. Human omental neutrophils (ON) - Omental tissue was digested with type 1 collagenase (Worthington, 1 mg / mL) at 37°C for 75 minutes with shaking at 130 rpm to obtain stromal vascular cells (SVC). ON was isolated from SVC using CD16 microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Human omental fibroblasts - Human primary fibroblasts were isolated from omental adipose tissue and cultured in DMEM (HyClone) containing 20% ​​heat-inactivated FBS (Gemini Bio Products) and 1% penicillin / streptomycin (Gibco) as previously described (Kenny et al., Int J Cancer, 2007).

[0128] Primary mouse cells. Unless otherwise indicated, cells were isolated from 6-8 week-old C57BL / 6 mice. Mouse bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow stem cells using L cell-conditioned medium as previously described (Kratz et al., Cell Metab., 2014). Mouse splenocytes were isolated as previously described (Reardon et al., Cell Rep., 2018). The resulting cells consisted of approximately 5% neutrophils, approximately 2% monocytes, approximately 38% T cells, and approximately 53% B cells. Mouse primary keratinocytes were isolated and cultured in E low calcein medium containing 15% heat-inactivated FBS and 1% penicillin / streptomycin as previously described (Wu et al., Cell, 2008). Mouse bone marrow-derived neutrophils (BMDN)—BMDN were purified using Histopaque 1119 (Sigma) and Histopaque 10771 (Sigma) density gradient centrifugation as previously described (Swamydas et al., Curr Protoc Immunol., 2015). For PMA activation, BMDN were treated with PMA (100 nM, Abcam) for 15 minutes, washed, and cultured to collect conditioned medium. Mouse thioglycollate-induced peritoneal neutrophils (PN)—PN were isolated from the peritoneal cavity 7 hours after 4% thioglycollate injection (3 mL / mouse, Sigma) as previously described (Swamydas et al., Curr Protoc Immunol., 2015). Mouse tumor-associated neutrophils (TAN)—E0771 cells were injected into the mammary fat pad of C57BL / 6 mice. Tumors with a volume of approximately 500 mm were isolated. 3At the time of tumor mass, tumors were digested with type 4 collagenase (Worthington, 3 mg / mL) and hyaluronidase (Sigma, 1.5 mg / mL) for 45 minutes at 37°C with shaking at 200 rpm. TANs were purified using Ly6G microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Mouse lung neutrophils (LN)—Mouse LNs were isolated from 8-9 week-old MMTV-PyMT mice prior to metastatic dissemination. Lungs were digested using Liberase TL (Roche, 200 μg / mL) and DNase I (Sigma, 0.1 mg / mL) as previously described (Swamydas et al., Curr Protoc Immunol., 2015). Mouse LNs were purified using Ly6G microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

[0129] Neutrophil media collection. Freshly isolated human or mouse neutrophils were plated in serum-free DMEM for 24 hours. Conditioned media was collected and centrifuged at 500 × g for 5 minutes, and media protein levels were determined using a Bradford assay (Biorad).

[0130] PMN medium manipulation. For boiling, PMN medium was boiled at 95°C for 5 minutes and centrifuged at 15,000 × g for 10 minutes to remove precipitated proteins. For dialysis, PMN medium was placed in a Slide-a-Lyzer™ cassette (3.5 kDa cutoff, ThermoFisher Scientific) and dialyzed against 2 × 4 L of PBS at 4°C for 4 hours. For PMSF inactivation, PMN medium was treated with PMSF (1 mM, Sigma) or A1AT (42 nM, Athens Research & Technology) and incubated at room temperature for up to 2 hours. Residual PMSF was removed using a PD-10 desalting column (GE Healthcare Life Sciences). Inhibition of ELANE catalytic activity was confirmed using a chromogenic substrate activity assay (see below). For serum addition experiments, human, mouse, or fetal bovine serum (1% or 10%) was added to PMN medium before or after cancer cell exposure. For immunodepletion studies, ELANE or ECP was immunoprecipitated using anti-ELANE (N2C3, GeneTx) or anti-ECP (MBS2535165, MyBioSource) coupled to Pierce™ Protein A / G magnetic beads (ThermoFisher Scientific).

[0131] In vitro cell viability assays. Cancer cells or normal or non-cancer cells were plated in complete growth medium and grown to 80-90% confluence. Cells were washed with serum-free DMEM and treated with various therapeutic agents (e.g., PMN medium, ELANE, etc.) for 4-24 hours. Cell viability was assessed using several methods: calcein AM assay. After 4-24 hours of treatment, cells were incubated with calcein AM (ThermoFisher Scientific, 4 ng / mL), washed with serum-free DMEM, and fluorescence was measured at 495 nm / 516 nm using a Synergy HT multimode microplate reader (Biotek). CASP3 activity assay. After 6 hours of treatment, cell-associated CASP3 activity was measured using the Caspase-Glo® 3 / 7 assay system (Promega). Luminescence was measured using a Victor X3 luminometer (PerkinElmer). ANXA5 staining - Thirty minutes to six hours after treatment, cells were stained using the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen™) according to the manufacturer's protocol. Samples were analyzed using a FACSCanto™ II flow cytometer (BD Pharmingen™).

[0132] Western blot analysis. Cells were lysed with 1% SDS containing protease and phosphatase inhibitors (Sigma), and protein was quantified using a BCA protein assay kit (Pierce). Proteins (10–20 μg) were separated on 10%, 12.5%, 15%, or 20% SDS-PAGE gels (depending on the target protein), transferred to PVDF membranes (Millipore), blocked with 5% BSA (Sigma) in 0.1% TBS / Tween-20 for 2 hours at room temperature, stained with primary and secondary antibodies, and visualized using an ECL detection kit (Biorad) and a LI-COR imager. Antibodies against pERK (4370), ERK (4695), pNFκB (3033), NFκB (8242), pJNK (4668), JNK (9252), CASP3 (9662), PARP (9542), H2AX (2595), and TUBB (2125) were from Cell Signaling Technology. Antibodies against γH2AX (05-636-I, Millipore), ELANE (68672, Abcam), N-terminal CD95 (3070R, BioVision), and C-terminal CD95 (60196, Proteintech) were from Cell Signaling Technology.

[0133] Mitochondrial ROS measurement. Cells were treated with various doses of ELANE for 30 min, washed, and labeled with CM-H2DCFDA dye (ThermoFisher Scientific, 10 μM) for 30 min at 37 °C, and fluorescence was quantified by flow cytometry.

[0134] CD95 overexpression studies. Polycistronic adenoviral vectors were prepared (VectorBuilder) to express human and mouse CD95 sequences under the control of the cytomegalovirus (CMV) promoter, followed by an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRS) and dTomato sequence. Human and mouse cancer cells or normal or non-cancer cells were transduced to overexpress full-length CD95, N-terminal CD95 (human: aa 1-209; mouse: aa 1-204), C-terminal CD95 (human: aa 212-335; mouse: aa 204-327), or C-terminal CD95 mimicking ELANE cleavage at site 1 (human: aa 221-335), site 2 (human: aa 212-331), or both sites (aa 221-331). Cancer cells, normal cells, or non-cancer cells were transduced with adenovirus at an MOI of 50–250 depending on the cell type. Expression of dTomato and CD95 (human: 558814; mouse: 565130, BD Biosciences) was confirmed by flow cytometry. A vector encoding GFP (Vector Builders) was used as a control.

[0135] ELANE activity assay. Catalytic activity was measured using the chromogenic substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma, 100 μg / mL) according to the manufacturer's protocol. Absorbance was measured at 405 nm using an accuSkan GO UV / Vis microplate spectrophotometer (ThermoFisher Scientific). For inactivation, ELANE was incubated with PMSF (1 mM, Sigma) or A1AT (42 nM, Athens Research & Technology) for 2 hours. Residual PMSF was removed using a PD-10 desalting column (GE Healthcare Life Sciences). To monitor the effect of ECP on ELANE activity, ELANE (10 nM) was incubated with various substrate concentrations (0–1.7 mM) and various doses of ECP (0–180 nM).

[0136] Activation of recombinant mouse ELANE. Recombinant mouse ELANE (50 μg / mL) was activated with CTSC (50 μg / mL) according to the manufacturer's protocol (R&D Systems).

[0137] Shotgun proteomic analysis. PMN culture media from two independent donors were passed through a 0.22 μm filter (MilliporeSigma). Pre- and post-filtered media (50 μg) were digested with trypsin.

[0138] Identification of ELANE cleavage sites in CD95 by mass spectrometry. Recombinant human C-terminal CD95 (aa 212-335, MyBioSource, 10 μg) or recombinant N-terminal CD95 (aa 1-173, ThermoFisher Scientific, 10 μg) was digested with human ELANE (0.1 μg) at 37°C for 2 hours, and the reaction was stopped with SDS-PAGE loading buffer. Proteins were run on a 20% SDS-PAGE gel, stained with Coomassie blue (ThermoFisher Scientific), and bands for mass spectrometry analysis were excised. Proteins were extracted from the excised bands.

[0139] Mass spectrometry analysis of recombinant peptides. The recombinant peptide (10 μM) (ThermoFisher Scientific) corresponding to TIFF2026021440000004.tif12128 was incubated with ELANE (0.1–0.2 μM) for 15–30 min at 37°C. The reaction was stopped with 0.1% formic acid.

[0140] Tumor inoculation and treatment. MDA-MB-231 cells (2 × 10 6 cells), A549 cells (2 × 10 cells with type 3 matrix) 6 cells), or MEL888 cells (2 × 10 with type 3 matrix) 6M1 or 4195 cells (50,000 cells) were injected into NOD.SCID mice (JAX); and E0771 cells (0.5 × 10 6 ), LLC1 cells (0.5×10 6 cells), or B16F10 cells (1 × 10 6 Cells were injected into C57BL / 6 mice (JAX). For TNBC models (MDA-MB-231, E0771, M1, 4195), cells were injected into the right ventral fourth mammary fat pad. For all other models (A549, LLC1, MEL888, B16F10), cells were injected into the flank. When tumors reached approximately 100 mm 3 At that time, ELANE or PMSF-ELANE (11.6 μg / 100 μL) or neutrophil medium or HSA (50 μg / 100 μL) was delivered IT once daily for 5 days. Tumor volume was assessed by caliper, and tumors >1000 mm in control mice were excluded. 3 The experiment was terminated when this was reached.

[0141] Tumor immunohistochemistry. Tumors were fixed in 4% paraformaldehyde in PBS for 24 hours, embedded in paraffin blocks, and sectioned (5 μm). Slides were stained with cCASP3 antibody (9661) and cPARP antibody (9625) from Cell Signaling Technology. Signals were developed using the VECTASTAIN ABC kit (Vector Laboratories) or fluorescently labeled secondary antibodies (melanoma model). For TUNEL assays, staining was performed using the DeadEnd™ colorimetric or fluorescent TUNEL system (Promega). Cell nuclei were labeled with hematoxylin or Hoechst 33342 (ThermoFisher Scientific). Images were acquired using a Nikon Eclipse Ti2 microscope and analyzed using NIS-Elements software.

[0142] Tumor immune cell analysis. Tumors were digested with type 4 collagenase (Worthington, 3 mg / mL) and hyaluronidase (Sigma, 1.5 mg / mL) for 45 minutes (E0771) or 30 minutes (LLC1 and B16F10) at 37°C with shaking at 200 rpm. Cells were labeled with various antibodies and analyzed by flow cytometry. Data were quantified using FlowJo v.10.4.1. Antibodies included: CD45 (47-0451), CD11b (25-0112), MHCII (11-5321), CD4 (17-0041), CD8 (12-0081), CD44 (25-0441) from ThermoFisher Scientific; CD3 (560527), CD62L (561917) from BD Biosciences, and Ly6G (127614) from BioLegend.

[0143] Abscopal effect study. For the E0771 model, 0.5 × 10 6 Inject 0.4 × 10 cells (1° tumor) into the left ventral fourth mammary fat pad. 6 cells were injected (2° tumor). 1° tumor was approximately 100 mm 3 At the time of tumor volume reaching 0.5 × 10, ELANE or PMSF-ELANE (11.6 μg / 100 μL) was injected intraperitoneally into the 1° tumor once daily for 5 days, and the volumes of the 1° and 2° tumors were measured using a vernier caliper. For the B16F10 model, 0.5 × 10 6 0.2 × 10 cells were injected into the flank (1° tumor). After 7 days, 0.2 × 10 cells were injected into the flank (1° tumor). 6 2 cells were injected into the lateral tail vein to generate 2 lung metastases. 1 tumor grew to approximately 100 mm 3 At the time of tumor growth, ELANE or PMSF-ELANE (11.6 μg / 100 μL) was injected intratumorally into the 1° tumor once daily for 5 days, and the volume of the 1° tumor was measured using a caliper. Ten days after the last ELANE treatment, the lungs were excised and 2° lung metastases were counted. +For T cell depletion, anti-mouse CD8α (clone 2.43, Bio X Cells) or rat IgG2b (isotype control, Bio X Cells) was injected IV (200 μg / injection) 3 days before the first ELANE treatment and once a week after the last ELANE treatment. + T cell depletion was confirmed by flow cytometry.

[0144] For the "spillover control," 0.5 × 10 6 E0771 cells were injected into the left ventral fourth mammary fat pad (2° tumor). ELANE or PMSF-ELANE (11.6 μg / 100 μL) was injected into the tumor-free fourth mammary fat pad of the right ventral pad once daily for 5 days, and the volume of the 2° tumor was measured using a vernier caliper.

[0145] For the "specificity control", 0.5 x 10 6 E0771 cells were injected into the right ventral fourth mammary fat pad (1° tumor), and 7 days later, 1 × 10 6 B16F10 cells were injected into the flank (2° tumor). 1° tumor was approximately 100 mm 3 At this time, ELANE or PMSF-ELANE (11.6 μg / 100 μL) was injected into the IT of the 1° tumor once daily for 5 days, and the volumes of the 1° and 2° tumors were measured using a caliper.

[0146] Assessment of potential side effects. ELANE or PMSF-ELANE (11.6 μg / 100 μL) was injected once daily for 5 days into the fourth mammary fat pad of the right ventral pad of tumor-free C57BL / 6 mice. One day after the final injection, mice were examined for potential side effects. Body and spleen weights were measured. Mammary adipose tissue was isolated, fixed, and stained for TUNEL, cCASP3, and cPARP using the same method as described for tumors above to examine apoptosis at the injection site. Mammary adipose tissue immune cell populations at the injection site were determined by flow cytometry using the same method as described for tumors above. Liver function was assessed by measuring plasma ALT activity using an alanine transaminase colorimetric activity assay kit (Cayman Chemical).

[0147] In vivo testing. Two million Tet-on C1-2-transduced MDA-MB-231 cells were injected into the mammary glands of nude mice. When tumors reached approximately 80–100 mm3, the mice were placed on a doxycycline diet or intraperitoneally injected with doxycycline (50 mg / ml) every 5 days to induce the Tet-on system. Tumor growth was monitored. 21 days after doxycycline treatment, tumors were isolated and digested, tumor weights were measured, tumor cell counts were performed, and CD45+ cells were measured by flow cytometry.

[0148] Statistics. Except for proteomics studies, statistical significance was determined using a two-tailed unpaired Student's t-test. Linear regression, Michaelis-Menten, and hyperbolic fitting were performed using Prism v.7 software.

[0149] Sequence information SEQUENCE LISTING <110> The University of Chicago <120> METHODS AND COMPOSITIONS RELATED TO THERAPEUTIC PEPTIDES FOR CANCER THERAPY <150> US 62 / 782,690 <151> 2018-12-20 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 335 <212> PRT <213> Homo sapiens <400> 1 Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala 1 5 10 15 Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 20 25 30 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn 35 40 45 Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro 50 55 60 Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro 65 70 75 80 Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85 90 95 Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly 100 105 110 Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120 125 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 130 135 140 Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr 145 150 155 160 Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp 165 170 175 Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg 180 185 190 Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly 195 200 205 Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu 210 215 220 Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met 225 230 235 240 Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu 245 250 255 Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu 260 265 270 Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys 275 280 285 Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys 290 295 300 Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser 305 310 315 320 Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 325 330 335 <210> 2 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 2 Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu Ser Asp Val 1 5 10 15 Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met Thr Leu Ser 20 25 30 Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile 35 40 45 Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val 50 55 60 Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr 65 70 75 80 Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala 85 90 95 Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp Ser Glu 100 105 110 Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 115 120 <210> 3 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 3 Ala Ile Asn Leu Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile 1 5 10 15 Ala Gly Val Met Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn 20 25 30 Gly Val Asn Glu Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln 35 40 45 Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu 50 55 60 His Gly Lys Lys Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys 65 70 75 80 Ala Asn Leu Cys Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys 85 90 95 Asp Ile Thr Ser Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln 100 105 110 Ser Leu Val 115 <210> 4 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 4 Ser Pro Thr Leu Asn Pro Glu Thr Val Ala Ile Asn Leu Ser Asp Val 1 5 10 15 Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met Thr Leu Ser 20 25 30 Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile 35 40 45 Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val 50 55 60 Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr 65 70 75 80 Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala 85 90 95 Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp Ser Glu 100 105 110 Asn Ser Asn Phe Arg Asn Glu Ile 115 120 <210> 5 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 5 Ala Ile Asn Leu Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile 1 5 10 15 Ala Gly Val Met Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn 20 25 30 Gly Val Asn Glu Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln 35 40 45 Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu 50 55 60 His Gly Lys Lys Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys 65 70 75 80 Ala Asn Leu Cys Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys 85 90 95 Asp Ile Thr Ser Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile 100 105 110

Claims

1. A method for treating cancer, comprising administering to a subject having cancer an effective amount of a therapeutic composition comprising one or more peptides that are 90 to 100% identical to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof.

2. The method of claim 1, wherein the therapeutic composition is administered by injection.

3. The method of claim 1, wherein the therapeutic composition is administered intratumorally.

4. 10. The method of claim 1, wherein the cancer is bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain / nervous system cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer, or uterine cancer.

5. 2. The method of claim 1, wherein the peptide is present in a dose between 0.001 mg / kg body weight and 10 mg / kg body weight, preferably at least 0.1-5 mg / kg body weight, at most 0.1-5 mg / kg body weight, or about 0.1-5 mg / kg body weight, most preferably 0.5-1 mg / kg body weight.

6. 10. The method of claim 1, further comprising administering a second anti-cancer treatment.

7. 7. The method of claim 6, wherein the second anti-cancer treatment is chemotherapy, radiation therapy, immunotherapy, or anti-hormonal therapy.

8. 7. The method of claim 6, wherein the second anti-cancer treatment is ELANE protease.

9. A method for inducing apoptosis in cancer cells, comprising contacting the cancer cells with an effective amount of one or more peptides having 90 to 100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof.

10. 10. The method of claim 9, wherein the cancer cells are present in a tumor of the bladder, blood, bone, bone marrow, brain / nervous system, breast, colorectal, esophageal, gastrointestinal, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus.

11. An anti-cancer peptide composition comprising one or more peptide components having 90-100% identity to a peptide selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a functional segment thereof.

12. 12. The composition of claim 11, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:

2.

13. 12. The composition of claim 11, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:

3.

14. 12. The composition of claim 11, wherein the peptide has an amino acid sequence that is 90% identical to the amino acid sequence of SEQ ID NO:

4.

15. The composition of claim 11 , wherein the peptide component is bound to a substrate.

16. The composition of claim 15, wherein the substrate is a delivery vehicle.

17. 17. The composition of claim 16, wherein the delivery vehicle is a nanoparticle.

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