阅读说明：本技术 与用于癌症疗法的治疗肽相关的方法和组合物 (Methods and compositions related to therapeutic peptides for cancer therapy ) 是由 莱芙·贝克尔 崔畅 于 2019-12-20 设计创作，主要内容包括：本发明的实施方案提供了与平衡患者毒性与癌症疗法的广泛功效相关的问题的解决方案。特别地,实施方案涉及抗癌肽,其表现出广泛的抗癌功效,而对正常细胞或非癌细胞的毒性有限。(Embodiments of the present invention provide solutions to the problems associated with balancing patient toxicity with the broad efficacy of cancer therapies. In particular, embodiments relate to anti-cancer peptides that exhibit broad anti-cancer efficacy with limited toxicity to normal or non-cancerous cells.)

1. A method of treating cancer comprising administering to a subject having cancer an effective amount of a therapeutic composition comprising one or more than one peptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or a functional segment thereof has 90% to 100% identity.

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. The method of claim 1, wherein the cancer is bladder cancer, blood 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. The method of claim 1, wherein the peptide is present at a dose of 0.001mg/kg to 10mg/kg body weight, preferably at least, at most, or about 0.1 to 5mg/kg body weight, most preferably 0.5 to 1mg/kg body weight.

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

7. The method of claim 6, wherein the second anticancer therapy is chemotherapy, radiation therapy, immunotherapy, or anti-hormone therapy.

8. The method of claim 6, wherein the second anticancer therapy is ELANE protease.

9. A method of inducing apoptosis in a cancer cell comprising contacting the cancer cell with an effective amount of one or more than one peptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or a functional segment thereof has 90% to 100% identity.

10. The method of claim 9, wherein the cancer cell is in a bladder tumor, a blood tumor, a bone tumor, a myeloma, a brain/nervous system tumor, a breast tumor, a colorectal tumor, an esophageal tumor, a gastrointestinal tumor, a head tumor, a kidney tumor, a liver tumor, a lung tumor, a nasopharyngeal tumor, a neck tumor, an ovarian tumor, a pancreatic tumor, a prostate tumor, a skin tumor, a stomach tumor, a testicular tumor, a tongue tumor, or a uterine tumor.

11. An anti-cancer peptide composition comprising one or more than one peptide component that is substantially identical to a peptide selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or a functional segment thereof has 90% to 100% identity.

12. The composition of claim 11, wherein the amino acid sequence of the peptide is identical to SEQ ID NO: 2 has 90% identity.

13. The composition of claim 11, wherein the amino acid sequence of the peptide is identical to SEQ ID NO: 3 has 90% identity.

14. The composition of claim 11, wherein the amino acid sequence of the peptide has a sequence that is substantially identical to SEQ ID NO: 4 has 90% identity.

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

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

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

I. Field of the invention

Embodiments of the compositions and methods described herein relate generally to the fields of molecular biology, medicine, and cancer therapy. In particular, embodiments relate to cancer therapies and anti-cancer peptide compositions.

II. background of the invention

Cancer is a mutated proliferative disease that exhibits a high degree of spatial and temporal genetic heterogeneity (Stratton et al, Nature, 2009; Vogelstein et al, Science, 2013). In addition to overcoming this heterogeneity, eradication of tumor cells while retaining non-cancer cells remains a formidable task. For these reasons, it has been challenging to identify agents that have broad efficacy against cancer types and that retain specificity to limit host toxicity.

The broad efficacy and specificity is an integral property of innate immunity. The innate immune system is evolved to protect against a variety of infectious pathogens, including bacteria, fungi, and protozoa, with genetic diversity far exceeding that of cancer. As a key effector of innate immunity, neutrophils eliminate genetically diverse pathogens and therefore may ideally 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 their delivery as Cell therapies. Despite the growing interest in human PMNs, the mechanism by which they kill cancer cells is not fully understood.

In contrast to the anticancer function of PMNs, many studies have shown that tumor-associated neutrophils (TAN) promote tumorigenesis. This discrepancy may be due to the source and activation status 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 Patholog, 2015). For example, mouse studies have shown that tumor cells hijack neutrophils to release molecules to promote metastatic spread (Coffelt et al, Nature, 2015; Finisgura et al, Nature, 2015). In addition, increased TAN accumulation is a poor prognostic marker for many Cancer types (Coffelt et al, Nat Rev Cancer, 2016; Shen et al, PLoS One, 2014; Powell et al, Immunol., 2016).

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.

Background

Disclosure of Invention

Embodiments of the present invention provide solutions to the problems associated with balancing patient toxicity with the broad efficacy of cancer therapies. In particular, embodiments relate to anti-cancer peptides that exhibit broad anti-cancer efficacy with limited toxicity to normal or non-cancerous cells.

Given that human PMNs release extracellular factors that kill a variety of pathogens, the present inventors sought to explore whether these factors have a similar ability to kill cancer cells. Using this strategy, ELANE was identified as the major anticancer protein released by human PMNs, whose mechanism of action was implicated at V²²⁰/A²²¹And I³³¹/Q³³²Human CD95(hCD95) was cleaved to release a proteolytic fragment containing the Death Domain (DD) that selectively kills cancer cells. ELANE has been shown to have broad anti-cancer efficacy and selectivity in a variety of models.

ELANE has several interesting properties in preclinical models. First, its ability to kill a variety of cancer cells can be performed without knowledge of its genetics. Second, its specificity for cancer versus non-cancer cells can limit potential toxicity. Again, preliminary studies indicate that it is challenging for cancer cells to acquire resistance to ELANE. Mechanistically, it has been suggested that these properties stem from the ability of ELANE to target CD 95. Indeed, the killing program, broad efficacy, specificity and resistance of ELANE closely mimic those reported in cancer cells treated with shRNA to reduce CD95 levels (l.chen et al, Nature, 2010), but with one important difference. The proposed mechanism involves gain of function (i.e. release of DD-containing fragments) but not loss of function (i.e. CD95 knockdown) of CD 95. CD95 is known to exert both pro-and anti-apoptotic effects depending on the physiological environment (Martin-vilalba, Liorens-Bobadilla, wolfny. trends Mol Med, 2013) and studies have identified ELANE as a potential therapeutic approach that can exploit its pro-apoptotic function.

The inventors' studies indicate that ELANE proteolytically releases CD95 DD to selectively kill a wide range of cancer cells. Indeed, transient overexpression of full-length CD95(aa 1-335) or C-terminal CD95 peptide containing two cleavage sites (aa.212-335) accelerated ELANE-mediated killing in human cancer cells, while transient overexpression of the N-terminal domain (aa 1-209) had no effect. However, transient overexpression of these hCD95 proteins/peptides in the absence of ELANE was not sufficient to kill cancer cells. Induction of apoptosis in the absence of ELANE requires expression of a C-terminal hCD95 peptide that mimics cleavage of ELANE at one or two sites (site 1: aa 221-335; site 2: aa 212-331; two sites: aa 221-331). These hCD95 peptides were shown to kill many types of human cancer cells without damaging non-cancer cells, supporting a method of delivering specific CD95 peptides or DNA encoding CD95 peptides for the treatment of many cancers.

The data show that some human CD95 peptides (which contain ELANE cleavage sites) are not toxic to cancer cells and only make ELANE more effective. On the other hand, in the absence of ELANE, transient expression of human CD95 peptide mimicking ELANE cleavage at one or two sites killed cancer cells. The C peptide is not toxic, but C1 (mimicking cleavage at site 1), C2 (mimicking cleavage at site 2), and C1-2 (mimicking cleavage at both sites) are toxic. In addition, the N-terminal domain peptides of CD95 and full-length CD95 were not toxic to cancer cells. Finally, the results indicate that expression of C1-2 peptide in MDA-MB-231 cells induces the same killing mechanism as treatment of these cells with ELANE (i.e., inhibition of survival pathways, and induction of DNA damage, mitochondrial ROS, and apoptotic effectors).

To investigate whether expression of CD95C 1-2 peptide could attenuate tumor growth in vivo, researchers constructedMDA-MB-231 cancer cells (TNBC) were established which stably expressed CD95C or C1-2 peptide under the control of a doxycycline inducible promoter. In vitro studies, doxycycline treatment induced the expression of both proteins. Consistent with the transient expression system, cells induced to express C1-2 peptide underwent apoptosis, whereas those induced to express C peptide did not. Furthermore, doxycycline-induced expression of C1-2 peptide in MCF10a cells (non-cancer cells) failed to induce apoptosis. Analysis of the different C1-2 peptide expressing colonies from MDA-MB-231 cells showed that the degree of apoptosis was significantly positively correlated with the expression level of C1-2. MDA-MB-231 cells expressing doxycycline-inducible C1-2 peptide were injected into mammary fat of mice to grow tumors to about 100mmw size. At this time, doxycycline was administered to mice by intraperitoneal injection or by food. The results indicate that induction of C1-2 expression in MDA-MB-231 cells reduces tumor growth and increases CD45 in tumors⁺The number of immune cells.

Certain embodiments relate to therapeutic or anti-cancer compositions comprising various combinations of anti-cancer peptides or variants thereof, expression vectors, or expression cassettes encoding the same. Compositions in certain aspects may include one or more than one peptide comprising, consisting essentially of, or consisting of: amino acid sequence SPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGF VRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIkDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 2), or segment AINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAE QKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 3), and/or SPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEI (SEQ ID NO: 4) and/or AINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEI (SEQ ID NO: 5). Certain embodiments relate to one or more than one peptide, or variant thereof, having an amino acid sequence that is identical to SEQ ID NO: 2, more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2930, 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, 119, 113, 115, 113, 114, 122, 124, 122, 124, 122, and/or 124, including all ranges therebetween 92. 94, 96, 98, 99 to 100% identity, including all values and ranges therebetween. The functional segment of the anti-cancer peptide can be derived from SEQ ID NO: 2, 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, 2930, 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, 119, 110, 111, 112, 111, 114, 6, 114, 6, 9, 114, 6, 9, 6, or 6, 23, 6, or 7, 6, or 7, 6, or 6, 72, 6, or 6, or more amino acids, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2930, 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, 124 or 124 bits. In certain aspects, the anticancer peptides described herein can be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by including heterologous peptide sequences at the amino or carboxy terminus of the peptide.

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

Certain embodiments relate to one or more than one anti-cancer peptide and/or compositions comprising the same. In certain aspects, one or more than one peptide component is identical to a peptide selected from SEQ ID NOs: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5 or a functional segment thereof has 90% to 100% identity. In certain aspects, the peptide has an amino acid sequence identical to SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4 has an amino acid sequence with at least 90% identity. The peptide component may be coupled to a substrate. In certain aspects, the substrate is a delivery vehicle. The delivery vehicle can be a nanoparticle (e.g., a liposome).

Certain embodiments relate to a method of treating cancer comprising administering to a subject having cancer an effective amount of a therapeutic composition comprising one or more than one peptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or a functional segment thereof has 90% to 100% identity. The therapeutic composition may be administered by injection. In certain aspects, the therapeutic composition is administered intratumorally (e.g., by intratumoral injection). The cancer may be bladder cancer, blood 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. In certain aspects, the peptide is present at a dose of 0.001mg/kg to 10mg/kg body weight, preferably at least, up to or about 0.1 to 5mg/kg body weight, most preferably 0.5 to 1mg/kg body weight. In other aspects, the method can comprise administering a second anti-cancer therapy. The second anti-cancer therapy may be chemotherapy, radiation therapy, immunotherapy (e.g., checkpoint inhibitors), or anti-hormone therapy.

Certain embodiments relate to a method of inducing apoptosis in a cancer cell comprising contacting the cancer cell with an effective amount of one or more than one peptide that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or a functional segment thereof has 90% to 100% identity. The cancer cell may be in a bladder tumor, a blood tumor, a bone tumor, a myeloma, a brain/nervous system tumor, a breast tumor, a colorectal tumor, an esophageal tumor, a gastrointestinal tumor, a head tumor, a kidney tumor, a liver tumor, a lung tumor, a nasopharyngeal tumor, a neck tumor, an ovarian tumor, a pancreatic tumor, a prostate tumor, a skin tumor, a stomach tumor, a testicular tumor, a tongue tumor, or a uterine tumor.

The anti-cancer peptide described herein was found to be a fragment of human CD95 isoform 1, having the amino acid sequence of MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 1, accession number NP-000034.1, which was incorporated herein by reference since the filing date of the present application). Amino acids 1 to 25 are signal peptides, and the mature form comprises amino acids 26 to 335. Certain embodiments relate to one or more than one peptide, or variant thereof, having an amino acid sequence that is identical to SEQ ID NO: 1, greater than 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) have 90, 92, 94, 96, 98, 99, to 100% identity, including all values and ranges therebetween. In certain aspects, the anticancer peptides described herein can be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by including heterologous peptide sequences at the amino or carboxy terminus of the peptide. The N-terminus referred to herein is from amino acids 1 to 209 (SEQ ID NO: 1), and the C-terminus referred to herein is from amino acids 212 to 335(SEQ ID NO: 1).

The compositions described herein can kill a variety of cancer cells, regardless of cancer cell genetics. 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 (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, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (e.g., melanoma), gastric cancer, testicular cancer, tongue cancer, or uterine cancer. The compositions described herein are toxic to cancer cells, but are non-toxic or have limited toxicity to non-cancer cells.

Certain embodiments relate to methods of killing cancer cells by contacting the 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 having cancer. In certain aspects, the cancer is bladder cancer, blood 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 can further comprise or be administered in combination 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 prior to administration of the polypeptide composition; during the period; then; before and during; before and after; during and after; or before, during and after. In certain aspects, the compositions described herein can be administered prior to administration of immunotherapy, chemotherapy, anti-hormone therapy, or radiation therapy; during the period; then; before and during; before and after; during and after; or before, during and after. In certain aspects, the peptide compositions described herein are administered in combination with chemotherapy, such as doxorubicin and/or paclitaxel.

Certain embodiments relate to expression vectors or cassettes encoding one or more than one anti-cancer peptide. Such vectors or cassettes may be administered to a subject for the purpose of expressing one or more than one anti-cancer peptide in or near a target cancer or tumor.

The term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

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

"therapeutically effective amount" with respect to treating cancer refers to an amount capable of causing one or more than one of the following effects: (1) inhibiting cancer or tumor growth to some extent, including slowing growth or completely arresting growth; (2) a reduction in the number of cancer or tumor cells; (3) reduction of tumor volume; (4) inhibiting (i.e., reducing, slowing, or completely stopping) the infiltration of cancer or tumor cells into peripheral organs; (5) inhibit (i.e., reduce, slow, or completely stop) metastasis; (6) enhance an anti-tumor immune response, which may, but need not, result in tumor regression or rejection, or (7) alleviate one or more symptoms associated with the cancer or tumor to some extent. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of one or more anti-cancer agents to elicit a desired response in the individual. A "therapeutically effective amount" is also an amount at which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

The phrases "treating cancer" and "treatment of cancer" refer to reducing, or inhibiting the replication of cancer cells; reducing, reducing or inhibiting the spread of cancer (forming metastases); reducing tumor size; reduction in tumor number (i.e., reduction in tumor burden); reducing or decreasing the number of cancer cells in vivo; preventing cancer recurrence following surgical resection or other anti-cancer therapy; or ameliorating or alleviating the symptoms of a disease caused by cancer.

The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing nucleic acid sequences that direct and/or control (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. The expression construct may include, but is not limited to, the following sequences: which affects or controls the transcription, translation, and, if present, splicing of RNA of a coding region to which it is operably linked.

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

The terms "inhibit," "reduce," or "prevent," or any variation of these terms, when used in the claims and/or specification includes any measurable decrease or complete inhibition to achieve a desired result.

The absence of a quantitative term preceding a word may mean "one" when used in conjunction with the term "comprising" in the claims and/or the specification, but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

Throughout this application, the term "about" is used to indicate a value that includes an inherent variation from error for a device, method used to determine the value, or a variation that exists between study objects.

The term "or" as used in the claims is intended to mean "and/or" unless explicitly indicated to refer to alternatives only or to alternatives being mutually exclusive, although the present disclosure supports the definition of alternatives and "and/or" only. It is also contemplated that any of the contents listed using the term "or" may also be explicitly excluded.

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

In any of the embodiments discussed herein, the term "consisting of … …" or "consisting essentially of … …" may be substituted for the term "comprising".

It is contemplated that any embodiment discussed herein may be practiced with respect to any method or composition, and vice versa. In addition, compositions and kits can be used to implement the methods.

Other embodiments are discussed throughout this application. Any embodiments discussed with respect to one aspect are also applicable to the other aspect, and vice versa. The embodiments in the examples section are to be understood as embodiments applicable in all aspects. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. .

Drawings

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 illustrative embodiments presented herein.

Elane cleaves CD95 to selectively kill cancer cells. (A) The method comprises the following steps Heatmap summary of ELANE effects on survival, stress and apoptotic pathways in cancer and healthy cells. The quantitative results are shown in FIG. 2. (B-C): cancer cells were treated with ELANE (100nM) and CD95 cleavage was assessed by western blotting using anti-N-terminal (B) or anti-C-terminal (C) antibodies. CD95 fragment of lower molecular weight. (D) The method comprises the following steps Cancer or non-cancer cells were transduced to overexpress CD95 protein and the effect on ELANE-mediated apoptosis was quantified by ANXA5 at 30 min. n is 2/group. Transduction and CD95 overexpression see figure 1I. (E) The method comprises the following steps Cleavage of recombinant human CD 95N-terminal (aa 1-173) or C-terminal (aa 212-335) protein by ELANE was assessed by SDS-PAGE and Coomassie blue staining. (F) The method comprises the following steps The band from (E) was digested with trypsin and analyzed by mass spectrometry to identify a putative ELANE cleavage site (i.e., a peptide with a non-trypsin terminus). The major non-tryptic peptides were quantified by ion chromatograms. (G) The method comprises the following steps Schematic representation of the ELANE cleavage site in CD 95. Heatmap shows the overlap between the 2 CD95 cleavage sites and ELANE sequence specificity (located at URL web. (H) The method comprises the following steps Cleavage of the recombinant peptides corresponding to aa 214-231 and aa317-335 in CD95 by ELANE was monitored by mass spectrometry. (I) The method comprises the following steps Cancer or non-cancer cells were treated with fluorescein-labeled ELANE for 10 minutes and uptake was visualized by immunofluorescence. (J) The method comprises the following steps ELANE catalytic activity in cancer cell lysates after 30 min exposure to ELANE in the presence and absence of Dynasore (60 μ M, an inhibitor of endocytosis). Effect of Dynasore on cancer cell killing ability of ELANE (measured by calcein-AM). n is 6/group. (K) The method comprises the following steps Cancer or non-cancer cells were transduced to overexpress various CD95 proteins, and cell viability in the absence of ELANE was determined by calcein-AM. n is 10/group. P < 0.05 student t-test.

FIGS. 2A-2D overexpression of CD95 protein promotes ELANE killing of cancer cells. Polycistronic adenoviral vectors were prepared to express the human and mouse CD95 sequences, followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site and dTomato sequence under the control of the Cytomegalovirus (CMV) promoter. Human and murine 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) The method comprises the following steps Transduction efficiency quantified by dTomato (left). Levels of CD95 in dtomat-and dtomat + cells were quantified by geometric Mean (MFI) (middle). Levels of ANXA5 in dTomato-and dTomato + cells after 30 min treatment with ELANE (40nM) (right). Representative data for a549 cells are shown. (B-C): transduction efficiency of cancer and non-cancer cells (B) and quantification of relative CD95 overexpression (C). (D) The method comprises the following steps Quantification of ANXA5 levels after ELANE treatment (40nM, 30 min) of mouse cancer cell lines (E0771, B16F 10). P < 0.05 student t-test.

FIG. 3C 1-2 peptide efficacy. Thermography of the effects of C1-2 expression on survival, stress and apoptotic pathways in cancer and non-cancer cells.

FIG. 4 inducible expression System. A scheme for expressing C (aa.157-335) or C1-2(aa.221-331) by a doxycycline inducible Tet-on system. C1-2 (fragment containing DD) after doxycycline addition (in vitro and in vivo).

FIGS. 5A-5C C1-2 expression and confirmation of cancer cell selective killing. (A) Tet-on C or C1-2 transduced MDA-MB-231 cells were treated with 2. mu.g/mL doxycycline (2. mu.g/mL) for 24 hours. Cell lysates were collected for western blotting. Confirming the expression of C or C1-2 expression. (B) Tet-onC or C1-2 transduced MDA-MB-231 cells were treated with 0.2 or 2. mu.g/mL doxycycline for 72 hours. Cell viability was measured by calcein AM. Expression of C1-2 instead of C resulted in MDA-MB-231 cell death. (C) Tet-on C1-2 transduced MCF10A cells were treated with 2. mu.g/mL doxycycline. Cell viability was measured by calcein AM at different time points. Expression of C1-2 in MCF10A cells did not result in cell death.

FIG. 6A-6C C1-2 in vitro test for MDA-MB-231 cell death after induction. (A) Each single colony of Tet-on C1-2 transduced MDA-MB-231 cells was treated with 2. mu.g/mL doxycycline (2. mu.g/mL) for 72 hours. Cell viability was measured by calcein AM. Different colonies differed in their susceptibility to death following induction by C1-2. (B) C1-2 expression of various single colonies of Tet-on C1-2 transduced MDA-MB-231 cells after doxycycline treatment was measured by Western blotting (C), and the level at 24 hours correlated with the percent mortality (normalized to full length). C1-2 expression was associated with MDA-MB-231 cell death. (C) Immunoblots of C1-2 and full-length (FL) CD95 expression of various colonies.

FIG. 7A-7C C1-2 post-induction in vivo testing for MDA-MB-231 tumor regression. (A) Tumor weight. (B) Tumor cell (million) counts (post-ficoll gradient). (C) The percentage of immune cells in the tumor (mouse CD45+) was measured by flow cytometry.

Detailed Description

ELANE cleaves CD95 to release proteolytic fragments that selectively kill a variety of cancer cells. These results, combined with previous work (Chen et al, Nature, 2010; Hadji et al, CellRep., 2014; Peter et al, Cell Death Diifier., 2015), underscore the critical selective importance of CD95 on cancer Cell viability. From a therapeutic perspective, delivery of the CD95 fragment to tumors may overcome the protective mechanisms and kill cancer cells through a genotype independent mechanism with a broad therapeutic window. CD95 degradation (generation of CD95 fragment) has been identified as a mechanism of action for ELANE to kill cancer cells and further suggests that a broad range of proteases can mimic the CD95 degradation and cancer cell killing properties of ELANE. Aspects of the invention are based, in part, on the following observations. Treatment of ELANE or neutrophil conditioned media with alpha-1-antitrypsin or PMSF (two irreversible, noncompetitive ELANE inhibitors) protects cancer cells from apoptosis. ELANE degradation purified the C-terminal domain of CD 95. Importantly, this cleavage pattern is different from that produced by MMP7, MMP7 was previously shown to cleave the extracellular N-terminal domain of CD95, thereby protecting cancer cells from FASL-mediated apoptosis (Strand et al, Oncogene, 2004).

Certain embodiments relate to therapeutic or anti-cancer compositions comprising various combinations of anti-cancer peptides or variants thereof, expression vectors or expression cassettes encoding the same. In certain aspects a peptide composition can include one or more peptides comprising, consisting essentially of, or consisting of: amino acid sequence SPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 2), or segment AINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEIQSLV

(SEQ ID NO: 3), and/or SPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEI (SEQ ID NO: 4) and/or AINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLCTLAEKIQTIILKDITSDSENSNFRNEI (SEQ ID NO: 5). Certain embodiments relate to one or more than one peptide, or variant thereof, having an amino acid sequence that is identical to SEQ ID NO: 2, more than 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, 119, 115, 112, 113, 112, 113, 114, 122, and 122, including all ranges therebetween 92. 94, 96, 98, 99 to 100% identity, including all values and ranges therebetween. The functional segment of the anti-cancer peptide can be derived from SEQ ID NO: 2, 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, 2930, 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, 119, 110, 111, 112, 111, 114, 6, 114, 6, 9, 114, 6, 9, 6, or 6, 23, 6, or 7, 6, or 7, 6, or 6, 72, 6, or 6, or more amino acids, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2930, 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, 124 or 124 bits. In certain aspects, the anticancer peptides described herein can be modified by chemical modification of amino acid side chains (e.g., cross-linking, glycosylation, etc.) or by including heterologous peptide sequences at the amino or carboxy terminus of the peptide.

I. Polypeptide compositions and formulations

In certain embodiments, the polypeptides and peptides include peptides having the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 and/or SEQ ID NO: 5 and functional segments thereof. "polypeptide" refers to any peptide or protein comprising amino acids linked by peptide bonds or modified peptide bonds. "polypeptide" may include short chain polypeptides, including peptides, oligopeptides, or oligomers, as well as long chain polypeptides, including proteins. A polypeptide may contain amino acids other than the 20 gene-encoded amino acids. "polypeptide" includes amino acid sequences modified 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 the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. It is understood that the same type of modification may be present to the same or different degrees at several sites in a given polypeptide. In addition, a given polypeptide may contain multiple types of modifications. Modifications include terminal fusions (N-and/or C-terminal), 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 phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer of RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination.

A variety of detectable labels can be attached to the polypeptides and variants thereof. For flow cytometer applications for extracellular and intracellular detection, commonly used fluorophores can be Fluorescein Isothiocyanate (FITC), Allophycocyanin (APC), R-Phycoerythrin (PE), apigenin 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-Cy 7. Other fluorophores include, inter alia, Alexa350、Alexa488、Alexa 25532、Alexa546、Alexa568、Alexa594、Alexa647 (monoclonal antibody labeling kit, 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, BODIPY630/650, BODIPY 650/665, Cascade Blue (Cascade Blue), Cascade Yellow (Cascade Yellow), Dansyl chloride (Dansyl), lisamine rhodamine B, marine Blue, Oregon green 488, Oregon green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, pt., Eugene, USA, Eugene 466G, Cy 4835, streptavidin 5, Cy5, streptavidin 5, Cy) using a secondary detection of avidin (Cyavidin labeling protein). It may be useful to label the polypeptide with biotin. The polypeptide may be labelled with a radioisotope, e.g.³³p、³²P、³⁵S、³H and¹²⁵I. as another example, when the polypeptide is useful for targeted radiation therapyThe mark may be³H、²²⁸Th、²²⁷Ac、²²⁵Ac、²²³Ra、²¹³Bi、²¹²pb、²¹²Bi、²¹¹At、²⁰³pb、¹⁹⁴Os、¹⁸⁸Re、¹⁸⁶Re、¹⁵⁵Sm、¹⁴⁹Tb、¹³¹I、¹²⁵I、¹¹¹In、¹⁰⁵Rh、⁹⁹mTc、⁹⁷Ru、⁹⁰Y、⁹⁰Sr、⁸⁸Y、⁷²Se、⁶⁷Cu or⁴⁷Sc。

The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material or culture medium from which it was originally derived (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Furthermore, an isolated polypeptide refers to a polypeptide 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 chromatographic column or embedded in a gel. In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally occur as a fragment and/or is not normally in a functional state.

The term "amino acid" or "residue" is understood to mean a compound containing an amino group (NH)₂) A carboxylic acid group (COOH) and any of a variety of pendant groups, having the basic formula NH₂CHRCOOH, and are linked together by peptide bonds to form proteins. The amino acids can be, for example, acidic, basic, aromatic, polar or derivatized. Non-standard amino acids may be referred to as "non-canonical" amino acids. Amino acids occur naturally in the alpha and L-forms, however, amino acids in the beta and D-forms can also be prepared.

One-letter abbreviation systems are commonly used to identify the twenty "canonical" amino acid residues commonly incorporated into naturally occurring peptides and proteins, and these names are well known in the art. Such single letter abbreviations may be fully interchanged in meaning with the three letter abbreviated or non-abbreviated amino acid names. The 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.

Some embodiments also include variants of the polypeptides described herein. Variants of the disclosed polypeptides may be produced by amino acid additions or insertions, amino acid deletions, amino acid substitutions and/or chemical derivatization of amino acid residues within the polypeptide sequence. One of skill in the art can determine the desired amino acid substitutions (whether conservative or non-conservative) based on the guidance provided herein for increasing stability while maintaining or enhancing the potency of the polypeptide. In some embodiments, conservative amino acid substitutions may encompass non-naturally occurring amino acid residues that are typically introduced by chemical peptide synthesis rather than by biological system synthesis.

Conservative modifications may produce peptides with similar functional, physical and chemical properties as the peptides subjected to such modifications. In contrast, substantial modification of the functional and/or chemical properties of the peptide can be achieved by selecting substitutions in the amino acid sequence that have a significantly different effect in maintaining (a) the structure of the molecular scaffold in the region of the substitution, e.g., as an alpha 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 the replacement of a natural amino acid residue with a non-natural residue, such that there is little effect on the polarity or charge of the amino acid residue at that position.

Recombinant DNA and/or RNA mediated protein expression and protein engineering techniques, or any other method of producing a peptide, can be used to produce a polypeptide disclosed herein or to express a polypeptide disclosed herein in a target cell or tissue. The term "recombinant" is understood to mean that a material (e.g., a nucleic acid or polypeptide) has been altered, either manually or synthetically (i.e., not naturally) by human intervention. Changes may be made within or removed from the natural environment or state of nature of the material. For example, a "recombinant nucleic acid" is made by recombining a nucleic acid, e.g., in a cloning, DNA shuffling, or other well-known molecular biological process. Examples of such Molecular biological methods can be found in Maniatis et al, Molecular cloning.A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982. A "recombinant DNA molecule" is composed of DNA fragments joined together by such molecular biological techniques. As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed using a recombinant DNA molecule. A "recombinant host cell" is a cell that contains and/or expresses a recombinant nucleic acid.

The polypeptide may be prepared 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, sequences encoding peptides can be excised from DNA using suitable restriction endonucleases. Any of a wide variety of available and well known host cells may be used in the practice of the various embodiments. The choice of a particular host depends on a number of factors including, for example, compatibility with the chosen expression vector, toxicity of the polypeptide encoded by the DNA molecule, transformation rate, ease of recovery of the polypeptide, expression characteristics, biological safety and cost, etc. It is recognized that not all hosts may have equivalent effects on the expression of a particular DNA sequence, and a balance should be struck between these factors. In these general guidelines, microbial host cells useful in culture include bacteria (e.g., E.coli), yeast (e.g., Saccharomyces) and other fungal cells, insect cells, plant cells, 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 codons more compatible with the host cell of choice. For E.coli, optimized codons are known in the art. Codons can be replaced to eliminate restriction sites or to include silent restriction sites, which can aid in processing DNA in a selected host cell. Next, the transformed host is cultured and purified. The host cell may be cultured under conventional fermentation conditions to express the desired polypeptide. In addition, the DNA optionally also encodes a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed polypeptide 5' to the coding region of the fusion protein.

Polypeptides may also be prepared by synthetic methods. Solid phase synthesis can be used as a technique for preparing individual polypeptides, as it is the most cost-effective method for preparing small peptides. For example, well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid 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. patent 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," G.A.Grant, Ed., W.H.Freeman & Company, New York, N.Y., 1992; "Advanced Chemtech Handbook of Combinatorial & Solid Phase Organic Chemistry," W.D. Bennet, J.W.Christensen, L.K.Hamaker, M.L.Peterson, M.R.Rhodes, and H.H.Saneii, eds., 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; fields et al, Synthetic Peptides: AUser's Guide, 77-183, 1990.

Compositions comprising a polypeptide, carrier (e.g., carrier) or half-life extending moiety covalently attached, attached or bound to another peptide, either directly or indirectly through a linker moiety, are "conjugates" or "conjugated" molecules, whether conjugated by chemical means (e.g., post-translational or post-synthetic) or by recombinant fusion. Conjugation of the polypeptide may be performed through the N-terminus and/or C-terminus of the polypeptide, or may be performed in the middle of the primary amino acid sequence of the peptide. Due to the specificity of the polypeptide for cancer cells, the polypeptide can be conjugated 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 generate fusion proteins that allow the introduction of additional moieties to enhance killing or localization of the polypeptide. Specific moieties of interest may include chemotherapeutic agents, pro-apoptotic factors, targeted therapeutic agents (e.g., kinase inhibitors, etc.), or other agents that promote killing.

In some embodiments, 1, 2, 3, or 4 polypeptides are conjugated or encapsulated in the same or different delivery vehicles, such as carriers (e.g., particles) or liposomes. In some embodiments, the coupling of the one or more than one polypeptide to the carrier comprises one or more than one covalent and/or non-covalent interaction. In one embodiment, the support is a metal or polymer particle. In one embodiment, the carrier is a liposome. The size of the particles may be micro-or nano-sized. In some aspects, the particles have a diameter of at least, at most, or about 0.1 μm to at least, at most, or about 10 μm. In another aspect, the particles have an average diameter of at least, at most, or about 0.3 μm to at least, at most, or about 5 μm, 0.5 μm to at least, at most, or about 3 μm, or 0.2 μm to at least, at most, or about 2 μm. In some aspects, the average diameter of the particles may be at least, up to or about 0.1 μm, or at least, up to or about 0.2 μm, or at least, up to or about 0.3 μm, or at least, up to or about 0.4 μm, or at least, up to or about 0.5 μm, or at least, up to or about 1.0 μm, or at least, up to or about 1.5 μm, or at least, up to or about 2.0 μm, or at least, up to or about 2.5 μm, or at least, up to or about 3.0 μm, or at least, up to or about 3.5 μm, or at least, up to or about 4.0 μm, or at least up to including about 4.5 μm, or at least, up to or about 5.0 μm, including all values and ranges therebetween.

In some embodiments, the charge (e.g., positive, negative, neutral) of the carrier is selected to confer an application-specific benefit (e.g., physiological compatibility, beneficial surface-peptide interactions, etc.). In some embodiments, the carrier has a net neutral charge or a net negative charge (e.g., to reduce non-specific binding to the cell surface that typically carries a net negative charge). In some cases, the carrier is coupled to multiple polypeptides and may have 2, 3, 4, 5, 6, 7, 8, 9, 10 … … 20, 20 … … 50 … … 100 or more than 100 copies or combinations of polypeptides of certain polypeptides exposed to the surface. In some embodiments, the vector displays a single type of polypeptide. In some embodiments, the carrier displays a plurality of different polypeptides on the surface.

As used herein, the terms "package," "encapsulate," and "encapsulate" refer to the introduction of or association of a polypeptide in a liposome or similar carrier. The polypeptide may be associated with a lipid bilayer or present in the aqueous interior of a liposome, or both.

Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The choice of lipid is often guided by considerations such as the size of the liposome and the stability of the liposome in the bloodstream. Various types of lipids are used to produce liposomes. For example, amphiphilic lipids found to be useful are zwitterionic, acidic lipids or cationic lipids. Examples of zwitterionic amphiphilic lipids are phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and the like. Examples of acidic amphiphilic lipids are phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and the like. Examples of cationic amphiphilic lipids are diacyltrimethylammonium propane, diacyldimethylammonium propane, stearylamine and the like. Examples of neutral lipids include diglycerides, such as glycerol dioleate, glycerol monopalmitoleate and mixed caprylic-capric glycerides; triglycerides, such as triolein, monopalmitin, triolein, tricaprylin and trilaurin; and combinations thereof. Additionally, in some embodiments, cholesterol or phytosterols are used, for example, to prepare multivesicular liposomes.

Liposomes can be prepared using a variety of methods, such as, for example, Szoka et al, ann.rev.biophysis.bioeng.9: 467(1980), us patent 4235871, 4501728 and 4837028, the entire contents of which are incorporated herein by reference. One method produces multilamellar vesicles of non-uniform size. 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 thin lipid film. Alternatively, the lipids can be dissolved in a suitable solvent, such as t-butanol, and then lyophilized to form a more homogeneous lipid mixture in a more easily hydrated powdered form. The film or powder is covered with an aqueous buffer solution and is hydrated, usually with stirring, within 15 to 60 minutes. The size distribution of the resulting multilamellar vesicles can be shifted to smaller sizes by hydrating the lipids under more vigorous stirring conditions or by adding solubilizing agents such as deoxycholate.

Multilamellar liposomes are formed, for example, by stirring the dispersion, preferably by using a thin film evaporator device or by shaking or vortex mixing. The unilamellar vesicles are formed by applying shear forces to an aqueous dispersion of a lipid solid phase, for example by sonication or using a microfluidization device such as a homogenizer or a french press. Shear forces may also be applied by injection, freeze-thawing, dialysis of detergent solutions from lipids, or other known methods for preparing liposomes. The size of the liposomes can be controlled using a variety of known techniques, including controlling the duration of shear forces.

A "unilamellar liposome", also known as a "unilamellar vesicle", is a spherical vesicle comprising one lipid bilayer membrane defining a single closed aqueous compartment. Bilayer membranes comprise two layers of lipids (or "lipid monolayers)", an inner layer and an outer layer. The hydrophilic head of the outer layer of the lipid molecule faces the external aqueous environment and the hydrophobic tail points inward to the interior of the liposome. The inner layer of lipids is located directly below the outer layer, with the head of the lipids facing the aqueous interior of the liposome and the tail facing the tail of the outer layer of lipids.

"multilamellar liposomes," also known as "multilamellar vesicles" or "multilamellar vesicles," comprise more than one lipid bilayer membrane defining more than one closed aqueous compartment. The membranes are arranged concentrically such that the different membranes are separated by an aqueous compartment, like an onion.

Pharmaceutical formulations and administration

Embodiments relate to compositions comprising 1, 2, 3, 4, or more than 4 anti-cancer peptides, or variants or functional segments thereof, and one or more than one of: a pharmaceutically acceptable diluent; a carrier; a solubilizer; an emulsifier; and/or preservatives. Such compositions may comprise an effective amount of at least one anti-cancer agent or complex. Thus, the use of one or more than one anticancer agent as described herein in the preparation of a pharmaceutical composition is also included. Such compositions may be used to treat a variety of cancers.

The anti-cancer agents can be formulated into therapeutic compositions in various dosage forms such as, 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 particular disease to be addressed. The composition also preferably comprises a pharmaceutically acceptable carrier, vehicle or adjuvant as is well known in the art.

Acceptable formulation components for pharmaceutical formulations are non-toxic to recipients at the dosages and concentrations employed. In addition to the anti-cancer agents provided, the compositions can also include components for modifying, maintaining or maintaining the composition, such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); an antibacterial agent; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (e.g., acetate, borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; a disaccharide; and other sugars (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); coloring, flavoring and diluting agents; an emulsifier; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate 80, triton, trimethylamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancing agents (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); a delivery vehicle; a diluent; excipients and/or pharmaceutical adjuvants. (see Remington's Pharmaceutical Sciences, 18 th edition, (A.R. Gennaro eds.), 1990, Mack Publishing Company), which is incorporated herein by reference.

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

Pharmaceutical compositions for in vivo administration are typically sterile. Sterilization may be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization may be performed before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution form. In some embodiments, the parenteral composition is placed in a container having a sterile access port, e.g., an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle, or a sterile pre-filled syringe ready for injection.

The above compositions may be administered using conventional means of delivery, including but not limited to intravenous administration, intraperitoneal administration, oral administration, intralymphatic administration, subcutaneous administration, intraarterial administration, intramuscular administration, intrapleural administration, intrathecal administration, and infusion via local catheters. Local administration (e.g., intratumoral administration) to the tumor in question is also contemplated. When the composition is administered by injection, it may be administered by continuous infusion or by single or multiple doses. For parenteral administration, the anti-metastatic agent may be administered in the form of a pyrogen-free parenterally acceptable aqueous solution comprising the desired anti-cancer agent in a pharmaceutically acceptable carrier. A particularly suitable carrier for parenteral injection is sterile distilled water in which one or more than one anticancer agent is formulated as a sterile isotonic solution, suitably preserved.

Once the pharmaceutical composition is formulated, it can be stored in sterile vials in the form of solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. Such formulations may be stored in a ready-to-use form or in a reconstituted (e.g., lyophilized) form prior to administration.

If desired, stabilizers conventionally used in pharmaceutical compositions, such as sucrose, trehalose or glycine, may be used. Typically, such stabilizers will be added in minor amounts, e.g., at least, up to, or about 0.1% to at least, up to, or about 0.5% weight/volume. Surfactant stabilizers may also be added in conventional amounts, e.g.Or(ICI Americas，Inc.，Bridgewater，N.J.，USA)。

The components used to formulate the pharmaceutical composition are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least national food (NE) grade, typically at least analytical grade, more typically at least pharmaceutical grade). Furthermore, compositions intended for in vivo use are typically sterile. If a given compound must be synthesized prior to use, the resulting product is generally substantially free of any potentially toxic agents. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

For the compounds described herein (alone or as part of a pharmaceutical composition), such dosage is at least, at most, or about 0.001mg/kg to 10mg/kg body weight, preferably at least, at most, or about 1 to 5mg/kg body weight, most preferably 0.5 to 1mg/kg body weight, including all values and ranges therebetween.

A therapeutically effective dose can be readily determined by one of skill in the art and will depend upon the severity and course of the disease, the health and response of the patient to the treatment, the age, weight, height, sex, past medical history of the patient, and the judgment of the attending physician.

In some methods, the cancer cell is a tumor cell. The cancer cells may be in the patient. The patient may have a solid tumor. In such a case, embodiments may also include performing surgery on the patient, such as resecting all or a portion of the tumor. The composition may be administered to the patient before, after, or simultaneously with the surgery. In additional embodiments, the administration to the patient can also be direct, endoscopic, intratracheal, intratumoral, intravenous, intralesional, intramuscular, intraperitoneal, regional, transdermal, topical, intraarterial, intravesical, or subcutaneous. The therapeutic composition 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 than 20 times; and the composition may be administered every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

The method may further comprise administering to the subject a second cancer therapy selected from chemotherapy, radiation therapy, immunotherapy, hormone therapy (or other targeted therapies), or gene therapy. The method may further comprise administering 1, 2, 3, 4, or all 5 polypeptides or variants thereof to the subject more than once. In certain aspects, the second cancer therapy can be the administration of ELANE or an analogous protease in combination with an anti-cancer peptide described herein.

The method of treating cancer may further comprise administering chemotherapy or radiation therapy to the patient, which may be administered more than once. Chemotherapy includes, but is not limited to, Cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, actinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, paclitaxel, carboplatin, 5-fluorouracil, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan, or any analog or derivative variant thereof. Radiotherapy includes, but is not limited to, X-ray irradiation, UV irradiation, gamma irradiation, electron beam irradiation, or microwaves. In addition, as part of the method, a microtubule stabilizing agent, including but not limited to a taxane, can be administered to the cell or patient. It is specifically contemplated that any compound or derivative or analog may be used with such combination therapies.

In some aspects, other therapeutic agents useful in cancer therapy in combination with the polypeptides described herein include anti-angiogenic agents. A number of anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, platelet response protein-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptor, placental-proliferator-related proteins, 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, a VEGFR tyrosine kinase inhibitor, and any combination thereof (e.g., anti-hVEGF antibody a4.6.1, bevacizumab, or ranibizumab).

Immunotherapy or biological response modifier therapy may be used in combination with the therapies described herein. These therapies utilize the immune system to combat disease. Immunotherapy can help the immune system recognize, 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 typically uses immune system components produced in vitro.

Examples of active immunotherapy include, but are not limited to, vaccines, including cancer vaccines, tumor cell vaccines (autologous or allogeneic), viral vaccines, dendritic cell vaccines, antigen vaccines, anti-idiotypic vaccines, DNA vaccines, or Tumor Infiltrating Lymphocyte (TIL) vaccines with interleukin 2(IL-2), or Lymphokine Activated Killer (LAK) cell therapy.

Examples of passive immunotherapy include, but are not limited to, monoclonal antibodies and targeted therapies containing toxins. Monoclonal antibodies include naked antibodies and conjugated antibodies (also referred to as labeled, labeled or loaded antibodies). Naked monoclonal antibodies do not have a drug or radioactive substance bound, while conjugated monoclonal antibodies bind to, for example, a chemotherapeutic drug (chemical label), a radioactive particle (radiolabel), or a toxin (immunotoxin).

In some embodiments, passive immunotherapy, such as a naked monoclonal antibody drug, may be used in combination with the polypeptide compositions described herein to treat cancer. Examples of such naked monoclonal antibody drugs include, but are not limited to, the anti-CD 20 antigen antibody rituximab (Rittman), whichFor the treatment of, for example, B-cell non-hodgkin's lymphoma; the antibody trastuzumab (herceptin) against the HER2 protein for use in the treatment of, for example, advanced breast cancer; the anti-CD 52 antigen antibody alemtuzumab (kanpase) for use in the treatment of, for example, B-cell chronic lymphocytic leukemia (B-CLL); the anti-EGFR protein antibody cetuximab (erbitux), for example in combination with irinotecan, for use in the treatment of, for example, advanced colorectal cancer and head and neck cancer; bevacizumab (avastin) as an anti-angiogenic therapy against VEGF proteins, in combination with e.g. chemotherapy, for the treatment of e.g. metastatic colorectal cancer. Other examples of therapeutic antibodies that may be used include, but are not limited to:(trastuzumab) (Genentech, CA), which is a humanized anti-HER 2 monoclonal antibody for the treatment of metastatic breast cancer patients;(abciximab) (Centocor), an anti-glycoprotein IIb/IIIa receptor on platelets, for preventing clot formation;(daclizumab) (Roche Pharmaceuticals, Switzerland), which is an immunosuppressive humanized anti-CD 25 monoclonal antibody for the prevention of acute kidney transplant rejection; panorex (R) D. C. A. C. A. B. A. C. A. B. C. A. C. B. A. C. A. C. B. C. A. C. A. C. A. C. B. A. C. A. B. A. C. A. B. C. B. A. C. A. C. A. C. A. C. A. C. A. B. A. B. A. B. A. B^TMA murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2, a murine anti-idiotypic (GD3 epitope) IgG antibody (ImClone system); IMC-C225, which is a chimeric anti-EGFR IgG antibody (Imclone system); VITAXIN (vitamin A)^TMHumanized anti- α V β 3 integrin antibody (Applied Molecular Evolution/medimmunee); campath 1H/LDP-03, which is a humanized anti-CD 52 IgG1 antibody (Leukosite); SmartM195, a humanized anti-CD 33 IgG antibody (protein design laboratory/Kanebo); RITUXAN^TMA chimeric anti-CD 20 IgG1 antibody (IDEC Pharm/Genetech, Roche/Zettyaku); LYMPHOCIDE^TMHumanized anti-CD 22 IgG antibodies (immunolamedics); LYMPHOCIDE^TMY-90 (Immunomedics); lymphosocan (Tc-99m label; radioimaging; Immunomedics); nuvion (for CD 3; Protein Design Labs); CM3, which is a humanized anti-ICAM 3 antibody (ICOS Pharm); IDEC-114, which is a primatized anti-CD 80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN^TMA radiolabeled murine anti-CD 20 antibody (IDEC/ScheringAG); IDEC-131, which is a humanized anti-CD 40L antibody (IDEC/Eisai); IDEC-151, which is a primatized anti-CD 4 antibody (IDEC); IDEC-152, which is a primatized anti-CD 23 antibody (IDEC/Seikagaku); SMART anti-CD 3, which is humanized anti-CD 3 igg (protein Design lab); 5G1.1, which is a humanized anti-complement factor 5(C5) antibody (mexion pharm); D2E7, which is a humanized anti-TNF-a antibody (CAT/BASF); CDP870, which is a humanized anti-TNF-ized Fab fragment (Celltech); IDEC-151, which is a primatized anti-CD 4IgG 1 antibody (IDEC Pharm/Smith Kline Beecham); MDX-CD4, which is a human anti-CD 4IgG antibody (Metarex/Eisai/Genmab); CD 20-streptavidin (+ biotin-yttrium 90; NeoRx); CDP571, which is a humanized anti-TNF- α IgG4 antibody (Celltech); LDP-02, which is a humanized anti- α 4 β 7 antibody (Leukosite/Genetech); orthodone OKT4A, a humanized anti-CD 4IgG antibody (Ortho Biotech); ANTOVA^TMHumanized anti-CD 40L IgG antibody (Biogen); ANTEGREN^TMA humanized anti-VLA-4 IgG antibody (Elan); and CAT-152, which is a human anti-TGF-. beta.2 antibody (Cambridge AbTech).

In some embodiments, passive immunotherapy, such as conjugated monoclonal antibodies, can be used in combination with the polypeptide compositions described herein to treat cancer. Examples of such conjugated monoclonal antibodies include, but are not limited to, the radiolabeled antibody ibritumomab (Zevalin), which delivers radioactivity directly to cancerous B lymphocytes for treatment of, for example, B-cell non-hodgkin lymphoma; the radiolabeled antibody tositumomab (Bexxar), which is used for the treatment of, for example, some types of non-hodgkin lymphoma; and the immunotoxin gemtuzumab ozogamicin (Mylotarg) containing calicheamicin for the treatment of, for example, Acute Myeloid Leukemia (AML). BL22 is a conjugated monoclonal antibody for the treatment of e.g. hairy cell leukemia, immunotoxins for the treatment of e.g. leukemia, lymphoma and brain tumors, and radiolabeled antibodies such as OncoScint for e.g. colorectal and ovarian cancer, ProstaScint for e.g. prostate cancer.

In some embodiments, targeted therapies containing toxins may be used in combination with the polypeptide compositions described herein to treat cancer. Targeted therapies comprising toxins are toxins linked to growth factors, or in particular embodiments to polypeptides described herein, and do not comprise antibodies.

Some embodiments further include the use of adjunctive immunotherapy in combination with the polypeptide compositions described herein, such adjunctive immunotherapy including, but not limited to, cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Macrophage Inflammatory Protein (MIP) -1-alpha, 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-alpha), and interferons (including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide (alum); bacillus Calmette-Guerin (BCG); keyhole Limpet Hemocyanin (KLH); freund's incomplete adjuvant (IFA); QS-21; DETOX; levamisole; dinitrophenyl phenol (DNP) and combinations thereof, such as combinations of interleukins (e.g., IL-2) with other cytokines such as IFN-alpha.

Some embodiments further include the use of hormone therapy (anti-hormonal agents) in combination with the polypeptide compositions described herein. Anti-hormonal agents include, but are not limited to, drugs that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (includingTamoxifen), raloxifene, droloxifene, 4-hydroxyttamoxifen, troxifene, keoxifene, LY117018, onasterone and FARESTON toremifene; aromatase inhibitors inhibiting aromatase which regulate the production of adrenal estrogens, e.g. 4(5) -imidazole, aminoglutarimide,Pregnansterol acetate,Exemestane, formestane, fadrozole,A chlorazol,Letrozole andanastrozole; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin; and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in cell proliferation, such as PKC- α, Ralf, and H-Ras; ribozymes, e.g., VEGF expression inhibitors (e.g.Ribozymes) and inhibitors of HER2 expression; vaccines, e.g. gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine;Ril-2；a topoisomerase 1 inhibitor;rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

In some embodiments, the cancer to which the compositions described herein are administered can be bladder cancer, blood 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 cell cancer.

Expression and expression vectors

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

The expression cassette, the expression vector and the sequences in the expression cassette or expression vector 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 manner. For example, a heterologous promoter can be a promoter that is not naturally associated with the nucleic acid to be expressed, or a promoter that has been introduced into a cell by a cell transformation procedure. Heterologous nucleic acids or promoters also include nucleic acids or promoters that occur naturally in an organism but have been altered in some manner (e.g., placed in a different chromosomal location, mutated, added in multiple copies, linked to non-native promoter or enhancer sequences, etc.). The heterologous nucleic acid can comprise a sequence comprising cDNA. For example, a heterologous coding region can be distinguished from an endogenous coding region by virtue of its association with a nucleotide sequence that includes regulatory elements, such as a promoter, not found naturally associated with the coding region, or by virtue of its association with a chromosomal segment that is not found in nature (e.g., a gene that is expressed in a locus where the protein encoded by the coding region is not normally expressed). Similarly, a heterologous promoter may be a promoter linked to a coding region, where the native linkage of the promoter to the coding region is not present.

Viral vectors that may be used include those associated with lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polio viruses, AIDS viruses, neurotrophic viruses, Sindbis and other viruses. Also useful are any viral families with these viral properties that make them suitable for use as vectors as well. Retroviral vectors that may be used include those described in Verma, I.M., Retroviral vectors for gene transfer in Microbiology-1985, American Society for Microbiology, pp.229-232, Washington, (1985). For example, such retroviral vectors may include the maloney murine leukemia virus, MMLV, and other retroviruses that express desired properties. Typically, viral vectors contain a nonstructural early gene, a structural late gene, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and a promoter that controls transcription and replication of the viral genome. When viruses are engineered into vectors, one or more of the early genes are typically removed, and a gene or gene/promoter cassette is inserted into the viral genome in place of the removed viral nucleic acid.

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

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells) may also contain sequences necessary to terminate transcription, which may affect the expression of mRNA. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region also contains a transcription termination site. Preferably, the transcriptional unit further comprises a polyadenylation region. One advantage of this region is that it increases the probability that the transcribed unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. Preferably, homologous polyadenylation signals are used in the expression construct.

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 may 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, e.g., CMV, SV40 and RSV promoters, and regulatable promoters, such as inducible promoters or repressible promoters, e.g., the tet promoter, hsp70 promoter, and synthetic promoters under the control of CRE. Vectors for bacterial expression include pGEX-5X-3, while vectors for eukaryotic expression include pCIneo-CMV.

The expression cassette or vector may comprise a nucleic acid sequence encoding a marker product. The marker product is used to determine whether the gene has been delivered to the cell and is expressed after delivery. Preferred marker genes are the E.coli lacZ gene encoding beta-galactosidase and green fluorescent protein. In some embodiments, the marker may be a selectable marker. When these selectable markers are successfully transferred into host cells, the transformed host cells can survive if placed under selective pressure. There are two widely used different classes of options. The first is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independently of supplemented media. The second category is dominant selection, which refers to selection schemes used in any cell type, and does not require the use of mutant cell lines. These protocols typically use drugs to prevent the growth of the host cell. Those cells with the novel gene will express a drug resistance-delivering protein 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 (Mullgan, R.C.and Berg, P.Science 209: 1422(1980)) or hygromycin (Sugden, B.et al, mol.cell.biol.5: 410-.

Gene transfer can be obtained by: direct transfer of genetic material using techniques including, but not limited to, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, and artificial chromosomes, or by transferring genetic material in cells or vectors such as cationic liposomes or viruses. Such methods are well known in the art and readily adapted for use with the methods described herein. The transfer vector may be any nucleotide construct useful for delivering a gene into a cell (e.g., a plasmid), or as part of a general strategy for delivering a gene, e.g., as part of a recombinant retrovirus or adenovirus (Ram et al, Cancer Res.53: 83-88, (1993)). Suitable means for transfection include viral vectors, chemical transfectants or physical mechanical methods such as electroporation and direct diffusion of DNA, e.g., Wolff et al, Science, 247, 1465-1468, (1990); and Wolff, Nature, 352, 815 + 818, (1991).

For example, the nucleic acid molecule, expression cassette encoding the protease, and/or vector can be introduced into the cell by any method, including but not limited to calcium-mediated transformation, electroporation, microinjection, lipofection, particle bombardment, and the like. The cells can be expanded in culture and then administered to a subject, e.g., a mammal, e.g., a human. The amount or number of cells administered may vary, but about 10 can be used⁶From one to about 10⁹The amount of individual cells. Cells are typically delivered in a physiological solution such as saline or buffered saline. The cells may also be delivered in a carrier such as a liposome, exosome or microvesicle.

The protease may be produced by a transgenic cell producing exosomes or microvesicles containing the protease. Exosomes and microvesicles mediate the secretion of multiple species of proteins, lipids, mrnas and micrornas, interacting with neighboring cells so that signals, proteins, lipids and nucleic acids can be transmitted between cells (see, e.g., 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).

Thus, transgenic cells having a heterologous expression cassette or expression vector that expresses one or more proteases can be administered to a subject, and exosomes produced by the transgenic cells deliver the proteases to the tumor and/or cancer cells of the subject.

In light of the above, the present disclosure relates to methods for producing vectors, in particular plasmids, cosmids, viruses and phages, conventionally used in genetic engineering and gene therapy, comprising a nucleic acid molecule encoding a polypeptide sequence of a protease as defined herein. In some cases, the vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus, or bovine papilloma virus can be used to deliver the polynucleotides or vectors to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant vectors. Alternatively, the nucleic acid molecule and vector can be reconstituted into a liposome for delivery to a target cell. Vectors containing the nucleic acid molecules of the present disclosure can be transferred into host cells by well-known methods, which vary depending on the type of cellular host.

Another aspect of the present invention relates to a gene therapy vector comprising the anti-cancer peptide construct. Gene therapy vectors are known in the art and include, but are not limited to, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids and the like. The construction of the gene therapy vector of the present invention can be performed by methods known in the art. In some aspects, the gene therapy vector can be about, up to or at least 10, 100, 1000, 1x10⁴1, 1 × 10⁵1, 1 × 10⁶1, 1 × 10⁷1, 1 × 10⁸1, 1 × 10⁹1, 1 × 10¹⁰1, 1 × 10¹¹1, 1 × 10¹²The amount of individual Virus Particles (VP) or Colony Forming Units (CFU), including all values and ranges therebetween, is administered.

As an example of a gene therapy vector, the expression cassette may be comprised in a lentiviral vector. The therapeutic vector can be transduced into cells in vitro and the cells delivered to the patient. Likewise, the therapeutic vectors of the present invention can be delivered directly to a patient.

Example IV

The following examples are included to illustrate preferred embodiments of the invention, along with the accompanying drawings. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Neutrophil elastase cleaves CD95 to kill cancer cells with broad safety

A. Results

Human neutrophils release factors that selectively kill cancer cells. Human peripheral polymorphonuclear neutrophils (PMNs) have a short half-life in vivo (about 8 hours) and undergo rapid apoptosis to release factors with potent antimicrobial properties extracellularly (daney 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/murine cancer cells were treated with serum-free media conditioned by purified PMNs (PMN media) circulating to apoptosis. PMN medium effectively killed all cancer cell lines tested within 24 hours. In sharp contrast, PMN medium did not kill any of the 6 human/murine normal cells or non-cancer cells tested. Conditioned medium of Omentum Neutrophils (ON) from healthy subjects also selectively kills cancer cells. However, murine neutrophils from various sources and activation states lack this cancer cell killing ability.

The killing ability of cancer cells in PMN medium is inhibited by culturing cancer cells in fetal bovine, murine or human serum. However, if serum is delivered after 5 minutes of exposure to PMN medium under serum-free conditions, the serum is unable to rescue the cancer cells, indicating that the serum contains an inhibitor that directly antagonizes one or more than one anti-cancer factor in the PMN medium (see below)). The dependence of PMN media on serum-free conditions may explain why previous studies in the presence of serum have shown that human neutrophils require intercellular contact to kill cancer cells (Yan et al, Oncoimmunology, 2014).

To begin understanding the mechanism by which PMN media kill cancer cells, representative studies focused on three cancer types with different mutation profiles: melanoma (MEL888, B16F10), lung cancer (A549, LLC1) and triple negative breast cancer (MDA-MB-231, E0771). PMN medium was found to kill all these cancer cells by inducing apoptosis.

Studies were performed to determine whether PMN media could induce apoptosis of cancer cells in vivo. Mice were injected with various cancer cells in a syngeneic model (E0771, LLC1, B16F10) and a PDX model of triple negative breast cancer (TNBC, 4195) to produce approximately 100mm³Size of the tumor. PMN media is delivered Intratumorally (IT) because serum antagonizes the cancer killing ability of PMN media in vitro. Tumors were injected daily with Human Serum Albumin (HSA) or PMN medium once a day for 5 days and examined for apoptosis by staining of TUNEL, cleaved parp (cparp) and cleaved CASP3(cCASP 3). In each test model, PMN medium attenuated tumor growth and induced cancer cell apoptosis. The effect in the syngeneic model is not due to the injection of human proteins into immunocompetent mice, but rather because inactivation of bioactive factors in PMN media prevents their anti-tumor effects. In contrast, media derived from mouse bone marrow-derived neutrophils (BMDN) failed to attenuate tumorigenesis in vivo, consistent with their inability to kill cancer cells in vitro. Consistent with its lack of toxicity to normal or non-cancerous cells in vitro, injection of PMN medium into the mammary fat pad of tumor-free C57BL/6 mice (once a day for 5 days) did not induce apoptosis at the injection site, nor did it affect body weight, spleen weight, or liver function.

ELANE is the major anticancer protein released by human PMNs. The results of the study show that PMNs release factors that selectively kill a variety of cancer cells in vitro and in vivo. To identify responsible factors, a quantitative cancer cell killing assay was developed to follow bioactive factors and to demonstrate the rationality of finding proteins using boiling, dialysis and micro concentrator (centricon) experiments. Next, studies were performed to purify the responsible protein. The PMN medium was clarified through a 0.22 μm filter and prepared for fractionation. Surprisingly, this step abolished the cancer killing activity of PMN medium from 2 independent donors without reducing the total protein level, indicating selective consumption of bioactive proteins.

Shotgun proteomic analysis identified 890 proteins (. gtoreq.2 peptides, FDR < 1%) in PMN medium, of which only 2 out of the two donors were significantly reduced by filtration (G test, p < 0.05, Bonferroni correction): elastase (ELANE) and Eosinophilic Cationic Protein (ECP). ELANE is a serine protease, which has been shown in previous studies to promote tumorigenesis (Houghton et al, Nat med., 2010), while ECP is a pore-forming protein toxic to both cancer and healthy cells (Young et al, Nature, 1986).

Two methods were used to determine whether ELANE and/or ECP were responsible for selective cancer killing activity of PMN media. First, ELANE or ECP were immunodepleted from PMN media and it was found that depletion of either protein attenuated the ability of PMN media to kill MDA-MB-231 cells. Second, cancer cells or normal or non-cancer cells were treated with purified ELANE or ECP and cell viability was monitored. ELANE kills MDA-MB-231 cells in a dose-dependent manner, and this effect is selective because it does not kill human monocyte-derived macrophages (HMDM). In contrast, ECP killed MDA-MB-231 cells only at doses that were also toxic to HMDM.

Next, MDA-MB-231 cells were treated with a concentration of ELANE (0.25. mu.g/mL) and ECP (0.05. mu.g/mL) in PMN medium. Although ECP's PMN media levels did not kill these cells alone, they significantly enhanced the selective killing ability of ELANE, indicating a synergistic effect between the two proteins.

Since ELANE affects biological pathways by proteolysis (Pham, Nat Rev immunol, 2006), it is hypothesized that its anti-cancer function may require catalytic activity, and ECP may enhance this activity to support synergistic killing. ELANE was treated with PMSF or alpha-1-antitrypsin (A1AT), inactivation was confirmed by chromogenic substrate activity assay, and catalytically inactivated ELANE was found to no longer kill cancer cells. PMSF or A1AT treatment also abolished the ability of PMN media to kill cancer cells in vitro and in vivo. These results indicate that ELANE is the major anticancer factor in PMN media. Indeed, the ELANE catalytic activity in PMN medium from 9 healthy donors was closely related to its ability to kill MDA-MB-231 cells.

Understanding the anti-cancer function of human neutrophils via ELANE also helps explain why murine neutrophils lack this ability. Although murine ELANE kills cancer cells and murine neutrophils contain ELANE that is catalytically active within the cell and release ELANE during apoptosis, this released ELANE is not catalytically active.

A study was conducted to further determine if ECP can enhance the catalytic activity of ELANE. ELANE was incubated with increasing concentrations of ECP, which was found to be a type II allosteric activator of ELANE, with ECP having high affinity (K)_D17nM) bound ELANE and catalytically converted (k)_cat) An increase of about 12 fold. Co-immunoprecipitation experiments demonstrated that ECP binds to ELANE in human PMN medium. These results may explain why consumption of ECP from PMN medium (and also ELANE) attenuated its killing activity, even though ECP was not toxic to cancer cells at the doses present in PMN medium. Furthermore, since 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) in PMN media. Looking to the future, ELANE is the focus because it is both effective and safe in vitro and can mimic ECP's ability to enhance its catalytic activity by increasing the concentration of ELANE. Indeed, higher doses of ELANE (3 μ g/ml) induced apoptosis effectively in all cancer cell lines tested, but were not toxic to all normal or non-cancerous cells tested.

ELANE selectively kills cancer cells by cleaving CD 95. How does ELANE selectively kill cancer cells? Research has focused on the CD95 pathway, as it is critical for the survival of a variety of cancer cells, but largely dispensable for the survival of normal or non-cancer cells (Chen et al, Nature, 2010), with unique properties reflecting the broad anticancer and low-toxicity characteristics of ELANE. Furthermore, CD95 function can be regulated by proteolysis (Strand et al, Oncogene, 2004). Previous studies have shown that using shRNA to reduce CD95 in cancer cells activates a powerful killing program characterized by inhibition of survival pathways, and 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 procedure was not induced in Cd 95-/-normal or non-cancerous cells. ELANE is therefore hypothesized to kill cancer cells through a mechanism involving CD95 cleavage. To begin to test this hypothesis, a study was conducted to investigate whether ELANE could mimic the unique killing program caused by CD95 knockdown in cancer cells (Chen et al, Nature, 2010). ELANE treatment (i) inhibited at least one survival pathway (ERK, nfkb or JNK phosphorylation (p)), (ii) induced DNA damage (γ H2AX), (iii) increased MT ROS, and (iv) activated proapoptotic pathways (cCASP3 and cprp) in all 6 cancer cell lines tested (fig. 1 a). Consistent with its lack of toxicity to normal or non-cancerous cells, ELANE did not induce this procedure in all tested normal or non-cancerous cells (fig. 1 a).

In view of the ELANE mimicking the killing program observed in CD95null cancer cells (CD95null cancer cells), it was determined whether ELANE could cleave CD95 in cancer cells and, if so, whether this resulted in the loss of CD 95. Cancer cells were treated with ELANE and a lower molecular weight CD95 band was observed shortly before cell death (FIGS. 1b-1 c). Although ELANE cleaves CD95 in all cancer cells tested, this cleavage did not result in loss of full-length CD95, suggesting that ELANE cleavage of CD95 may kill cancer cells through gain-of-function rather than loss-of-function processes. Consistent with this explanation, overexpression of human CD95(hCD95) or mouse CD95(mCD95) in cancer cells did not prevent ELANE-mediated killing, but accelerated killing (fig. 1d, fig. 2).

To identify the ELANE cleavage site in hCD95, the recombinant protein hCD95, corresponding to the N-terminal (aa.1-173 of SEQ ID NO: 1) or C-terminal (aa.212-335 of SEQ ID NO: 1) domain of hCD95, was incubated with ELANE. The results show that ELANE preferentially cleaves the C-terminal domain (fig. 1e), and mass spectrometry identifies two major cleavage sites, site 1: v²²⁰And A²²¹Position 2: i is³²¹And Q³³²In turn, resulted in proteolytic release of the Death Domain (DD) of CD95 (fig. 1 f). Both sites mapped well to the sequence specificity of ELANE and studies with synthetic peptides confirmed these findings (fig. 1g-1 h).

Since CD95 is a plasma membrane protein and its C-terminal domain is intracellular, it was concluded that its anti-cancer function should require ELANE internalization. In fact, cancer cells rapidly internalize fluorescein-labeled ELANE, while internalized ELANE retains its catalytic activity (fig. 1i-1 j). Endocytosis inhibitor Dynasore treatment attenuated ELANE uptake and protected cancer cells from apoptosis (fig. 1 j). Dynasore also protects cancer cells from PMN medium-mediated killing. Furthermore, overexpression of the C-terminal domain of CD95 comprising two cleavage sites (aa.212-335 of SEQ ID NO: 1) accelerated ELANE-mediated killing in human and murine cancer cells, while overexpression of the N-terminal domain (aa 1-209 of SEQ ID NO: 1) had NO effect (FIG. 1d, FIG. 2). These results indicate that cleavage of the released C-terminal proteolytic fragment of CD95 by ELANE may be sufficient to induce apoptosis in cancer cells.

To further test this possibility, C-terminal hCD95 proteins that mimic ELANE cleavage at position 1 (aa.221-335 of SEQ ID NO: 1) and/or position 2 (aa.212-331 of SEQ ID NO: 1) were overexpressed, and expression of either of these proteins in the absence of ELANE was found to induce cancer cell death (FIG. 1 k). In sharp contrast, overexpression of these same C-terminal hCD95 proteins in MCF10A cells or human retinal adipose tissue fibroblasts from healthy subjects did not induce toxicity (fig. 1 k). Thus, while normal or non-cancerous cells internalize ELANEs, and the internalized ELANEs are catalytically active, the proteolytic fragment of CD95 is not toxic to them (fig. 1a, 1d, 1i, 1 k). These findings potentiate the specificity of this killing mechanism for cancer cells.

IT-delivered ELANE attenuated tumorigenesis. ELANE has been shown to selectively kill cancer cells in vitro, and IT was investigated whether IT-delivered ELANE could slow tumor progression in vivo. To establish the treatment conditions, various doses of ELANE were injected into the E0771 TNBC model and 12 μ g/day was found to provide reproducible good therapeutic effects. Inactivation of ELANE with PMSF proved to eliminate this therapeutic benefit and PMSF-ELANE did not produce undesirable deterioration. Finally, the results show that the injection of ELANE (12 μ g/day) into the mammary fat pad of non-tumor bearing C57BL6 mice did not produce significant side effects.

Next, the therapeutic potential of ELANE was explored in TNBC, lung cancer and melanoma models. Injecting MDA-MB-231, A549 or MEL888 cells (xenograft model) into athymic nude mice; SCID mice with M1 or 4195 tumors (TNBC PDX model) and C57BL/6 mice with E0771, LLC1, or B16F10 cells (syngeneic model) to generate tumors of approximately 100mm3 size. At this time, IT was injected with PMSF-ELANE or ELANE (12 μ g/day) and monitored for effects on tumor growth.

ELANE attenuated tumor growth in all models tested. Immunohistochemistry showed increased staining of TUNEL, cPARP and cCASP3 in all ELANE treated tumors, indicating that ELANE induced cancer cell apoptosis 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 mechanism work, indicating that the antitumor effect of ELANE is both targeted and safe.

To explore whether cancer cells could acquire resistance to ELANE, a combination of in vitro and in vivo approaches were used. MDA-MB-231 cells were not rendered resistant by repeated exposure to ELANE in vitro (7 exposures to yield approximately 90% death/exposure). Likewise, repeated treatment of E0771 or MEL888 tumors with ELANE in vivo (once a day for 7 days) did not diminish the ability of ELANE to kill isolated cancer cells ex vivo. These findings suggest that resistance to ELANE may be challenging, similar to what was previously reported in CD95 knockdown cancer cells (Murmann et al, Oncotarget, 2017).

IT-delivered ELANE induced CD8+ T cells to attack distant tumors. The dependence of ELANE on ITs catalytic activity for ITs anti-cancer function, as well as the abundance of serine protease inhibitors in the blood, limits ITs therapeutic delivery to IT pathways and is a barrier to entry into metastatic sites. This obstacle can be overcome if the action of ELANE on the primary tumour can induce/activate immune cells to attack distant tumours (this property is known as the ectopic effect (Ngwa et al, Rev Cancer, 2018)).

To test this possibility, studies were conducted to examine whether ELANE could increase tumor immunityA cell. In all three immunocompetence models tested, ELANE increased Dendritic Cells (DCs), CD8⁺T cells and CD8⁺T effector cells (CD 8)⁺T_eff) The number of the cells. No increase in immune cells was observed if ELANE was injected into the mammary fat pad of tumor-free mice, and no increase in immune cells was observed if ELANE was injected into the opposite site (i.e., tumor-opposite) of tumor-bearing mice, where it failed to attenuate tumorigenesis. Thus, ELANE increases both innate and adaptive immune cells in tumors, an effect that is not due to an immune response to human ELANE.

A study was conducted to determine whether treatment of primary tumors with ELANE produced an immune response to reduce tumorigenesis at distant sites. E0771 cells were injected into the left (1 ° tumor) and right (2 ° tumor) mammary fat pads to produce genetically identical tumors. Treatment of 1 ° tumors with ELANE (12 μ g/day for 5 days) attenuated tumorigenesis in both sites, and this effect was specific, since treatment of 1 ° E0071 tumors with ELANE did not affect tumorigenesis of 2 ° B16F10 tumor, and was not due to ELANE overfill. To test this in another model, 1 ° B16F10 tumor was generated in the flank and B16F10 cells were injected into the tail vein to generate 2 ° lung metastases. Treatment of 1 ° tumors with ELANE (12 μ g/day for 5 days) reduced the number of lung metastases. Importantly, depletion of CD8+ T cells attenuated the ectopic effect of ELANE in both models.

Notably, ELANE therapy produced "craters" in approximately 40% of tumors in all tested immunocompetent models. No pits were observed in tumor-free mice, nor were they observed in any immunocompromised cancer model, including CD8^-T cell-depleted C57BL/6 mice. These data indicate that ELANE-mediated cancer cell killing, combined with a subsequent adaptive immune response, forms a "pit" in the tumor. However, the underlying mechanism of pit formation and its importance for therapeutic efficacy require further investigation.

In vitro and in vivo expression of peptides. FIG. 3 illustrates the effect of C1-2 expression on survival, stress and apoptotic pathways in cancer (e.g., MDA-MB-231, MEL888 and A549) and non-cancer cells (MCF10A and fibroblasts). FIG. 4 illustrates the scheme for expressing C (aa.157-335) or C1-2(aa.221-331) in the doxycycline inducible Tet-on system. C1-2 (fragment containing DD) after doxycycline addition (in vitro and in vivo). C1-2 expression selectively killed cancer cells (FIGS. 5A-5C). In vitro testing confirmed that C1-2 induced cell death in MDA-MB-231 cells (FIGS. 6A-6C). In vivo testing demonstrated C1-2-induced MDA-MB-231 tumor regression (FIGS. 7A-7C).

B. Method of producing a composite material

Human studies have been approved by the institutional review board of chicago university (IRB 16-0321). The animal studies were approved by the institutional animal care and use committee of chicago university (ACUP72209, 72504). Cancer cell lines and virus studies were approved by the institutional biosafety committee (IBC 1503).

A cell line. Ovarian cancer cell lines-CAOV 3, OVCAR3, OVCAR4, OVCAR5, A2780/CP70, HeyA8, TykNu, SKOV3, ID8, and ID8p 53-/-were gifted by Dr Ernst Lengyel university of Chicago. Breast cancer cell lines-MDA-MB-231, MDA-MB-231.BM1, MCF-7, M6C, E0771 and E0771.LMB were gifted by Dr Marsha Rosner, university of Chicago. The colon cancer cell line RKO, the glioblastoma cancer cell line T98G, the osteosarcoma cell lines U-2OS and Saos-2, and the hepatocellular carcinoma cell line HepG2 were offered by doctor Kay McLeod, university of Chicago. The lung cancer cell line NCI-H552 was obtained from ATTC as a gift from doctor Stephanie Huang, Chicago university, A549 and LLC1 cells. Melanoma cell lines-B16F 10, Mel888, Mel1106 and SK-MEL-28 cells were complimentary to doctor Thomas Gajewski, university of Chicago. The pancreatic cancer cell line PANC1 was hewed by Yamuna Krishnan doctor, university of chicago. Leukemia cell line K562 was offered by Dr.W. Amittha Wickrema, university of Chicago. Cells were cultured in Dubecco modified Eagles medium (DMEM; HyClone) containing 10% heat-inactivated FBS (Gemini Bio products) and 1% penicillin/streptomycin (Gibco).

Prostate cancer cell lines-CWR 22Rv1, LAPC4, and LNCaP were hewed by Dr doctor Donald Vander Griend, university of Chicago. The glioblastoma cell lines SK-N-BE (2) and NBL-WN cells were gifted by the university of Chicago Lucy Godley doctor. Cells were cultured in RPMI 1640 medium (Hyclone) containing 10% heat-inactivated fbs (gemini Bio products) and 1% penicillin/streptomycin (Gibco).

The mammary epithelial cell line MCF10A was offered by Dr. Marsha Rosner, Chicago university. Cells were incubated in the presence of 10% heat-inactivated FBS (Gemini Bio products), 20ng/mL EGF (Peprotech), 0.5mg/mL hydrocortisone (Sigma), 100ng/mL cholera toxin (Sigma), 10. mu.g/mL insulin (Sigma), 1% penicillin/streptomycin (Gibco).

Primary human blood-derived cells. Human peripheral blood was donated from healthy volunteers with approval from the institutional review board of chicago (IRB16-0321) and after written consent was obtained. Blood was collected into an EDTA-coated collection tube (BD Vacutainer), and cells were separated with Ficoll Paque Plus (GE Healthcare) to obtain a buffy coat (containing monocytes and lymphocytes) and a bottom coat (containing neutrophils and erythrocytes). Human peripheral blood neutrophils (PMNs) -as previously described (Kuhns et al, Curr protocol immunol, 2015), PMNs were purified from the bottom layer by repeated RBC dissection. HMDM-monocytes were purified from buffy coats using CD14 microbeads (miltenyl biotec) and differentiated into HMDM using human M-CSF (125ng/mL, R & D Systems) as previously described (Kratz et al, Cell metab., 2014). Human lymphocytes-human lymphocytes were isolated from buffy coats by collecting streams from CD14 and CD16 microbeads (Miltenyi Biotec). The resulting cell population is compromised by about 90% T cells and about 10% B cells.

Primary human omentum adipose-derived cells. Human retinal adipose tissue was obtained from healthy volunteers after approval by the institutional review board of chicago and written consent. Human retinal neutrophils (ON) -retinal tissue was digested with collagenase type 1 (Worthington, 1mg/mL) at 37 ℃ for 75 minutes with shaking at 130rpm to obtain Stromal Vascular Cells (SVC). The ON was isolated from SVC using CD16 microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Human omentum fibroblasts-human primary fibroblasts were isolated from omentum adipose tissue and cultured in dmem (hyclone) containing 20% heat-inactivated fbs (gemini Bio products) and 1% penicillin/streptomycin (Gibco) as previously described (Kenny at al, Int J Cancer, 2007).

Primary mouse cells. Unless otherwise stated, cells were isolated from 6-8 week old C57BL/6 mice. Murine bone marrow-derived macrophages (BMDM) -BMDM was differentiated from bone marrow stem cells using L Cell conditioned media as previously described (Kratz et al, Cell mate., 2014). Mouse splenocytes-mouse splenocytes were isolated as described previously (rerdon et al, Cell Rep, 2018). The resulting cells consisted of about 5% neutrophils, about 2% monocytes, about 38% T cells, about 53% B cells. Mouse primary keratinocytes-mouse primary keratinocytes were isolated and cultured in E low calcein medium containing 15% heat-inactivated FBS and 1% penicillin/streptomycin, as described previously (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 described previously (Swamydas et al, Curr Protoc Immunol. 2015). For PMA activation, BMDN was treated with PMA (100nM, Abcam) for 15 min, washed and cultured to collect conditioned medium. Murine thioglycolate-induced Peritoneal Neutrophil (PN) -PN was isolated from the peritoneal cavity 7 hours after injection of 4% thioglycolate (3 mL/mouse, Sigma), as previously described (Swamydas et al, Curr protocol immunol., 2015). Murine Tumor Associated Neutrophils (TAN) -E0771 cells were injected into the mammary fat pad of C57BL/6 mice. When the tumor volume reaches about 500mm³In this case, the tumor was digested with collagenase type 4 (Worthington, 3mg/mL) and hyaluronidase (Sigma, 1.5mg/mL) at 37 ℃ for 45 minutes with shaking at 200 rpm. TAN was purified using Ly6G microbeads (Miltenyi Biotec) according to the manufacturer's protocol. Murine Lung Neutrophils (LN) -murine LNs were isolated from 8-9 weeks (time point before metastatic spread) of large MMTV-PyMT mice. Lungs were digested with Liberase TL (Roche, 200. mu.g/mL) and DNase I (Sigma, 0.1mg/mL) as previously described (Swamydas et al, Curr Protoc Immunol. 2015). Murine LN was purified using Ly6G microbeads (Miltenyi Biotec) according to the manufacturer's protocol.

A collection of neutrophil media. Freshly isolated human or murine neutrophils were seeded in serum-free DMEM for 24 hours. Conditioned media were collected, spun at 500Xg for 5 minutes, and media protein levels were determined by Bradford assay (Biorad).

PMN media manipulations. For boiling, PMN medium was boiled at 95 ℃ for 5 minutes, and then spun at 15000 x g for 10 minutes to remove precipitated proteins. For dialysis, PMN medium was placed in Slide-a-Lyzer^TMIn a cassette (3.5kDa cut-off, ThermoFisher Scientific) and dialyzed against 2X4L PBS for 4 hours at 4 ℃. For PMSF inactivation, PMN medium was either PMSF (1mM, Sigma) or A1AT (42nM, Athens Research)&Technology) and incubated at room temperature for up to 2 hours. The residual PMSF was removed using a PD-10 desalting column (GE Healthcare Life). Inhibition of ELANE catalytic activity was confirmed using a chromogenic substrate activity assay (see below). For serum spiking experiments, human, mouse or fetal bovine serum (1% or 10%) was added to PMN medium before or after exposure of cancer cells. For the immunodepletion study, Pierce was used^TMProtein A/G magnetic beads (ThermoFisher Scientific) coupled anti-ELANE (N2C3, GeneTx) or anti-ECP (MBS2535165, MyBioSource) immunoprecipitated ELANE or ECP.

And (4) measuring the in vitro cell viability. Cancer cells or normal or non-cancer cells are seeded in complete growth medium and grown to 80-90% confluence. Cells were washed with serum-free DMEM, treated with various therapeutic agents (e.g., PMN medium, ELANE, etc.) for 4-24 hours, and cell viability was assessed using several methods: calcein-AM assay. 4-24 hours after treatment, cells were incubated with calcein-AM (ThermoFisher Scientific, 4ng/mL), washed with serum-free DMEM, and fluorescence was measured at 495nm/516nm using a Synergy HT Multi-Mode Microplate Reader (Biotek). CASP3 Activity assay-useThe 3/7 assay system (Promega) measures cell-associated CASP3 activity 6 hours after treatment. Luminescence was measured using a Victor X3 luminometer (PerkinElmer). ANXA5 staining-30 min-6 h after treatment, FITC Annexin V apoptosis detection kit (BD Pharmingen) was used according to the manufacturer's protocol^TM) Cells were stained. Using FACSCANTO^TMII flow cytometer (BD Pharmingen^TM) The samples were analyzed.

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

Mitochondrial ROS measurement. Cells were treated with different doses of ELANE for 30 minutes, washed, labeled with CM-H2DCFDA dye (ThermoFisher Scientific, 10 μ M) for 30 minutes at 37 ℃, and fluorescence quantified by flow cytometry.

CD95 overexpression studies. Polycistronic adenoviral vectors were prepared to express the human and mouse CD95 sequences, followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site and dTomato sequence under the control of the Cytomegalovirus (CMV) promoter (VectorBuilder). Human and murine 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 that mimics cleavage of ELANE at site 1 (human: aa221-335), site 2 (human: aa 212-331), or both sites (aa 221-331). Cancer cells or normal cells or non-cancer cells were transduced with adenovirus at an MOI of 50-250 depending on the cell type, and the expression of dTomato and CD95 (human: 558814; mouse, mouse: 565130, BD Biosciences) was confirmed by flow cytometry. Vectors encoding GFP (vector Builders) were used as controls.

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

Recombinant mouse ELANE activation. Recombinant mouse ELANE (50. mu.g/mL) was activated with CTSC (50. mu.g/mL) according to the manufacturer's protocol (R & D Systems).

Shotgun proteomics analysis. PMN medium from 2 independent donors was passed through a 0.22 μm filter (MilliporeSigma). Pre-and post-filtration media (50. mu.g) were digested with trypsin.

The ELANE cleavage site in CD95 was identified by mass spectrometry. Recombinant human C-terminal CD95(aa212-335, MyBioSource, 10. mu.g) or recombinant N-terminal CD95(aa 1-173, ThermoFisher Scientific, 10. mu.g) were digested with human ELANE (0.1. mu.g) at 37 ℃ for 2 hours and the reaction was stopped with SDS-PAGE loading buffer. Proteins were run on 20% SDS-PAGE gels, stained with Coomassie blue (ThermoFisher Scientific), and bands were excised for mass spectrometry. Protein was extracted from the excised band.

The recombinant peptides were analyzed by mass spectrometry. Recombinant peptides (10. mu.M) corresponding to aa214- (TLNPETVAINLSDVDLSK) -231(SEQ ID NO: 6) or 317- (DITSDSENSNFRNEIQSLV) -335(SEQ ID NO: 7) of human CD95(ThermoFisher Scientific) were incubated with ELANE (0.1-0.2. mu.M) at 37 ℃ for 15-30 minutes. The reaction was stopped with 0.1% formic acid.

Tumor inoculation and treatment. MDA-MB-231 cells (2X 10)⁶) A549 cells having type 3 Matrix (2X 10)⁶) Or MEL888 cells (2X 10)⁶) Injection into athymic nude mice (Charles River); injecting M1 or 4195 cells (50000) into nod. scid mice (JAX); e0771 cells (0.5X 10)⁶) LLC1 cell (0.5X 10)⁶) Or B16F10 cells (1X 10)⁶) Injection into C57BL/6 mice (JAX). For the TNBC model (MDA-MB-231, E0771, M1, 4195), cells were injected right ventrallyThe 4 th mammary fat pad of (1). For all other models (a549, LLC1, MEL888, B16F10), cells were injected into the flank. Once the tumor reaches about 100mm³Intrathecal (IT) delivery of ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L), or neutrophil medium or HSA (50. mu.g/100. mu.L), once a day for 5 days. Tumor volume was assessed with calipers when tumor volume in control mice reached > 1000mm³The experiment was terminated.

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(9661) and cprp (9625) antibodies from Cell Signaling Technology. Signals were displayed using VECTASTAIN ABC kit (Vector Laboratories) or fluorescently labeled secondary antibody (melanoma model). For TUNEL analysis, DeadEnd was used^TMStaining was done using colorimetric or fluorescent TUNEL system (Promega). Nuclei were labeled with hematoxylin or Hoechst 33342(ThermoFisher Scientific). Images were obtained using a Nikon Eclipse Ti2 microscope and analyzed using NIS-Elements software.

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

And (5) researching the far-field effect. For the E0771 model, 0.5x10 would be used⁶Cells were injected into the 4 th mammary fat pad on the right ventral side (1 ℃ tumor) of C57BL/6 mice, and 0.4X10 cells were injected⁶Individual cells were injected into the 4 th mammary fat pad on the left ventral side (2 ° tumor) of C57BL/6 mice. Once 1 ° tumor reaches about 100mm³ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L) IT was injected into 1 ℃ tumors once a day for 5 days and the 1 ℃ and 2 ℃ tumor volumes were measured by calipers. For the B16F10 model, 0.5x10⁶Individual cells were injected into the flank (1 ° tumor). After 7 days, 0.2X10⁶Individual cells were injected into the lateral tail vein to generate 2 ° lung metastases. Once 1 ° tumor reaches about 100mm³ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L) IT was injected into 1 ℃ tumors once a day for 5 days, and the 1 ℃ tumor volume was measured by calipers. Lungs were excised 10 days after the last ELANE treatment and 2 ° lung metastases were counted. For CD8⁺T cell depletion, anti-mouse CD8 a (clone 2.43, Bio X cells) or rat IgG2b (isotype control, Bio X cells) (200 μ g/injection) was injected Intravenously (IV) 3 days before the first ELANE treatment and once a week after the last ELANE treatment. CD8⁺T cell depletion was confirmed by flow cytometry.

For "overfill control", 0.5x10 would be⁶One E0771 cell was injected into the 4 th mammary fat pad on the left ventral side (2 ° tumor). ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L) was injected into the tumor-free 4 th mammary fat pad of the right flank pad once a day for 5 consecutive days, and 2 ℃ tumor volume was measured with a caliper.

For "specificity control", 0.5 × 10⁶One E0771 cell was injected into the 4 th mammary fat pad on the right ventral side (1 ° tumor), and 7 days later, 1x10 cells were injected⁶Individual B16F10 cells were injected into the flank (2 ° tumor). Once 1 ° tumor reaches about 100mm³ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L) was injected into 1 ℃ tumors once a day for 5 days, and the 1 ℃ and 2 ℃ tumor volumes were measured by calipers.

Evaluation of potential side effects. ELANE or PMSF-ELANE (11.6. mu.g/100. mu.L) was injected into the 4 th mammary fat pad of the right flank pad of the tumor-free C57BL/6 mice once a day for 5 days. One day after the last injection, mice were studied for potential side effects. Body and spleen weights were measured. Apoptosis at the injection site was studied by isolating, fixing and staining TUNEL, cCASP3 and cPARP of mammary adipose tissue using the same method as the above tumors. The breast adipose tissue immune cell population at the injection site was determined by flow cytometry using the same method as the above tumor. Liver function was assessed by measuring plasma ALT activity using an alanine transaminase colorimetric activity assay kit (Cayman Chemical).

And (4) testing in vivo. Mammary glands of nude mouse models were injected with 200 ten thousand Tet-on C1-2 transduced MDA-MB-231 cells. Once the tumor reaches about 80 to 100mm³Mice received a doxycycline diet every 5 days or were injected intraperitoneally with doxycycline (50mg/ml) to induce the Tet-on system. Tumor growth was monitored. Tumors were isolated and digested on day 21 post doxycycline treatment, tumor weight was measured, tumor cell number was counted, and CD45+ cells were measured by flow cytometry.

And (6) counting. Statistical significance was determined by student two-tailed unpaired t-test, except for proteomic studies. Linear regression, Michaelis-Menten and hyperbolic fitting were performed using Prism v.7 software.