Phosphatidylserine Complex

Abstract

Provided herein, in certain embodiments, are complexes capable of binding phosphatidylserine (PS) and Toll-like receptors (TLRs), and their use for the treatment of selected diseases and disorders, such as cancer.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 295,462, filed December 30, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] SUMMARY OF THE DISCLOSURE Embodiments of the present invention relate to phosphatidylserine-binding (PS-binding) complexes capable of activating or stimulating Toll-like receptors (TLRs) and methods of using these complexes to treat cancer. Summary of the Invention

[0003] One aspect of the present disclosure relates to a phosphatidylserine-binding (PS-binding) complex, the complex comprising at least one PS-binding domain, and the complex comprising at least one Toll-like receptor-binding (TLR-binding) domain.

[0004] Another aspect of the present disclosure relates to a method of treating a subject having cancer comprising administering to the subject a conjugate disclosed herein. [Brief description of the drawings]

[0005] [Figure 1] 1 is a graph depicting exemplary growth curves of syngeneic subcutaneous tumors of the CT26 colon cancer cell line in BALB / c mice treated with one or more cycles of cisplatin, CBP501, anti-CTLA-4 antibody, TSA005, and / or vehicle.

[0006] [Diagram 2] 1 is a graph depicting exemplary growth curves of CT26 colon cancer cell line, syngeneic subcutaneous tumors in BALB / c mice treated with one cycle of cisplatin, CBP501, anti-CTLA-4 antibody, TSA005, TSA009, or vehicle.

[0007] [Diagram 3]1 is a flow cytometry plot showing an exemplary analysis of the cyanine-5.5 (Cy5.5) versions of TSA005 and TSA006 stained with FITC-Annexin V. The data in this figure show dual staining of Annexin V with TSA005 or TSA006.

[0008] [Figure 4] Figure 4 shows an exemplary effect of TSA005 or TSA006 on the CT-26 mouse colon cancer cell line implanted subcutaneously in syngeneic BALB / c mice. Figure 4A shows an exemplary growth curve of the CT-26 mouse colon cancer cell line implanted subcutaneously in syngeneic BALB / c mice. This figure shows the tumor growth suppression effect of the addition of TSA005 or TSA006 on cisplatin + CBP501 + anti-CTLA4 treatment. In this figure, "ip" stands for "intraperitoneal injection" and "iv" stands for "intravenous injection". Figures 4B-4D show the individual tumor growth curves of the experiment shown in Figure 4A (Figure 4B: CDDP + 501 + aCTLA4 + saline treatment, Figure 4C: CDDP + 501 + aCTLA4 + TSA005 treatment, Figure 4D: CDDP + 501 + aCTLA4 + TSA006 treatment).

[0009] [Diagram 5] 1 shows the distribution of the Cy5.5 version of TSA006 (TSA013) following treatment of syngeneic BALB / c mice implanted subcutaneously with the CT26 murine colon carcinoma cell line with cisplatin, CBP501, and TSA013. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The success of immune checkpoint inhibitors, namely anti-CTLA4, anti-PD1, and anti-PDL1 antibodies, has established immunotherapy as the fifth pillar of cancer fighting, in addition to surgery, radiation therapy, chemotherapy, and molecular targeted therapy (Reference 1). Antibodies and genetically modified T cells that exploit T cell functions are being investigated. Some of these treatments have already been approved or will soon be approved by the US FDA as anticancer drugs (Reference 2).

[0011] The effects of these immunological drugs that utilize T cells tend to be robust and have been shown to significantly improve survival and patient outcomes. However, these treatments appear to be effective in only a minority of patients. Thus, there remains a large unmet medical need (Reference 1).

[0012] T cells belong to one of the two major immune systems in mammals and humans: the adaptive immune system. The adaptive immune system, also called the acquired immune system, is a subsystem of the immune system that consists of specialized, systemic cells and processes that eliminate pathogens or prevent their proliferation. The acquired immune system is one of the two major immune strategies found in vertebrates (the other being the innate immune system). Like the innate immune system, the adaptive immune system contains both humoral and cell-mediated immune components to destroy invading pathogens. Unlike the innate immune system, which is preprogrammed to respond to general, broad categories of pathogens, the adaptive immune system is highly specific for each particular pathogen encountered by the body.

[0013] The innate immune system is a defense strategy that has evolved over a relatively long time. Its main functions are to recruit immune cells to the site of infection by producing chemical factors, including chemical mediators such as cytokines, to recognize bacteria, activate cells, and promote the clearance of antibody complexes and dead cells by activating the complement cascade, to recognize and remove foreign bodies in organs, tissues, blood, and lymphatic fluids by specialized white blood cells, and to activate the adaptive immune system through antigen presentation.

[0014] The innate immune system tends to be activated prior to the adaptive immune system, initiating and / or enhancing its activity. Among the components of the innate immune system, Toll-like receptors (TLRs) have been considered as interesting candidates to induce the activation of the immune system against cancer. To date, many compounds have been studied in basic research, many of which have been subjected to human clinical trials (Reference 3). Although many TLR agonists show promising anticancer effects in animal models, many of them have not yet shown efficacy in clinical trials. Some of the reasons for the failure of TLR agonists in clinical trials are systemic toxicity and / or lack of activity at tolerated doses (Reference 4).

[0015] Targeting tumors with antibodies has been successful in many cancer types, but these methods have proven less effective when treating solid tumors, thought to be because these large molecules tend not to penetrate the dense stromal tissue (7).

[0016] To overcome this problem, the present invention provides compounds containing phosphatidylserine (PS)-binding peptides that target apoptotic cells and deliver TLR agonists to tumor sites. Upon binding, such complexes trigger phagocytosis of apoptotic cells by antigen-presenting cells (APCs). This allows anti-cancer drugs to act against a variety of tumors, either alone or in combination with other anti-tumor drugs that are expected to induce apoptosis of tumor cells and / or act via the adaptive immune system.

[0017] Phosphatidylserine (PS) is primarily retained in the inner leaflet of the plasma membrane within cells, but is externalized to the cell surface during apoptosis. Externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Such phagocytosis is thought to silence the immune system against antigens contained in the phagocytosed cells. It is also thought to be one of the mechanisms by which cancer cells escape immune surveillance. By being targeted for phagocytosis by macrophages, a large number of dying cancer cells would not provoke an antigen-specific response from the immune system (Reference 8).

[0018] Here, the present invention activates immune responses against antigens present on apoptotic and / or dying cells by conjugating TLR agonists to PS-binding peptides.

[0019] One aspect of the present disclosure relates to a phosphatidylserine-binding (PS-binding) complex, the complex comprising at least one PS-binding domain, and the complex comprising at least one Toll-like receptor-binding (TLR-binding) domain.

[0020] As used herein, the term "conjugated" when referring to two moieties means that the two moieties are attached, and the bond connecting the two moieties can be covalent or non-covalent. In some embodiments, the two moieties are covalently bonded to each other (e.g., directly or through a covalently bonded intermediate). In some embodiments, the two moieties are non-covalently bonded (e.g., ionic bond, van der Waals bond / interaction, hydrogen bond, polar bond, or combination or mixture thereof).

[0021] In some embodiments, the PS binding domain is a peptide sequence, non-limiting examples of which include LIKKPF (SEQ ID NO: 1), PGDLSR (SEQ ID NO: 2), CLIKKPF (SEQ ID NO: 3), CPGDLSR (SEQ ID NO: 4), FNFRLKAGAKIRFG (SEQ ID NO: 5), FXFXLKXXXKXR (SEQ ID NO: 6), TLVSSL (SEQ ID NO: 7), CLSYYPSYC (SEQ ID NO: 8), GEGKGGr (SEQ ID NO: 9), gegkggr (SEQ ID NO: 10), GEGr (SEQ ID NO: 11), gegr (SEQ ID NO: 12), GE, ge, RGEGR (SEQ ID NO: 13), rgegr (SEQ ID NO: 14), and Cyclo(RKKKWFGC) (SEQ ID NO: 15); where upper case letters indicate L-amino acids, lower case letters indicate D-amino acids, and "X" indicates any L-amino acid. In some embodiments, the PS binding peptide sequence is GEGKGGr (SEQ ID NO: 9).

[0022] In some embodiments, the structure of TSA006 is (GEGKGGr)-K-K-Cys(MI-CL264)-NH. In some embodiments, the structure of TSA029 is (RGEGR)-K-K-Cys(MI-CL264)-NH.

[0023] In some embodiments, the PS-binding peptide sequence is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to TLVSSL (SEQ ID NO:7).

[0024] In some embodiments, the PS-binding peptide sequence is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to GEGKGGr (SEQ ID NO: 9), where uppercase letters indicate L-amino acids and lowercase letters indicate D-amino acids.

[0025] As used herein, the term "capable of binding" or "binding" refers to a moiety (e.g., a compound described herein) that can measurably bind to a target (e.g., phosphatidylserine or a Toll-like receptor protein). In certain embodiments, when a moiety is capable of binding to a target, the moiety is capable of binding with a Kd of less than about 10 μM, 5 μM, 1 μM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.

[0026] In some embodiments, the TLR binding domain is a TLR agonist, non-limiting examples of which include Pam3Cys, PAM3CSK4, PAM3CSK4, SMP-105, CBLB612, IPH 3102, ARNAX, MPLA, MALP-2, zymosan, poly(I:C), poly-ICLC, poly-IC12U, GLA-SE, BNT411, AS04, AS15, OK-432, CBLB502, M-VM3, bistriazolyl, VTX1463, MGN1703, CpG-7909, IMO2055, dSLIM, SD-101, KSK-CpG, ODN M362, CpG-1826, LPS, flagellin, imiquimod, motolimod, lintatorimod, CL264, imidazoquinoline, resiquimod, tilsotolimod, UC-1V150, CADI-05, GNKG168, RO7119929, SHR2150, TransCon, CMP-001, and CpG ODN.

[0027] As used herein, the terms "Toll-like receptor" and "TLR" refer to a class of proteins that play an important role in the innate immune system. TLRs are single-pass transmembrane receptors typically expressed on sentinel cells, such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microorganisms. When these microorganisms breach physical barriers, such as the skin or intestinal mucosa, they are recognized by the TLRs and activate immune cell responses. TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. Humans lack the genes for TLR11, TLR12, and TLR13 [1], and mice lack a functional gene for TLR10. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 are present on the cell membrane, whereas TLR3, TLR7, TLR8, and TLR9 are present in intracellular vesicles as they are sensors of nucleic acids.

[0028] The terms "agonist," "activator," "upregulator," and the like refer to an agent that can detectably increase the expression or activity of a given gene or protein. An agonist can increase expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of agonist. In some embodiments, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more over expression or activity in the absence of agonist.

[0029] In some embodiments, the complex comprises two or more PS binding peptide sequences. In some embodiments, the complex comprises three or more PS binding peptide sequences. In some embodiments, the complex comprises a dimer of the same PS binding sequence. In some embodiments, the complex comprises a trimer of the same PS binding sequence. In some embodiments, the complex comprises a tetramer of the same PS binding sequence. In some embodiments, the complex comprises a pentamer of the same PS binding sequence. In some embodiments, the complex comprises a hexamer of the same PS binding sequence. In some embodiments, the complex comprises a heptamer of the same PS binding sequence. In some embodiments, the complex comprises an octamer of the same PS binding sequence. In some embodiments, the complex comprises a 9mer of the same PS binding sequence. In some embodiments, the complex comprises a 10mer of the same PS binding sequence. In some embodiments, the complex comprises an 11-mer of the same PS binding sequence. In some embodiments, the complex comprises a 12-mer of the same PS binding sequence. In some embodiments, the complex comprises a tetramer of TLVSSL (SEQ ID NO: 7), where uppercase letters indicate L-amino acids and lowercase letters indicate D-amino acids. In some embodiments, the complex comprises a tetramer of GEGKGGr (SEQ ID NO: 9), where uppercase letters indicate L-amino acids and lowercase letters indicate D-amino acids.

[0030] In some embodiments, the complex comprises one or more retro-inverso of any of the PS-binding peptides disclosed herein.

[0031] The term "retro-inverso" refers to linear peptides with an inverted amino acid sequence and inverted α-center chirality of the amino acid subunits. Typically, this type of peptide is designed by including D-amino acids in the inverted sequence to maintain a similar side chain topology as the original L-amino acid peptide and to make it more resistant to proteolysis. Other synonyms for these peptides reported in the scientific literature are: retro-inverso peptides, all-D-retropeptides, retroenantiopeptides, retro-inverso analogs, retro-inverso analogs, retro-inverso derivatives, retro-inverso isomers. D-amino acids are the conformational mirror images of the natural L-amino acids found in natural proteins present in biological systems. Peptides containing D-amino acids are superior to peptides containing only L-amino acids. In general, this type of peptide is less susceptible to proteolysis and has a longer shelf life when used as a pharmaceutical. Furthermore, by inserting D-amino acids into selected sequence regions, either as sequence blocks containing only D-amino acids or between L-amino acids, peptide-based drugs can be designed that are not only resistant to proteolysis but also biologically active and have improved bioavailability. Furthermore, when properly designed, retro-inverso peptides can have binding properties similar to L-peptides. Retro-inverso peptides are useful candidates for studying protein-protein interactions by designing peptidomimetics that mimic the shape of peptide epitopes, protein-protein, or protein-peptide interfaces. Retro-inverso peptides are an attractive alternative to L-peptides used as pharmaceuticals. These peptides have been reported to induce a lower immunogenic response compared to L-peptides. In this disclosure, L-amino acids are represented in uppercase letters and D-amino acids in lowercase letters.

[0032] In some embodiments, the TLR agonist is selected from the group consisting of: In some embodiments, the TLR agonist is CL264 In some embodiments, the TLR binding domain is the TLR agonist CL264.

[0033] In some embodiments, the complex comprises a linker moiety connecting the PS-binding domain and the TLR-binding domain. In some embodiments, the linker moiety is a short amino acid sequence (or "amino acid linker") that is naturally produced to separate multiple domains in a protein. Most of the amino acid linkers are rigid and function to prevent unwanted interactions between the separated domains, while other linkers, such as "Gly-rich" linkers, are flexible and connect various domains in a protein without interfering with the function of each domain. Many amino acid linkers are known in the art, for example in the publication by Reddy Chichili VP et al. ("Linkers in the structural biology of protein-protein interactions" Protein Sci. 2013;22(2):153-167.doi:10.1002 / pro.2206). Modifications to known amino acid linkers are also possible, such as the use of non-natural or non-proteinogenic amino acids. For example, the linker moiety can include ornithine, 2,4-diaminobutyric acid, and / or 2,3-diaminopropanoic acid. In some embodiments, the linker moiety does not comprise any amino acids.

[0034] Another aspect of the present disclosure relates to a method of treating a subject having cancer comprising administering to the subject a conjugate disclosed herein.

[0035] In some embodiments, the cancer is a solid tumor. Non-limiting examples of solid tumors include sarcoma, carcinoma, and lymphoma. Solid tumors can also be identified based on the organ in which they originate, such as the subject's brain, lung, esophagus, stomach, pancreas, or liver. In some embodiments, the solid tumor is mesothelioma. In some embodiments, the cancer is a blood-based cancer. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is leukemia.

[0036] The term "cancer" as used herein refers to all types of cancer, neoplasms or malignant tumors found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that can be treated with the compounds or methods provided herein include brain cancer, glioma, neuroblastoma, prostate cancer, bladder cancer, urothelial cancer, gallbladder cancer, colorectal cancer, pancreatic cancer, medulloblastoma, skin cancer, melanoma, cervical cancer, gastric cancer, esophageal cancer, liver cancer, endometrial cancer, ovarian cancer, lung cancer, head and neck cancer, medulloblastoma, skin cancer, melanoma, cervical cancer, gastric cancer, esophageal cancer, liver cancer, endometrial cancer, ovarian cancer, lung cancer, head and neck cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds or methods provided herein include cancer of the thyroid, endocrine system, brain, breast, cervical, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, esophagus, stomach, and uterus. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive carcinoma of the breast, lung adenocarcinoma, squamous cell carcinoma of the lung, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, soft tissue sarcoma, osteosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulinoma, neuroendocrine tumors, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer.

[0037] The term "leukemia" broadly refers to progressive, malignant diseases of the blood-forming organs, generally characterized by distorted proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are generally classified clinically based on (1) the duration and character of the disease-acute or chronic, (2) the type of cells involved-myeloid (myeloid), lymphoid (lymphoid), or monocytic; and (3) the increased or non-increased number of abnormal cells in the blood-leukemic or non-leukemic (subleukemic). Exemplary leukemias that may be treated with the compounds or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, nonleukemic leukemia, nonleukemic leukemia, basal leukemia, blastic leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonic leukemia, eosinophilic leukemia, gross leukemia, hairy cell leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic ... tic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloblastic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Leder cell leukemia, Schilling leukemia, stem cell leukemia, hypoleukemia, or anaplastic cell leukemia.

[0038] The methods and uses of the present invention include administering an effective amount of the conjugate disclosed herein to treat tumors or cancer. In certain embodiments, the methods or uses inhibit or reduce the recurrence, growth, progression, worsening, or metastasis of tumors or cancer; partial or complete destruction of the cell mass, volume, size, or cell number of a neoplasm, tumor, cancer, or malignant tumor, stimulating, inducing, or increasing the necrosis, lysis, or apoptosis of cells of a neoplasm, tumor, cancer, or malignant tumor, reducing the volume size, cell mass of a neoplasm, tumor, cancer, or malignant tumor, inhibiting or preventing the progression or increase of the volume, volume, size, or cell number of a neoplasm, tumor, cancer, or malignant tumor, or extending the life span; reducing or decreasing the severity, duration, or frequency of adverse symptoms or complications associated with or resulting from a neoplasm, tumor, cancer, or malignant tumor, or reducing or decreasing pain, discomfort, nausea, weakness, or lethargy, or improving energy, appetite, athletic performance, or psychological well-being.

[0039] As used herein, the term "apoptosis" refers to programmed cell death and associated changes in cell physiology, such as nucleic acid fragmentation, caspase activation, etc., as understood in the art. The term "catastrophe" refers to cell death resulting from errors in the mitotic process, in which features characteristic of apoptosis, such as caspase activation, chromosome condensation, etc., are largely absent.

[0040] The term "treating" or "treatment" refers to an indication of success in treating or ameliorating an injury, disease, pathology, or condition, and includes any objective or subjective parameter, such as remission; relief, reduction of symptoms, or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or debilitation; making the end point of degeneration less debilitating; improving the physical or mental well-being of the patient. The treatment or amelioration of symptoms can be based on objective or subjective parameters, including the results of a physical exam, a neuropsychiatric exam, and / or a psychiatric evaluation. The term "treating" and conjugations thereof can include prevention of an injury, pathology, condition, or disease. In some embodiments, treating is preventing. In some embodiments, treating does not include preventing.

[0041] As used herein (and well understood in the art), "treating" or "treatment" broadly includes any approach to obtain beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stabilization (i.e., not worsening) of a disease state, prevention of disease transmission or spread, delay or slowing of disease progression, amelioration or relief of a disease state, reduction in recurrence of disease, and remission, whether partial or total, whether detectable or undetectable, and the like. In other words, as used herein, "treatment" includes the cure, amelioration, or prevention of any disease. Treatment can prevent the onset of disease, inhibit the spread of disease, alleviate symptoms of disease, completely or partially eliminate the underlying cause of disease, shorten the duration of disease, or a combination thereof.

[0042] As used herein, "treating" and "treatment" also include prophylactic treatment. The treatment method includes administering a therapeutically effective amount of an active agent to the subject. The administration step may consist of a single administration or may include a series of administrations. The length of the treatment period will depend on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used in the treatment, or a combination thereof. It will also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease over the course of a particular treatment or prevention regimen. Changes in dosage may occur and be evident by standard diagnostic assays known in the art. In some instances, administration over a long period of time may be required. For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient. In some embodiments, the treating or treatment is not a prophylactic treatment.

[0043] In some embodiments, the subject is a mammal, hi some embodiments, the subject is a human.

[0044] As used herein, the terms "patient," "subject," or "subject in need thereof" refer to a living organism suffering from or susceptible to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, bovine, rats, mice, dogs, monkeys, goats, sheep, cows, deer, cats, and other non-mammalian animals. In some embodiments, the subject is a human.

[0045] In some embodiments, the method further comprises administering to the subject an anti-cancer drug, and / or an immunological drug. In some embodiments, the method further comprises administering to the subject an anti-cancer drug and an immunological drug. In some embodiments, the method further comprises administering to the subject an anti-cancer drug or an immunological drug.

[0046] As used herein, the term "immunological drugs" refers to drugs or compounds that can modify immune responses by enhancing or suppressing the immune system. They are used to fight infections and prevent and treat certain diseases. Immunological drugs include drugs used for immunosuppression to prevent rejection of transplants. They can be used as cancer chemotherapy drugs.

[0047] As used herein, "anti-cancer agent" or "anticancer agent" refers to a molecule (e.g., a compound, a peptide, a protein, or a nucleic acid) that is used to treat cancer through the destruction or inhibition of cancer cells or tissues. Anti-cancer agents may act selectively on a particular cancer or on a particular tissue. In some embodiments, the anti-cancer agents herein include epigenetic inhibitors and multikinase inhibitors. In some embodiments, the anti-cancer agent is a serine-threonine kinase inhibitor.

[0048] 15 (Abbott), HTI-286 (i.e., SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), resberastatin sodium phosphate, BPR-OY-007 (National Health Research Institute) Institutes), SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin releasing hormone agonists (GnRH) such as goserelin or leuprolide, corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethylstilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., bacillascalmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.). Monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), immunotherapy (e.g., cellular immunotherapy, antibody therapy, cytokine therapy, combination immunotherapy, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated with l11In, 90Y, or 131I, etc.), immune checkpoint inhibitors (e.g., CT LA4 blockers, PD-1 inhibitors, PD-L1 inhibitors, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) targeted therapies or treatments (e.g., gefitinib (Iressa)商標 ), erlotinib (Tarceva 商標 ), cetuximab (Erbitux 商標 ), lapatinib (Tykerb 商標 ), panitumumab (Vectibix 商標 ), vandetanib (Capressa 商標 ), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, or the like.

[0049] In some embodiments, the anti-cancer drug is a serine / threonine kinase inhibitor, non-limiting examples of which are ((d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 16), (d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6 -F)(d-Cha) (SEQ ID NO: 17), (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg) (SEQ ID NO: 18), (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha) (SEQ ID NO: 19), (d-Cha)(d -Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(SEQ ID NO:20),(d-Arg)(d-Arg)(d-Arg)( d-G1n)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO: 21), (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d- Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 22), (d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg)(d-Arg)(d-Cha)( d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO: 23), (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(SEQ ID NO: 24), (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Ser)(d-Trp)(d-Ser)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 25), (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 26), (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 27), (d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 28)(d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(SEQ ID NO: 29)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 30), (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 31), (d-Arg)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Trp)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 32), (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Trp)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Arg)(SEQ ID NO: 33), (d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg)(d-Phe-2,3,4,5,6-F)(d-Cha)(SEQ ID NO: 34), and (d-Cha)(d-Phe-2,3,4,5,6-F)(d-Arg)(d-Arg)(d-Arg)(d-Bpa)(d-Arg)(d-Arg)(d-Arg) (SEQ ID NO: 35).

[0050] In some embodiments, the serine / threonine kinase inhibitor is the peptide compound "CBP501" comprising (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 16). In some embodiments, CBP501 is a pharmaceutical salt. In some embodiments, CBP501 is H-(D)p-benzoylphenylalanyl-(D)seryl-(D)tryptophanyl-(D)seryl-(D)pentafluorophenylalanyl-(D)cyclohexylalanyl-(D)arginyl-(D)arginyl-(D)glutaminyl-(D)arginyl-(D)arginine acetate.

[0051] In some embodiments, the method or use uses T cell activating drugs.Non-limiting examples of T cell activating drugs include drugs that target CD28 (cell differentiation antigen 28, also known as Tp44, T cell specific surface glycoprotein, CD28 antigen CD28 molecule), OX40 (tumor necrosis factor receptor superfamily, member 4, TNFRSF4, also known as OX40L receptor, OX40 antigen, TXGP1L), GITR (glucocorticoid-inducible tumor necrosis factor receptor), CD137 (also known as 4-1BB), CD27 (also known as TNFRSF7, Tp55) and HVEM (herpes virus entry mediator, also known as CD270, TNFRSF14).

[0052] Representative T cell activating agents include ligands that bind to such targets, such as CD28, OX40, GITR, CD137, CD27, and HVEM ligands. Representative T cell activating agents also include antibodies that bind to such targets, such as anti-CD28 antibodies, anti-OX40 antibodies, anti-GITR antibodies, anti-CD137 antibodies, anti-CD27 antibodies, and anti-HVEM antibodies.

[0053] In some embodiments, there is a method or use of immune checkpoint inhibitors. Non-limiting examples of immune checkpoint inhibitors include drugs that target CTLA-4 (cytotoxic T-lymphocyte antigen 4, also known as CD152), PD1 (programmed cell death 1, also known as CD279, SLEB2, HPD-1, HSLE1), PDL1 (programmed cell death ligand 1, also known as CD274, B7-H1 (B7 homolog 1), programmed cell death 1 ligand 1, PDCD1 ligand 1), PDL2 (programmed cell death 1 ligand 2), VISTA (V domain Ig T-cell activation inhibitor, also known as B7-H5, Gi24, Diesl, and SISP1), TIM3 (T-cell immunoglobulin-mucin domain 3), LAG-3 (lymphocyte activation gene 3, also known as CD223), or BTLA (B-lymphocyte and T-lymphocyte attenuator, also known as CD272).

[0054] Representative immune checkpoint inhibitors include ligands that bind to such targets, such as CTLA-4, PD1, PDL1, PDL2, VISTA, TIM3, LAG-3, and BTLA ligands. Representative immune checkpoint inhibitors, i.e., immunological agents, also include antibodies that bind to such targets, such as anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PDL1 antibodies, anti-PDL2 antibodies, anti-VISTA antibodies, anti-TIM3 antibodies, anti-LAG-3 antibodies, and anti-BTLA antibodies.

[0055] In some embodiments, the immunological agent is an antibody. In some embodiments, the immunological agent is an antibody that binds to an immune checkpoint inhibitor, including, but not limited to, an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-PDL2 antibody, an anti-VISTA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, or an anti-BTLA antibody. In some embodiments, the antibody is an anti-CTLA-4 antibody.

[0056] In some embodiments, the method further comprises administering a nucleic acid damaging or anti-proliferative drug, non-limiting examples of which include platinum-containing drugs such as cisplatin, carboplatin, nedaplatin, mitaplatin, satraplatin, picoplatin, triplatin, miriplatin, or oxaliplatin. In some embodiments, the platinum-containing drug is cisplatin.

[0057] In some embodiments, the methods and uses include the use of platinum-containing drugs, cisplatin, carboplatin, oxaliplatin, pemetrexed, gemcitabine, 5-fiurocil (5-FU), rebeccamycin, adriamycin (ADR), bleomycin (Bleo), pepleomycin, cisplatin, cisplatinum, or cis-diamminedichloroplatinum (II) (CDDP), oxaliplatin, or camptothecin (CPT), cyclophosphamide, azathioprine, cyclosporine A, prednisolone, melphalan, chlorambucil, mechlorethamine, The present invention includes or consists of the administration of busulfan, methotrexate, 6-mercaptopurine, thioguanine, 5-fluorouracil, cytosine arabinoside, AZT, 5-azacytidine (5-AZC) or 5-azacytidine related compounds, actinomycin D, mithramycin, mitomycin C, carmustine, lomustine, semustine, streptozotocin, hydroxyurea, cisplatin, mitotane, procarbazine, dacarbazine, taxanes, vinblastine, vincristine, doxorubicin, dibromomannitol, radiation or radioisotopes. Particular non-limiting examples of radiation include UV radiation, IR radiation, X-rays, or alpha, beta, or gamma radiation. Non-limiting examples of radiation include UV radiation, IR radiation, X-ray radiation, alpha, beta, or gamma radiation. Specific non-limiting examples of radioisotopes include I131, I125, Sr89, Sm153, Y90, or Lu177.

[0058] The methods and uses of the present invention can be applied to cell proliferation or hyperproliferative disorders or unwanted cell proliferation. In certain embodiments, the cell proliferation disorder comprises a tumor or cancer. In further particular embodiments, the cell proliferation disorder comprises a metastatic tumor or cancer.

[0059] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration will be the concentration of active compound that achieves the methods described herein, as measured using the methods described herein or known in the art.

[0060] As is well known in the art, the therapeutically effective amount for use in humans can also be determined from animal models.For example, the dosage for humans can be formulated to be at a concentration that has been confirmed to be effective in animals.The dosage for humans can be adjusted by monitoring the effectiveness of the compound and adjusting the dosage upwards or downwards, as described above.Based on the above and other methods, it is within the capabilities of a person of ordinary skill in the art to adjust the dosage to achieve maximum effectiveness in humans.

[0061] The term "therapeutically effective amount" as used herein refers to an amount of a treatment agent sufficient to improve a disorder as described above. For example, for a given parameter, a therapeutically effective amount may show at least a 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least a 100% increase or decrease. The therapeutic effect may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

[0062] Dosage can vary depending on the patient's needs and the compound used. In the context of this disclosure, the dose administered to the patient should be sufficient to provide a beneficial therapeutic response to the patient over time. The size of the dose will also be determined by the presence, nature, and extent of side effects. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with a lower than optimal dose of the compound. Thereafter, the dosage is gradually increased until the optimum effect is achieved for the situation. Dosage and administration intervals can be individually adjusted to provide a level of the administered compound that is effective for the particular clinical indication being treated. This will provide a treatment regimen that is tailored to the individual's condition.

[0063] In some embodiments, the amount administered to the subject is about 2 mg / kg or less every 3 weeks. In some embodiments, the amount administered to the subject is 2 mg / kg or less every 3 weeks. In some embodiments, the amount administered to the subject is at least 2 mg / kg every 3 weeks. In some embodiments, the amount administered to the subject is 2 mg / kg every 3 weeks. In some embodiments, the amount administered to the subject is about 0.1 to about 10 mg / kg every 3 weeks. In some embodiments, the amount administered to the subject is about 1 mg / kg or less every 2 weeks. In some embodiments, the amount administered to the subject is 1 mg / kg or less every 2 weeks. In some embodiments, the amount administered to the subject is at least 1 mg / kg every 2 weeks. In some embodiments, the amount administered to the subject is 1 mg / kg every 2 weeks. In some embodiments, the amount administered to the subject is about 0.1 to about 10 mg / kg every 2 weeks. In some embodiments, the amount administered to the subject is 1 mg / kg or less every week. In some embodiments, the dose administered to the subject is at least 1 mg / kg every week. In some embodiments, the dose administered to the subject is 1 mg / kg every week. In some embodiments, the amount administered to a subject is about 0.1 to about 10 mg / kg weekly.

[0064] The term "administering" as used herein refers to oral administration, administration as a suppository, topical contact, intravenous administration, parenteral administration, intraperitoneal administration, intramuscular administration, intraarticular administration, intrathecal administration, intranasal administration or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, into a subject. Administration may be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, and transdermal patches. In certain embodiments, administration does not include administration of any active agent other than the conjugate. In some embodiments, administration includes the conjugate and an immunological agent. In some embodiments, administration includes the conjugate and an anti-cancer agent. In some embodiments, administration includes the conjugate, an immunological agent, and an anti-cancer agent. In some embodiments, the administration includes a conjugate, an immunological agent, an anti-cancer agent, and a platinum-containing agent.

[0065] In some embodiments, the complex is administered intravenously. In some embodiments, the complex is administered intraperitoneally.

[0066] In some embodiments, the complex is administered to the subject daily. In some embodiments, the complex is administered to the subject every other day. In some embodiments, the complex is administered to the subject every third day. In some embodiments, the complex is administered to the subject every fourth day. In some embodiments, the complex is administered to the subject every fifth day. In some embodiments, the complex is administered to the subject every sixth day. In some embodiments, the complex is administered to the subject every seventh day. In some embodiments, the complex is administered to the subject every eighth day. In some embodiments, the complex is administered to the subject every ninth day. In some embodiments, the complex is administered to the subject every tenth day. In some embodiments, the complex is administered to the subject every eleventh day. In some embodiments, the complex is administered to the subject every twelfth day. In some embodiments, the complex is administered to the subject every thirteenth day. In some embodiments, the complex is administered to the subject every fourteenth day. In some embodiments, the complex is administered to the subject at least every fourteenth day. In some embodiments, the complex is administered to the subject monthly. In some embodiments, the complex is administered to the subject at least monthly. In some embodiments, the complex is administered to the subject every two months. In some embodiments, the complex is administered to the subject at least every two months.

[0067] "Co-administration" refers to administration of the compositions described herein simultaneously with, immediately before, or immediately after administration of one or more additional therapies. The compounds provided herein can be administered alone or co-administered to a patient. Co-administration refers to simultaneous or sequential administration of the compounds alone or in combination (two or more compounds). Thus, the formulations can also be combined with other active agents as desired (e.g., to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally by topical routes, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0068] In some embodiments, the cytotoxic agent is administered prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 1 day prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 2 days prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 3 days prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 4 days prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 5 days prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 6 days prior to the PS complex. In some embodiments, the cytotoxic agent is administered at least 1 week prior to the PS complex.

[0069] As used herein, "cell" refers to a cell that has sufficient metabolic or other functions to store or replicate genomic DNA. Cells can be identified by methods well known in the art, such as the presence of an intact membrane, staining with certain dyes, the ability to produce progeny, or, in the case of gametes, the ability to combine with a second gamete to produce viable progeny. Cells include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells of plant and animal origin, such as mammalian, insect (e.g., Spodoptera), and human cells. Cells may be useful if they are naturally non-adherent or have been treated to prevent them from adhering to surfaces, such as by trypsinization.

[0070] A cancer model organism, as used herein, is an organism that exhibits a phenotype that indicates cancer or the activity of a factor that causes cancer in the organism. The term cancer is defined as above. A wide variety of organisms can be used as cancer model organisms, including, for example, cancer cells and mammalian organisms such as rodents (e.g., mice or rats) and primates (e.g., humans). Cancer cell lines are widely understood by those skilled in the art as cells that exhibit a phenotype or genotype similar to cancer in vivo. As used herein, cancer cell lines include cell lines derived from animals (e.g., mice) and humans.

[0071] The term "immune response" and the like refers in its ordinary and conventional sense to a response by an organism to protect against disease, which response may be initiated by either the innate or adaptive immune system, as is well known in the art.

[0072] The term "modulating an immune response" and the like refers to a change in a subject's immune response as a result of administration of an agent, e.g., a compound disclosed herein, including embodiments thereof. Thus, the immune response can be activated or inactivated as a result of administration of an agent, e.g., a compound disclosed herein, including embodiments thereof.

[0073] "B cell" or "B lymphocyte" refer to standard usage in the art. B cells are lymphocytes, a type of white blood cell (leukocyte), that develop into antibody-producing plasma cells ("mature B cells"). "Immature B cells" are cells that can develop into mature B cells. Generally, pro-B cells undergo immunoglobulin heavy chain rearrangement to become pre-B cells, and further immunoglobulin light chain rearrangement to become immature B cells. Immature B cells include T1 and T2 B cells.

[0074] As used herein, "T cell" or "T lymphocyte" refers to a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presentation of T cell receptors on the cell surface. T cells include, for example, natural killer T (NKT) cells, cytotoxic T lymphocytes (CTL), regulatory T (Treg) cells, and T helper cells. Different types of T cells can be distinguished by using T cell detection agents.

[0075] "Memory T cells" are T cells that have encountered and responded to their cognate antigen before an infection, cancer, or a previous vaccination. Upon a second encounter with the cognate antigen, memory T cells can replicate (divide) to mount a faster and stronger immune response than the first time the immune system responded to the pathogen.

[0076] "Regulatory T cells" or "suppressor T cells" are lymphocytes that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease.

[0077] An amino acid residue in a protein "corresponds" to a residue if that amino acid residue occupies the same essential structural position in the protein as that residue.

[0078] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and those that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a compound with an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that do not occur in nature.

[0079] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Terminology Commission. Nucleotides may also be referred to herein by their commonly accepted one-letter codes.

[0080] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which in certain embodiments may be linked to moieties that are not composed of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. "Fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0081] The terms "fusion" or "chimeric" and grammatical variations thereof, when used in connection with a sequence, mean that the sequence contains one or more portions based on, derived from, or obtained or isolated from two or more different proteins. That is, for example, one portion of the sequence is based on or derived from one particular protein and another portion of the sequence is based on or derived from another particular protein. Thus, a fusion or chimeric polypeptide is a molecule in which different portions of the polypeptide are of different protein origin.

[0082] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" that replace amino acids with chemically similar amino acids. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present disclosure.

[0083] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins (1984)).

[0084] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0085] The term "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or specified region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection (see, e.g., the NCBI website at http: / / www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are said to be "substantially identical". This definition may also refer to or apply to the complement of a test sequence. This definition includes sequences that have deletions, additions, and substitutions. As described below, preferred algorithms can take into account gaps, etc. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0086] The "position" of an amino acid or nucleotide base is designated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its relative position from the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, determined by simple counting from the N-terminus, is generally not necessarily the same as the number of the corresponding position in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, there is no amino acid in the variant that corresponds to the position of the deletion site in the reference sequence. If there is an insertion in the aligned reference sequence, the insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence.

[0087] The term "numbered with reference to" or "corresponding to," when used in the context of the numbering of an amino acid sequence or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when comparing an amino acid sequence or polynucleotide sequence to a reference sequence.

[0088] The term "antibody" refers to a polypeptide or functional fragment thereof encoded by an immunoglobulin gene that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0089] The term "pharmaceutical acceptable salt" is meant to include salts of active compounds prepared with relatively non-toxic acids or bases depending on the specific substituents found in the compounds described herein. When the compounds of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds, either in the form of neat base compounds or base compounds dissolved in a suitable inert solvent, with a sufficient amount of the desired base, or by treating with an ion exchange resin. Non-limiting examples of pharmaceutical acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. In some embodiments, pharmaceutical acceptable base addition salts are compounds that contain a counterion of a complex. In some embodiments, the counterion to the conjugate is Na + , K + , Ca + , Mg 2+ , or NH4 +In the case where a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound, either as a neat acid compound or as an acid compound dissolved in a suitable inert solvent, with a sufficient amount of the desired acid, or by treatment with an ion exchange resin. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, and phosphoric acid, as well as those derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, oxalic acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginic acid, and salts of organic acids such as glucuronic acid or galactunoric acid (see, e.g., Berge et al., Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the disclosure contain both basic and acidic functional groups and can be converted into either base or acid addition salts. In some embodiments, the pharma-ceutically acceptable salt is a trifluoroacetic acid salt, obtained by contacting trifluoroacetic acid, a relatively low boiling point (72.4°C) acid, with the neutral base form of a pharma-ceutically active compound.

[0090] Therefore, the compounds of the present disclosure may exist as salts with pharma- ceutically acceptable acids and the like. The present disclosure also includes such salts. Non-limiting examples of such salts include hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, propionate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamic acid, quaternary ammonium salts (e.g., methyl iodide, ethyl iodide, and the like). These salts can be prepared by methods known to those skilled in the art.

[0091] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0092] In addition to salt forms, the present disclosure provides compounds in the form of prodrugs. Prodrugs of the compounds described herein are compounds that undergo easy chemical changes under physiological conditions to provide the compounds of the present disclosure. Prodrugs of the compounds described herein can be converted in vivo after administration. In addition, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods ex vivo, for example, when contacted with a suitable enzyme or chemical reagent.

[0093] Certain compounds of the present disclosure can exist not only in unsolvated form but also in solvated form, including hydrated form.In general, solvated form is equivalent to unsolvated form and is included within the scope of the present disclosure.Certain compounds of the present disclosure can exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the use contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0094] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids in the administration and absorption of an active agent by a subject and can be included in the compositions of the present disclosure without causing significant toxicological side effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxyesters, hydroxygenases, and the like, including flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose, starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorings, and the like. Such formulations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not deleteriously react with the compounds of the present disclosure. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0095] The term "preparation" is intended to include formulations of the active compound with an encapsulating material as a carrier to provide a capsule in which the active ingredient is surrounded by a carrier, with or without other carriers. Also included are cachets and lozenges. Tablets, powders, capsules, pills, cachets, lozenges, and the like can be used as solid dosage forms suitable for oral administration.

[0096] As used herein, the term "about" refers to a range of values ​​including the stated value that one of ordinary skill in the art would consider to be reasonably similar to the stated value. In some embodiments, it refers to within a standard deviation using measurements generally accepted in the art. In some embodiments, about refers to a range of ±10% of the stated value. In some embodiments, about includes the specified value.

[0097] As used herein, a "synergistic amount" refers to the sum of a first amount (e.g., an amount of a compound provided herein) and a second amount (i.e., a therapeutic agent, etc.) that results in a synergistic effect (i.e., an effect that is greater than an additive effect). Thus, the terms "synergistic effect," "synergistic," "synergistic," "combined synergistic amount," and "synergistic therapeutic effect," as used interchangeably herein, refer to a measured effect of compounds administered in combination that is greater than the sum of the individual effects of each compound provided herein administered alone as a single agent.

[0098] In certain embodiments, a synergistic amount is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 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, It may be 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, or 99%.In certain embodiments, a synergistic amount is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.1, 10.2, 10.3, 10.4, 10.5 .4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 ,8.0,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9.0,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10.0,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42, It may be 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, or 99%.

[0099] In this disclosure, "comprises," "comprising," "containing," "having," and the like, shall have the meaning assigned to them in United States patent law and may mean "includes," "including," and the like. "Consisting essentially of" or "consists essentially" likewise shall have the meaning assigned to them in United States patent law, and the term is open-ended, permitting the presence of more than is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than is recited. EXAMPLES

[0100] Example 1 Introduction There are numerous peptides known to bind to cell surface PS, such as LIKKPF (SEQ ID NO: 1), PGDLSR (SEQ ID NO: 2) (Reference 9), CLIKKPF (SEQ ID NO: 3), CPGDLSR (SEQ ID NO: 4) (Reference 10), FNFRLKAGAKIRFG (SEQ ID NO: 5) (Reference 11), FXFXLKXXXKXR (SEQ ID NO: 6) (Reference 12), TLVSSL (SEQ ID NO: 7) (Reference 13), CLSYYPSYC (SEQ ID NO: 8) (Reference 14), and Cyclo(RKKWFGC) (SEQ ID NO: 15) (Reference 19). However, the affinity of these peptides for PS is not high enough to make them ideal targeting delivery tools in themselves.

[0101] We reasoned that by creating multivalent versions of these we could overcome the problem of low affinity, yet still be small enough to penetrate solid tumors.

[0102] Our prediction was confirmed in that monomers or dimers of phosphoserine-binding peptides bound to TLR agonists showed no antitumor activity, whereas tetramers of TLVSSL (SEQ ID NO: 7) showed potent antitumor activity in mice without any apparent systemic adverse activity.

[0103] Materials and Methods Syngeneic mouse model, TSA005 (Figure 1) All animal experiments were performed in accordance with protocols approved by the Animal Care and Use Committee of Canvas Co., Ltd. CT26WT cell suspensions (5 × 10 5 After 10 days, the mice were divided into 5 groups (6-8 mice / group), and treatment was started on the first day.

[0104] The first cycle of treatment was from days 1 to 5, and the second cycle was from days 10 to 14, as follows: Cisplatin Cisplatin 4 mg / kg and 5% dextrose intravenously (iv) on days 1 and 10. Saline iv on days 2, 3, 11, and 12. Cisplatin + CBP501 Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on days 1 and 10. Saline iv on days 2, 3, 11, and 12. Cisplatin + CBP501 + TSA005 Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on days 1 and 10. TSA005 100 μg iv on days 2, 3, 11, and 12. Cisplatin + CBP501 + anti-CTLA-4 antibody : Anti-CTLA4 antibody 200ug intraperitoneally (ip) on days 1 and 5. Cisplatin 4mg / kg and CBP501 6mg / kg iv on day 2. Cisplatin + CBP501 + anti-CTLA-4 antibody + TSA005Anti-CTLA4 antibody 200 μg ip on days 1 and 5. Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on day 2. TSA005 100 μg iv twice on day 3 and once on day 4.

[0105] Diphenhydramine was administered ip at 10 mg / kg 15 min before CBP501 or 5% glucose treatment. Tumor size was measured three times weekly with a caliper and the volume (mm 3 )=[(width) 2 Tumor volume was calculated using the formula: [(mm) × length (mm)] ÷ 2.

[0106] FIG. 1 shows the growth curves of the CT26 colon cancer cell line, a syngeneic subcutaneous tumor in BALB / c mice treated with cisplatin, CBP501, anti-CTLA-4 antibody, TSA005, and / or vehicle.

[0107] CT26 colon cancer cells were inoculated into BALB / c mice at 5x10 5 The tumors were inoculated subcutaneously with 1000 cells / mouse. Treatment began on day 1, and the tumor size in each test group (6-8 mice) was approximately 200 mm on average. 3 The treatment schedule, dosage, and treatment route cycles are detailed below. Treatment Groups and Schedules Group 1: Days 1 and 10: Cisplatin 4 mg / kg and 5% glucose administered intravenously (iv) Days 2, 3, 11, and 12: saline, i.v. Group 2: Days 1 and 10: Cisplatin 4 mg / kg and CBP501 6 mg / kg iv Days 2, 3, 11, and 12: saline, i.v. Group 3: Days 1 and 10: Cisplatin 4 mg / kg and CBP501 6 mg / kg, iv Days 2, 3, 11, and 12: TSA005 100 μg, i.v. Group 4: Day 1: Anti-CTLA-4, intraperitoneal injection (ip) Day 2: Cisplatin 4 mg / kg and CBP501 6 mg / kg, iv Day 5: Anti-CTLA-4, i.p. Group 5: Day 1: Anti-CTLA-4, i.p. Day 2: Cisplatin 4 mg / kg and CBP501 6 mg / kg, iv Day 3: TSA005 100 μg, iv, twice Day 4: TSA005 100 μg, 1 dose, i.v. Day 5: Anti-CTLA-4, i.p.

[0108] peptide Table 1 below shows the structures of PS-binding or nonsense control peptides conjugated to TLR agonists or immunofluorescent molecules, in which capital letters indicate L-amino acids and lower case letters indicate D-amino acids. [Table 1]

[0109] Comparison of syngeneic mouse models - TSA005 and TSA009 (Figure 2) All animal experiments were performed according to protocols approved by the Animal Care and Use Committee of Canbus Co., Ltd. Six-week-old female BALB / c mice (The Jackson Laboratories Japan, Inc, Kanagawa, Japan) were subcutaneously inoculated with a CT26WT cell suspension (5x105 cells) into the flank. Ten days later, the mice were divided into three groups (6 mice / group), and treatment was initiated on day 1.

[0110] Cisplatin + CBP501 + anti-CTLA-4 antibody Anti-CTLA4 antibody 200 μg intraperitoneally (ip) on days 3 and 5. Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on day 2. Saline iv twice on day 3 and once on day 4.

[0111] Cisplatin + CBP501 + anti-CTLA-4 antibody + TSA005:Anti-CTLA4 antibody 200 μg ip on days 3 and 5. Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on day 2. TSA005 100 μg iv twice on day 3 and once on day 4.

[0112] Cisplatin + CBP501 + anti-CTLA-4 antibody + TSA009 Anti-CTLA4 antibody 200 μg ip on days 3 and 5. Cisplatin 4 mg / kg and CBP501 6 mg / kg iv on day 2. TSA005 100 μg iv twice on day 3 and once on day 4.

[0113] Diphenhydramine was administered ip at 10 mg / kg 15 min before CBP501 or 5% glucose treatment. Tumor size was measured three times weekly with a caliper and the volume (mm 3 )=[(width) 2 Tumor volume was calculated using the formula: [(mm) × length (mm)] ÷ 2.

[0114] Flow cytometry analysis of Cy5.5 versions of TSA005 and 006 stained with FITC-Annexin V (AnxV) (Figure 3). Cy5.5 (cyanine-5.5) is a fluorescent compound with an excitation peak at 683 nm and an emission peak at 703 nm. Cy5.5 (cyanine-5.5) is spectrally similar to TF6WS (Tide Fluor 6WS), Alexa Fluor 680, Alexa Fluor 700, and Rhodamine 800.

[0115] The Cy5.5 version of TSA005 (TSA012) and the Cy5.5 version of TSA006 (TSA013) are dendrimers in which CL264 in (TLVSSLr)4-K2-K-Cys(MI-CL264)-NH2 and (GEGKGGr)4-K2-K-Cys(MI-CL264)-NH2 is replaced by cyanine 5.5, respectively, and were synthesized at Peptide Institute, Inc., Osaka, Japan (MI: maleimide).

[0116] Human T cell line, Jurkat clone E6-1 (ATCC, TIB-152), was co-stained with vehicle, TSA012, or TSA013 and FITC-AnxV (BioLegend, CA, 640906, Lot No. B345098) and analyzed by flow cytometry. The following is a brief experimental procedure.

[0117] To induce apoptotic events, 5 μM camptothecin (Sigma, MO, C-9111) was added to 8 × 10 cells in a T-75 flask. 5 The cells were then washed once with 10 mL of PBS(-) and once with AnxV binding buffer, and 4x10 5 Cells were resuspended in 100 μL of AnxV binding buffer in a 1.5 mL tube. Cells were incubated with 5 μL of diluted FITC-AnxV, TSA012, or TSA013 for 15 min at room temperature in the dark. Then, 10 μL of 20 μg / mL propidium iodide (final concentration 1.7 μg / mL) was added and incubated on ice in the dark for 2 min. 300 μL of AnxV binding buffer was added to each tube before analysis on CytoFLEX (Beckman Coulter, IND). Acquired data were analyzed with FlowJo software.

[0118] Tumor growth curves of the CT-26 murine colon cancer cell line implanted subcutaneously in syngeneic BALB / c mice. (Figures 4A-4D) BALB / c mice were injected with 5 × 10 5 The mouse colon cancer cell line CT-26WT was implanted subcutaneously. The average tumor size was 205 mm 3 (132-279mm 3 At the time of the first injection, six mice were assigned to each treatment group. Mice weighing 19.5 to 24.5 g and aged 8 weeks were used for the treatment, and tumor size was measured three times a week.

[0119] Administration schedule ("ip": intraperitoneal injection, "iv": intravenous injection): Diphenhydramine (ip): 10 mg / kg (15 min before cisplatin / CBP501 on day 1) Cisplatin (iv): 4 mg / kg (day 1) CBP501(iv): 6 mg / kg (day 1) Anti-CTLA4 antibody (ip): 200 μg / mouse (day 2, day 4) TSA005, 006 saline (iv): 100 μg / mouse (2 days x 2, 1 day x 3) TSA015HEPES(iv): 100μg / mouse (2 days x 2, 1 day x 3) Vehicle: Saline (iv): 100 μL / mouse (Day 2 x 2, Day 3 x 1)

[0120] Distribution of the Cy5.5 version of TSA006 (TSA013) in BALB / c mice (Figure 5). CT-26WT was injected at 5x10 into 7-week-old female BALB / c mice. 5 Cells / mouse were implanted subcutaneously and tumors were grown to 114-190 mm 3 (Average size: 152mm 3 After treatment with 9-week-old mice weighing 23.0-24.0 g, Cy5.5 luminescence was detected on the 4th day using an IVIS LuminaX5 (filters: Ex: 660, Em: 710).

[0121] Treatment is as follows: Diphenhydramine (ip): 10 mg / kg (15 min before cisplatin / CBP501 on day 1) Cisplatin (iv): 4 mg / kg (day 1) CBP501(iv): 6 mg / kg (day 1) TSA013 saline (iv): 100 μg / mouse (day 3)

[0122] result The combination of CBP501 plus cisplatin inhibited the growth of subcutaneous tumors much better than cisplatin alone, as shown in Figure 1. Addition of TSA005 to the combination further inhibited tumor growth.

[0123] The addition of an anti-CTLA4 antibody, an immune checkpoint inhibitor, suppressed tumor growth for a longer period than the CBP501 plus cisplatin combination alone, even though the triple combination with the anti-CTLA4 antibody was only treated with one cycle compared with two cycles of the doublet combination of CBP501 and cisplatin.

[0124] TSA005 enhanced antitumor activity in doublet or triplet treatment regimens.

[0125] As shown in Figure 2, the addition of TSA005, but not TSA009, suppressed the growth of CT26 subcutaneous tumors more than the syngeneic tumor model of CBP501 + cisplatin + anti-CTLA4 antibody alone.

[0126] Structure of TSA005: (TLVSSLr)4-K2-K-Cys(MI-CL264)-NH2.

[0127] Structure of TSA009: (TLVSSLr)8-K4-K2-K-Cys(MI-CL264)-NH2.

[0128] With reference to Figure 2, a detrimental effect of TSA009 was observed when compared to CBP501+cisplatin+anti-CTLA4 antibody treatment with or without TSA005. One hypothesis for this observed effect of TSA009 would be that the complex acts as if it were reaching systemically but not apoptotic and / or cancer cells.

[0129] As shown in Figure 3, camptothecin treatment, known to induce apoptosis in Jurkat cells, increased AnxV and TSA012 or TSA013 double-positive cells in the upper right quadrant from 5.77 to 37.9 for Cy5.5 version of TSA005 (TSA012) and from 11.5 to 39.6% for Cy5.5 version of TSA006 (TSA013), respectively. As shown in the six right panels, when gating was performed on live cells only, the high intensity double-positive signals disappeared, so the high intensity double-positive signals are likely derived from dead cells. Nevertheless, AnxV and TSA012 or TSA013 double-positive cells were clearly increased by camptothecin treatment, for example, from 2.21 to 29% and from 5.1 to 31.3%, respectively. This data presented in Figure 3 suggests that TSA012 and TSA013 bind to AnxV-binding cells. These cells were found to be apoptotic or dead cells.

[0130] Data presented in Figure 4A show that treatment with cisplatin, CBP501, and anti-CTLA-4 inhibited CT26 tumor growth compared to vehicle, each single compound, or each dual combination (data not shown). Addition of TSA005 and TSA006 further inhibited tumor growth. Addition of these compounds did not change mouse body weight more than the triple combination (data not shown).

[0131] The data presented in Figures 4B-4D show that, unlike many cytotoxic drugs, immune-acting drugs variably suppress tumor growth under the same treatment conditions. Smaller tumors tended to be more suppressed than larger tumors. This data was in line with the expected immune-related mechanism of action of TSA005 and TSA006.

[0132] Data presented in Figure 5 show that TSA013 accumulated at the site of implanted tumors where apoptosis was induced by cisplatin and CBP501 treatment.

[0133] discussion We have shown that CBP501 may enhance the efficacy of platinum therapeutic drugs in cancer cells (15, 16) and induce immunogenic cell death (17). CBP501 also inhibits cytokine release by macrophages and suppresses the accumulation of cancer stem cells (18). As expected, CBP501 plus cisplatin inhibited tumor growth more than cisplatin alone, and the addition of TSA005 further inhibited tumor growth.

[0134] Similarly, TSA005 inhibited tumor growth more than the triple combination of CBP501, cisplatin, and anti-CTLA-4 antibody.

[0135] These data suggest that TSA005 acts through a mechanism distinct from those of cisplatin, CBP501, and anti-CTLA-4. Because TSA005 contains a phosphatidylserine-binding moiety and a Toll-like receptor agonist moiety, it is predicted that TSA005 is engulfed by phagocytes along with apoptotic cells, and that the engulfed Toll-like receptor agonist moiety would change the antigen presentation by phagocytes from negative to positive.

[0136] References 1. JA Marin-Acevedo1 et al., Next generation of immune checkpoint inhibitors and beyond. J. Hematol. Oncol., 2021 2. AD Waldman et al., A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat. Rev. Imm., 2020 3. S. Pahlavanneshan et al., Toll-Like Receptor-Based Strategies for Cancer Immunotherapy. J. Imm. Res., 2021 4. A. Keshavarz1 et al., Toll-like receptors (TLRs) in cancer; with an extensive focus on TLR agonists and antagonists. IUBMB Life. 2021 5. A. J. R. Gadd et al., Targeted Activation of Toll-Like Receptors: Conjugation of a Toll-Like Receptor 7 Agonist to a Monoclonal Antibody Maintains Antigen Binding and Specificity. Bioconjug Chem, 2015 6. S. E. Ackerman et al., Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nat. Can., 2020 7. G. M. Thurber et al., Antibody tumor penetration: Transport opposed by systemic and antigen-mediated clearance. Adv. Drug Deliv. Rev., 2008 8. W. Chang et al., Targeting phosphatidylserine for Cancer therapy: prospects and challenges. Theranostics, 2020 9. C. Burtea et al., Peptidic Targeting of Phosphatidylserine for the MRI Detection of Apoptosis in Atherosclerotic Plaques. Mol. Pharm., 2009 10. J. Kapty et al., Evaluation of Phosphatidylserine-Binding Peptides Targeting Apoptotic Cells. J. Bio. Scr., 2012 11. A. Perreault et al., Targeting Phosphatidylserine with a 64Cu-Labeled Peptide for Molecular Imaging of Apoptosis. Mol. Pharm., 2016 12. K. Igarashi et al., A Novel Phosphatidylserine- binding Peptide Motif Defined by an Anti-idiotypic Monoclonal Antibody. J.B.C., 1995 13. C. Laumonier et al., A New Peptidic Vector for Molecular Imaging of Apoptosis, Identified by Phage Display Technology. J. Bio. Scr., 2006 14. N. Thapa et al., Discovery of a phosphatidylserine-recognizing peptide and its utility in molecular imaging of tumour apoptosis. J. Cell. Mol. Med., 2008 15. S. Sha et al., Cell cycle phenotype-based optimization of G2 abrogating peptides yields CBP501 with a unique mechanism of action at the G2 checkpoint. Mol. Cancer Ther., 2007 16. N. Mine et al., CBP501-calmodulin binding contributes to sensitizing tumor cells to CDDP and BLM. Mol. Cancer Ther., 2011 17. K. Sakakibara et al., CBP501 induces immunogenic tumor cell death and CD8 T cell infiltration into tumors in combination with platinum, and increases the efficacy of immune checkpoint inhibitors against tumors in mice. Oncotarget, 2017 18. N. Mine et al., CBP501 suppresses macrophage induced cancer stem cell like features and metastases. Oncotarget, 2017 19. Barth et al., A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis. Nature Communications vol 11: 4027, 2020 Citation of any patent, patent application, publication, or any other document is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of such publications or documents.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0138] All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced with an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, a disclosed feature (e.g., an antibody) is an example of a genus of equivalent or similar features.

[0139] In this specification, all numerical values ​​or numerical ranges include integers within the range and fractions of integers within the numerical value or numerical range, unless the context clearly indicates otherwise. Furthermore, when a list of values ​​is described herein (e.g., about 50%, 60%, 70%, 80%, 85%, or 86%), the list includes all intermediate values ​​and fractions (e.g., 54%, 85.4%). Thus, reference to 80% or greater identity also includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, etc.

[0140] References to greater or smaller integers include every number greater or less than the referenced number, respectively. Thus, for example, less than 100 includes 99, 98, 97, etc., all numbers down to 1, and references to less than 10 include 9, 8, 7, etc., all numbers down to 1.

[0141] As used herein, all numerical values ​​or ranges include numbers and integer fractions within that range unless the context clearly indicates otherwise. Thus, a reference to a numerical range such as 1 to 10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, a reference to a range of 1 to 50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0142] A set of ranges includes ranges that combine the boundary values ​​of different ranges within that set of ranges. Thus, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, 2,500-3,000 References to the series of ranges 0, 3,000-3,500, 3,500-4,000, 4,000-4,500, 4,500-5,000, 5,500-6,000, 6,000-7,000, 7,000-8,000, or 8,000-9,000 include 10-50, 50-100, 100-1,000, 1,000-3,000, 2,000-4,000, etc.

[0143] Modifications can be made to the above without departing from the basic aspects of the present technology. Although the present technology has been described in substantial detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to the embodiments specifically disclosed herein, but these modifications and improvements are within the scope and spirit of the present technology.

[0144] The present invention is generally disclosed herein using positive language to describe a number of embodiments and aspects. The present invention also specifically includes embodiments in which certain subject matter, such as substances or materials, method steps and conditions, protocols or procedures, are excluded in whole or in part. For example, in certain embodiments or aspects of the present invention, materials and / or method steps are excluded. Thus, even if nothing is expressed herein as to what the present invention does not generally include, aspects of the present invention that are not expressly excluded are nevertheless disclosed herein.

[0145] Some embodiments of the technology described herein may be suitably practiced in the absence of any element not specifically disclosed herein. Thus, in some embodiments, the term "comprising" or "comprises" may be replaced with "consisting essentially of" or "consisting of," or grammatical variations thereof. "A" or "an" may mean one or more of the element it modifies (e.g., "a reagent" may mean one or more drugs). As used herein, the term "about" refers to values ​​within 10% (i.e., plus or minus 10%) of the underlying parameter, and the use of the term "about" at the beginning of the value string modifies each value (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). For example, a weight of "about 100 grams" may include a weight between 90 grams and 110 grams. As used herein, the term "substantially" refers to a value modifier meaning "at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and can include 100%. For example, a composition that is substantially free of X can contain less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% X, and / or X may be absent or undetectable in the composition.

Claims

1. A phosphatidylserine-binding complex comprising at least one phosphatidylserine-binding (PS-binding) domain and at least one Toll-like receptor-binding (TLR-binding) domain.

2. The complex of claim 1 , wherein the PS-binding domain is a peptide sequence.

3. The conjugate of claim 1 , wherein the TLR binding domain is a TLR agonist.

4. The complex of claim 2, comprising three or more PS-binding peptide sequences.

5. the PS-binding peptide sequence is selected from the group consisting of LIKKPF (SEQ ID NO: 1), PGDLSR (SEQ ID NO: 2), CLIKKPF (SEQ ID NO: 3), CPGDLSR (SEQ ID NO: 4), FNFRLKAGAKIRFG (SEQ ID NO: 5), FXFXLKXXXKXR (SEQ ID NO: 6), TLVSSL (SEQ ID NO: 7), CLSYYPSYC (SEQ ID NO: 8), GEGKGGr (SEQ ID NO: 9), gegkggr (SEQ ID NO: 10), GEGr (SEQ ID NO: 11), gegr (SEQ ID NO: 12), GE, ge, RGEGR (SEQ ID NO: 13), rgegr (SEQ ID NO: 14), and Cyclo (RKKKWFGC) (SEQ ID NO: 15); 5. The conjugate of claim 4, wherein capital letters indicate L-amino acids, lower case letters indicate D-amino acids, and "X" indicates any L-amino acid.

6. The complex of claim 5, wherein the PS-binding peptide sequence is GEGKGGr (SEQ ID NO: 9).

7. The complex of claim 6, comprising a tetramer of GEGKGGr (SEQ ID NO: 9).

8. TLR agonists include Pam3Cys, PAM3CSK4, SMP-105, CBLB612, IPH3102, ARNAX, MPLA, MALP-2, zymosan, poly(I:C), poly-ICLC, poly-IC12U, GLA-SE, BNT411, AS04, AS15, OK-432, CBLB502, M-VM3, bistriazolyl, VTX1463, MGN1703, CpG-7909, IMO2055, dSLIM, SD-101, KSK-CpG, and ODN. The complex of claim 3, wherein the complex is selected from the group consisting of M362, CpG-1826, LPS, flagellin, imiquimod, motolimod, lintatolimod, CL264, imidazoquinoline, resiquimod, tilsotolimod, UC-1V150, CADI-05, GNKG168, RO7119929, SHR2150, TransCon, CMP-001, and CpG ODN.

9. 9. The conjugate of claim 8, wherein the TLR agonist is CL264.

10. 8. The conjugate of claim 7, wherein the TLR binding domain is the TLR agonist CL264.

11. The conjugate of claim 1 , comprising a linker moiety connecting the PS-binding domain to the TLR-binding domain.

12. A pharmaceutical composition for treating a subject with cancer, comprising the complex of any one of claims 1 to 11.

13. The pharmaceutical composition of claim 12, wherein the cancer is a solid cancer or solid tumor.

14. 13. The pharmaceutical composition of claim 12, wherein the cancer is a blood-based cancer.

15. The pharmaceutical composition of claim 12, wherein the cancer is colon cancer.

16. The pharmaceutical composition of claim 14, wherein the cancer is leukemia.

17. The pharmaceutical composition of claim 12, wherein the subject is a human.

18. 13. The pharmaceutical composition of claim 12, further comprising or used in combination with an anti-cancer drug and / or an immunological drug.

19. 19. The pharmaceutical composition of claim 18, wherein the anticancer drug is a serine / threonine kinase inhibitor.

20. 20. The pharmaceutical composition of claim 19, wherein the serine / threonine kinase inhibitor is a peptide compound comprising (d-Bpa)(d-Ser)(d-Trp)(d-Ser)(d-Phe-2,3,4,5,6-F)(d-Cha)(d-Arg)(d-Arg)(d-Arg)(d-Gln)(d-Arg)(d-Arg) (SEQ ID NO: 16).

21. 19. The pharmaceutical composition of claim 18, wherein the immunological agent is an antibody.

22. 22. The pharmaceutical composition of claim 21, wherein the antibody is an anti-CTLA-4 antibody, an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-PDL2 antibody, an anti-VISTA antibody, an anti-TIM3 antibody, an anti-LAG-3 antibody, or an anti-BTLA antibody.

23. 23. The pharmaceutical composition of claim 22, wherein the antibody is an anti-CTLA-4 antibody.

24. 20. The pharmaceutical composition of claim 18, further comprising or used in combination with a platinum-containing drug.

25. 25. The pharmaceutical composition of claim 24, wherein the platinum-containing drug is cisplatin.

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