Immunomodulatory peptides

Abstract

Provision of immunomodulatory peptides. [Solution] This disclosure provides peptides that can be used for various therapeutic purposes (e.g., inhibiting the progression of hyperproliferative disorders (including cancer); treating infectious diseases; enhancing the response to vaccination; treating sepsis; and promoting hair repigmentation or reduction of pigmented skin lesions). In some embodiments, the disclosed peptides consist of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the disclosed peptides include the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the disclosed peptides essentially consist of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

Description

[Technical Field]

[0001] This application incorporates, by reference, the contents of a 1.52kb text file named "00047900276sequencelisting.txt", which is the sequence listing for this application.

[0002] Each scientific document, patent, and published patent application cited herein is incorporated herein by reference in its entirety.

[0003] Technical field This disclosure generally relates to immunomodulatory peptides. [Background technology]

[0004] background Programmed cell death-1 (PD1), and its ligands PD-L1 and PD-L2, are widely expressed and play many immunoregulatory roles in T cell activation, including attenuation of immunity against tumor cells and infectious agents. Therefore, PD1 is an attractive target for various therapeutic applications. The need for useful regulators of the immune checkpoint pathway continues. [Brief explanation of the drawing]

[0005] [Figure 1] Figure 1 is a graph showing the ability of various peptides to inhibit PD1 activity and recruit SHP1, as measured using the PATHHUNTER® checkpoint signaling assay (DiscoverX).

[0006] [Figure 2] Figure 2 is a graph showing how much the peptide increases the number of IFNγ-positive CD8 T cells, as measured by IFNγ-ELISPOT, when combined with the vaccine antigen (AdPyCS).

[0007] [Figure 3-1] Figure 3A shows a graph comparing MC38 tumor volumes in mice, normalized to the value on day 0 for each animal. The mouse cohort received intratumoral (IT) injection of 50 μg FoLD01 peptide or intraperitoneal injection of antibodies against CTLA4 and / or PD1. The median value for the control cohort (DMSO / PBS injected into the tumor) is shown as a thick line.

[0008] [Figure 3-2] Figure 3B shows a graph comparing normalized tumor growth dynamics for the indicated groups. Error bars represent the cohort mean and 95% confidence interval.

[0009] [Figure 3-3] Figure 3C shows a graph comparing normalized tumor growth at day 16. The p-value was determined using the non-parametric Mann-Whitney t-test.

[0010] [Figure 4-1] Figure 4A shows a graph comparing Pan02 tumor volumes in mice, normalized to the value on day 0 for each animal. The mouse cohort received intratumoral injection of 50 μg FoLD04 or intraperitoneal injection of antibodies against CTLA4 and / or PD1. The median value for the control cohort (intratumoral injection of DMSO / PBS) is shown as a thick line.

[0011] [Figure 4-2] Figure 4B, a graph comparing normalized tumor growth dynamics for the indicated groups. Error bars represent the cohort mean and 95% confidence interval.

[0012] [Figure 4-3] Figure 4C shows a graph comparing normalized tumor growth on day 21. The p-value was determined using the non-parametric Mann-Whitney t-test.

[0013] [Figure 4-4]Graph comparing normalized tumor growth on day 27 in Fig. 4D. The p-value was determined using the non-parametric Mann-Whitney t-test. **Mode for Carrying Out the Invention**

[0014] **Detailed Description** The present disclosure provides peptides that antagonize the activity of the checkpoint receptor "programmed death 1" (PD-1). The amino acid sequences of these peptides are shown below. **[Table 1]**

[0015] In some embodiments, the disclosed peptides consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the disclosed peptides comprise the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the disclosed peptides consist essentially of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0016] In some embodiments, the disclosed peptides are modified using chemical or recombinant methods to enhance their stability or other pharmacokinetic properties. See, for example, US 2017 / 0020956. Modifications include, but are not limited to, substitution of 1 or more L-amino acids with their corresponding D-forms, acetylation of C-terminal and / or N-terminal residues, amidation of C-terminal and / or N-terminal residues, cyclization, esterification, glycosylation, acylation, attachment of myristic acid or palmitic acid, addition of an N-terminal glycine, addition of a lipophilic moiety (e.g., a long-chain fatty acid), and PEGylation.

[0017] Peptides can be made by any method known in the art, including synthetic methods, recombinant methods, or both. Synthetic methods include solid-phase and solution methods and may involve the use of protecting groups. See, for example, Bodanszky et al. (1976), McOmie (1973), Merrifield (1963), Neurath et al. (1976), Stuart & Young (1984).

[0018] Recombinant production of unmodified peptides can be performed using any nucleotide sequence encoding the peptide in any suitable expression system. Nucleic acid molecules encoding one or more of the disclosed peptides can be incorporated into an expression cassette that includes control elements operably linked to its coding sequence. Control elements include, but are not limited to, initiators, promoters (including inducible, repressible, and constitutive promoters), enhancers, and polyadenylation signals. Signal sequences may be included. The above expression cassette can be provided in a vector that can be introduced into a suitable host cell for the production of the above peptide. Methods for constructing expression cassettes and expression vectors are well known. The expression vector can include one or more expression cassettes encoding one or more peptides that contain, consist essentially of, or consist of SEQ ID NO: 1, 2, 3, or 4.

[0019] In some embodiments, the disclosed peptide or a modified version thereof is conjugated to a moiety (e.g., albumin or transthyretin) to enhance the plasma half-life of the peptide. Methods for preparing such conjugates are well known in the art (e.g., Penchala et al., 2015; Kontermann, 2016; Zorzi et al., 2017).

[0020] In some embodiments, the disclosed peptide or a modified version thereof is conjugated to a partner molecule (e.g., a peptide or protein, such as an antibody intended to increase the half-life of the peptide or modified peptide in vivo and / or provide specific delivery to a target tissue or cell). Conjugation may be direct or via a linker. In some of these embodiments, the peptide or a modified version thereof may be altered by substituting one or more amino acids with amino acids used to conjugate the partner molecule (e.g., lysine), or by N-terminal extension of the peptide with, for example, one, two, three, or four glycine spacer molecules.

[0021] This disclosure also provides CAR-T cells expressing one or more of the disclosed peptides. Methods for preparing CAR-T cells are disclosed, for example, in U.S. Patent No. 9,328,156; U.S. Patent No. 9,845,362; and U.S. Patent No. 9,101,584.

[0022] This disclosure also provides oncolytic viruses comprising nucleic acid molecules encoding one or more of the disclosed peptides. See US 2017 / 0157188; Lawler et al., 2017; US 2015 / 0250837. Examples of oncolytic viruses include, but are not limited to, reoviruses, Seneca Valley virus, varicella stomatitis virus, Newcastle disease virus, herpes simplex virus, morbillivirus, retrovirus, influenza virus, Sindbis virus, poxvirus, and adenovirus.

[0023] An example of an oncolytic reovirus is REOLYSIN. (登録商標) Examples include (pelareorep) and the reovirus disclosed in US 2017 / 0049829.

[0024] An example of an oncolytic Seneca Valley virus is NTX-101 (Rudin et al., 2011).

[0025] Examples of oncolytic bullous stomatitis viruses are disclosed in Stojdl et al., 2000; and Stojdl et al., 2003.

[0026] Examples of oncolytic Newcastle disease viruses include the 73-T PV701 and HDV-HUJ strains (Phuangsab et al., 2001; Lorence et al.) See also 2007 and Freeman et al., 2006.

[0027] Examples of oncolytic herpes simplex viruses include NV1020 (Geevarghese et al., 2010) and T-VEC (Andtbacka et al., 2013).

[0028] Examples of oncolytic morbilliviruses include oncolytic measles virus (e.g., MV-Edm (McDonald et al., 2006) and HMWMAA (Kaufmann et al., 2006)). 2013)) is one example.

[0029] Examples of oncolytic retroviruses are disclosed in Lu et al., 2012.

[0030] Examples of oncolytic influenza viruses are disclosed, for example, in US 2018 / 0057594.

[0031] An example of oncolytic sindobisvirus is disclosed, for example, in Lundstrom, 2017.

[0032] Examples of oncolytic poxviruses are disclosed, for example, in Chan & McFadden, 2014.

[0033] Examples of oncolytic adenoviruses include ONYX-015 (Khuri et al., 2000) and H101 or Oncorine (Liang, 2018).

[0034] therapeutic use The disclosed peptides have numerous therapeutic applications, including treating hyperproliferative disorders (including cancer), treating infectious diseases, enhancing the response to vaccination, treating sepsis, promoting hair re-pigmentation, and promoting the reduction of pigmented skin lesions. (Examples include) having. "To treat" as used herein includes reducing or inhibiting the progression of one or more symptoms of a condition to which the peptide or a modified version thereof is administered.

[0035] "Administer" as used herein includes direct and indirect administration of the disclosed peptide or any modified version thereof.

[0036] In some embodiments, one or more of the disclosed peptides are administered directly. In some of these embodiments, a peptide carrier system is used. Many peptide carrier systems are known in the art, and these include microparticles, polymer nanoparticles, liposomes, solid lipid nanoparticles, hydrophilic mucosal adhesive polymers, thiolated polymers, polymer matrices, nanoemulsions, and hydrogels. See Patel et al. (2014), Bruno et al. (2013), and Feridooni et al. (2016). Any suitable system may be used.

[0037] In some embodiments, engineered T cells expressing and secreting one or more of the disclosed peptides may be used to deliver PD1 inhibition at the engagement site between the T cell receptor and the antigen. T cell-based therapies may, for example, be CAR-T cells expressing one or more of the disclosed peptides. Either inducible or constitutive expression may be used.

[0038] In some embodiments, oncolytic viruses may be used to deliver one or more of the disclosed peptides. Either inducible or constitutive expression may be used.

[0039] In other embodiments, one or more of the disclosed peptides are delivered using one or more nucleic acids (e.g., DNA, cDNA, PNA, RNA, or a combination thereof) encoding the peptides; see, for example, US 2017 / 0165335. Nucleic acids encoding one or more peptides may be delivered using various delivery systems known in the art. These nucleic acid delivery systems include, but are not limited to, gene guns; cationic lipids and cationic polymers; encapsulation in liposomes, microparticles, or microcapsules; electroporation; and virus-based and bacterial-based delivery systems. Virus-based systems include, but are not limited to, modified viruses (e.g., adenoviruses, adeno-associated viruses, herpesviruses, retroviruses, vaccinia viruses, or hybrid viruses containing one or more viral elements). US 2002 / 0111323 describes the use of "naked DNA," i.e., "non-infectious, non-immunogenic, non-integrated DNA sequences" that do not contain "transfection-promoting proteins, viral particles, liposomal formulations, charged lipids, and calcium phosphate precipitants," for the delivery of peptides. Bacteria-based delivery systems are disclosed, for example, in Van Dessel et al. (2015) and Yang et al. (2007).

[0040] In some embodiments, the peptide is administered via an RNA molecule encoding the peptide. In some embodiments, the RNA molecule is encapsulated in nanoparticles. In some embodiments, the nanoparticles include cationic polymers (e.g., poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan, poly(β-aminoester)). In some embodiments, the nanoparticles include cationic lipids or ionizable lipids. In some embodiments, the RNA molecule is conjugated to a bioactive ligand (e.g., N-acetylgalactosamine (GalNAc), cholesterol, vitamin E, antibody, cell-penetrating peptide). See, for example, Akinc et al. (2008), Akinc et al. (2009), A Anderson et al. (2003), Behr (1997), Boussif et al. (1995), Chen et al. (2012), Dahlman et al. (2014), Desigaux et al. (2007), Dong et al. (2014), Dosta et al. (2015), Fenton et al. (2016), Guo et al. (2012), Howard et al. (2006), Kaczmarek et al. (2016), Kanasty et al. (2013), Kauffman et al. (2015), Kozielski et al. (2013), Leus et al. (2014), Lorenz et al. (2004), Love et al. (2010), Lynn See & Langer (2000), Moschos et al. (2007), Nair et al. (2014), Nishina et al. (2008), Pack et al. (2005), Rehman et al. (2013), Schroeder et al. (2010), Tsutsumi et al. (2007), Tzeng et al. (2012), Won et al. (2009), Xia et al. (2009), and Yu et al. (2016).

[0041] In some embodiments, RNA molecules may be modified to reduce their chances of degradation or recognition by the immune system. Ribose sugars, phosphate bonds, and / or individual bases may be modified. See, for example, Behlke (2008), Bramsen (2009), Chiu (2003), Judge & MacLachlan (2008), Kauffman (2016), Li (2016), Morrissey (2005), Prakash (2005), Pratt & MacRae (2009), Sahin (2014), Soutschek (2004), and Wittrup & Lieberman (2015). In some embodiments, the above modifications include one or more of ribo-difluorotoluyl nucleotides, 4'-thio-modified RNAs, boranophosphate bonds, phosphorothioate bonds, 2'-O-methyl (2'-OMe) sugar substitutions, 2'-furanone (2'-F), 2'-O-methoxyethyl (2'-MOE) sugar substitutions, locked nucleic acids (LNAs), and L-RNAs.

[0042] In some embodiments, the administration is carried out in conjunction with one or more other treatments. "In conjunction with" includes the administration preceding or following the administration of one or more of the other treatments.

[0043] Pharmaceutical compositions, routes of administration, and devices One or more peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered in a pharmaceutical composition comprising a pharmaceutically acceptable vehicle, as considered above. The “pharmaceutically acceptable vehicle” may contain one or more substances that do not affect the biological activity of the peptides or modified versions thereof and do not cause adverse reactions when administered to a patient. The pharmaceutical composition may be a liquid or lyophilized. A lyophilized composition may be provided in a kit with a suitable liquid (typically, water for injection (WFI) for use in reconstituting the composition). Other suitable forms of the pharmaceutical composition include suspensions, emulsions, and tablets.

[0044] The pharmaceutical composition may be administered by any suitable route (including, but not limited to, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, epidural, intratumoral, percutaneous (e.g., US 2017 / 0281672), mucosal (e.g., intranasal or oral), pulmonary, and topical (e.g., US 2017 / 0274010) routes). See, for example, US 2017 / 0101474.

[0045] Administration may be systemic or local. In addition to local infusions and injections, implants may be used to achieve local administration. Examples of suitable materials include, but are not limited to, sialastic membranes, polymers, fibrous matrices, and collagen matrices.

[0046] Topical administration may be by cream, ointment, lotion, transdermal patch (e.g., microneedle patch) or other suitable form known in the art.

[0047] Administration may also be by controlled release, for example, using a microneedle patch, pump, and / or a suitable polymeric substance. Examples of suitable substances include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.

[0048] Devices containing any of the peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above include, but are not limited to, syringes, pumps, transdermal patches, spray devices, vaginal rings, and pessaries.

[0049] Treatment of hyperproliferative disorders (including cancer) In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered to a patient to inhibit the progression of hyperproliferative disorders (including cancer). Such inhibition may include, for example, reducing the proliferation of neoplastic or preneoplastic cells; destroying neoplastic or preneoplastic cells; and inhibiting tumor metastasis or reducing tumor size.

[0050] Examples of cancer include, but are not limited to, melanoma (including malignant melanoma of the skin or eye), kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endometrial cancer, uterine cancer, cervix cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, and Endocrine cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcomas, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia), lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors. ), brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, and T-cell lymphoma.

[0051] Combination cancer treatment In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered together with one or more other cancer treatments or immunotherapies (e.g., those described below).

[0052] In some embodiments, the second treatment includes a second agent that reduces or blocks the activity of PD1 (e.g., nivolumab, pembrolizumab, durvalumab) or a second agent that reduces or blocks the activity of CTLA-4 (e.g., ipilimumab, tremelimumab).

[0053] In some embodiments, the second treatment includes a drug that reduces or blocks the activity of PD-L1 (e.g., atezolizumab).

[0054] In some embodiments, the second treatment includes agents that reduce or block the activity of other inhibitory checkpoint molecules and / or molecules that suppress the immune system. These molecules include, but are not limited to, the following: 1. Lymphocyte-activation gene-3 (LAG-3; see He et al., 2016; Triebel et al., 1990); 2. V-domain immunoglobulin suppressor of T cell activation (VISTA (also known as c10orf54, PD1H, DD1α, Gi24, Dies1, and SISP1); US 2017 / 0334990, US 2017 / 0112929, Gao et al., 2017, Wang et al., 2011; Liu et al.) See 2015; 3. T-cell immunoglobulin domain and mucin domain (oglobulin domain and mucin domain)3 (TIM-3; see US 2017 / 0198041, US 2017 / 0029485, US 2014 / 0348842, Sakuishi et al., 2010); 4. Killer immunoglobulin-like receptor (KIR; see US 2015 / 0290316); 5. Drugs that inhibit indoleamine(2,3)-dioxygenase (IDO; see Mellemgaard et al., 2017); 6. B and T Lymphocyte Attenuator (BTLA; see US 2016 / 09222114); and 7. A2A adenosine receptor (A2AR; Beavis et al., 2015; US See 2013 / 0267515; US 2017 / 0166878; Leone et al., 2015; Mediavilla-Varela et al., 2017; Young et al., 2016.

[0055] Drugs that reduce or block LAG-3 activity include, but are not limited to, BMS-986016, IMP321, and GSK2831781 (He et al., 2016).

[0056] Drugs that reduce or block VISTA activity include, but are not limited to, small molecules such as CA-170 and antibodies (e.g., Le Mercier et al., 2014).

[0057] Drugs that reduce or block TIM-3 activity include, but are not limited to, antibodies (e.g., MBG453 and TSR-022; see Dempke et al., 2017).

[0058] Drugs that reduce or block KIR activity include, but are not limited to, monoclonal antibodies (e.g., IPH2101 and lirilumab (BMS-986015, formerly IPH2102); see Benson & Caligiuri, 2014).

[0059] Examples of agents that reduce or block IDO activity include, but are not limited to, epacadostat and the agents disclosed in US 2017 / 0037125.

[0060] Drugs that reduce or block BTLA activity include, but are not limited to, peptides (e.g., Spodzieja et al., 2017).

[0061] Drugs that reduce or block A2AR activity include, but are not limited to, small molecules such as CPI-444 and vipardenant.

[0062] In some embodiments, the second treatment includes a cytokine (e.g., interleukin-7).

[0063] In some embodiments, the second treatment described above includes an agonist of an irritant checkpoint molecule. These molecules include, but are not limited to, the following: 1. CD40; 2. OX40; 3. Glucocorticoid-induced tumor necrosis factor-related protein (GITR); and 4. Inducible T cell costimulatory molecule (ICOS).

[0064] CD40 agonists include, but are not limited to, CD40 agonist monoclonal antibodies (e.g., cp-870, 893, ChiLob7 / 4, dasetuzumab, and lucatumumab). See, for example, Vonderheide et al., 2007; Khubchandani et al., 2009; Johnson et al., 2010; Bensinger et al., 2012; Vonderheide and Glennie, 2013; Johnson et al., 2015.

[0065] OX40 agonists include, but are not limited to, OX40 agonist antibodies (e.g., MOXR0916, MED16469, MED10562, PF-045618600, GSK3174998, and INCCAGN01949) and OX40L-Fc fusion proteins (e.g., MEDI6383). See, for example, Huseni et al., 2014; Linch et al., 2015; Messenheimer et al., 2017. See also Shrimali et al., 2017.

[0066] MEDI1873 is one example of a GITR agonist, but it is not limited to this. See, for example, Schaer et al., 2012; Tigue et al., 2017.

[0067] Examples of ICOS agonists include, but are not limited to, the ICOS agonist antibodies JTX-2011 and GSK3359609. See, for example, Harvey et al., 2015; Michaelson et al., 2016.

[0068] In other embodiments, the second treatment described above includes 4-1BB agonists such as urelumab (Shindo et al., 2015); 4-1BB antagonists (see US 2017 / 0174773); inhibitors of anaplastic lymphoma kinases (ALK; Wang et al., 2014; US 2017 / 0274074) such as crizotinib, ceritinib, alectinib, PF-06463922, NVP-TAE684, AP26113, TSR-011, X-396, CEP-37440, and RXDX-101; and histone deacetylase (HDAC; US Inhibitors (see 2017 / 0327582); VEGFR inhibitors (e.g., axitinib, sunitinib, sorafenib, tivozanib, bevacizumab); and / or anti-CD27 antibodies (e.g., valrirumab).

[0069] In some embodiments, the second treatment described above includes a cancer vaccine (e.g., Duraiswamy et al., 2013). The “cancer vaccine” is an immunogenic composition intended to induce an immune response to a specific antigen in an individual to whom the cancer vaccine is administered. The cancer vaccine typically includes its tumor antigen, which can induce or stimulate an immune response to the tumor antigen. The “tumor antigen” is an antigen present on the surface of a target tumor. The tumor antigen may be a molecule not expressed by non-tumor cells, or it may be, for example, a modified version of a molecule expressed by non-tumor cells (e.g., a protein that is misfolded, shortened, or otherwise mutated).

[0070] In some embodiments, the second treatment described above includes chimeric antigen receptor (CAR) T cell therapy. See, for example, John et al., 2013; Chong et al., 2016.

[0071] In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered in conjunction with the CAR-T cell cancer treatment to enhance its efficacy.

[0072] In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered together with an oncolytic virus, for example, as disclosed in US 2017 / 0143780. Non-limiting examples of oncolytic viruses are described above.

[0073] Further therapeutic use In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered to a patient to treat infectious diseases (including, for example, chronic infections caused by viruses, fungi, bacteria, and protozoa, as well as helminths).

[0074] Examples of viral factors include human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), herpes simplex virus (HSV including HSV1 and HSV2), human papillomavirus (HPV), varicella-zoster virus (VSV), cytomegalovirus (CMV), and hepatitis A, B, and C viruses.

[0075] Examples of fungal factors include Aspergillus, Candida, Coccidioides, Cryptococcus, and Histoplasma capsulatum.

[0076] Examples of bacterial factors include Streptococcal bacteria (e.g., pyogenes, agalactiae, pneumoniae), Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis.

[0077] Examples of protozoa include Sarcodina (e.g., Entamoeba), Mastigophora (e.g., Giardia), Ciliophora (e.g., Balantidium), and Sporozoa (e.g., Plasmodium falciparum, Cryptosporidium).

[0078] Examples of worms include Platyhelminths (e.g., trematodes, tapeworms), Acanthocephalins, and Nematodes.

[0079] In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered as vaccine adjuvants to enhance the response to vaccination (e.g., by increasing effector T cells and / or reducing T cell exhaustion). The vaccine may be, for example, an RNA vaccine (e.g., US 2016 / 0130345, US 2017 / 0182150), a DNA vaccine, a recombinant vector, a protein vaccine, or a peptide vaccine. Such vaccines may be delivered using, for example, virus-like particles, as is well known in the art.

[0080] In some embodiments, one or more of the above-mentioned peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered to treat sepsis.

[0081] In some embodiments, one or more of the above-described peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses are administered to promote the re-pigmentation of hair color. In some embodiments, one or more of the peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described herein are administered to promote the reduction of pigmented skin lesions. [Examples]

[0082] Example 1. PATHHUNTER (登録商標) Checkpoint signaling assay Peptides, PATHHUNTER (登録商標) We used a checkpoint signaling assay (DiscoverX) to test their ability to inhibit the binding of PDL1 to PD1.

[0083] Jurkat cells expressing PD1 and SHP1 proteins (each fused to a fragment of the enzyme fragment complementation (EFC) system) were co-incubated with PD1-presenting U2OS cells. This resulted in PD1 activation and SHP1 recruitment to the PD1 receptor, bringing the two EFC fragments together to generate a photosignal. Inhibitory peptides or antibodies added to the culture reduced this photosignal.

[0084] Cells in the co-culture were incubated at room temperature (RT) for 2 hours (PD1 assay). The assay signal was then analyzed using PATHHUNTER. (登録商標) Bioassay The peptides were generated using a detection kit. FoLD01 (SEQ ID NO: 1), LD10 (SEQ ID NO: 5), and LD12 (SEQ ID NO: 6) peptides were tested in double denominations at two concentrations, 20 μM and 100 μM. The peptides were dissolved in water or DMSO and diluted with assay buffer.

[0085] Microplates for chemiluminescence signal detection using PerkinElmer ENVISION TM The signal was read after signal generation using the instrument. The percentage of inhibitory effectiveness was calculated using the following formula (where "RLU" means relative optical units):

number

[0086] The results indicate that the FoLD01 peptide inhibited the PD1-PDL1 interaction to a greater extent than peptides LD10 and LD12 (Figure 1).

[0087] Example 2. Evaluation of peptides in the AdPyCS mouse model Groups of five mice were immunized intramuscularly with recombinant replication-deficient adenovirus expressing Plasmodium yoelli perisporozoite protein (AdPyCSP), and then subcutaneously treated with peptide LD10da (SEQ ID NO: 6, 1 μg, with the first amino acid being a D-amino acid), FoLD01 (1 μg, 10 μg), or anti-PD1 monoclonal antibody (10 μg). Ten days after injection, the mice were euthanized and their individual spleens were removed. The number of IFNγ secretory antigen-specific CD8+ T cells was determined using the ELISPOT assay (Figure 2).

[0088] The results showed that peptide FoLD01 responded to antigen-specific CD8+ stimulation by antigen alone. We demonstrated an increase in T cell count (one-way ANOVA; statistical differences are for AdPyCS stimulation only).

[0089] Example 3. Evaluation of peptides in the MC38 tumor model On the mouse, 1x10 6 MC38 (colon cancer) cells were subcutaneously injected, and tumor volume was measured on days 2, 5, 9, 12, and 16 (end of the study), normalized to the value on day 0 for each animal. The average tumor size was approximately 80-120 mm. 3When the [parameter] was reached, the mice were randomized and treatment was initiated. The peptides were tested in the MC38 tumor model. The FoLD01 peptide was administered intratumorally twice a week (50 μg dose). The monoclonal antibodies were administered intraperitoneally twice a week (5 mg / kg (anti-CTLA4) and 10 mg / kg (anti-PD1) doses). Statistical test used: non-parametric, two-sided Mann-Whitney test without pairing.

[85] Control used: intratumoral injection of DMSO diluted in PBS (intratumoral injection for FoLD01). The results are shown in FIGS. 3(A-C). Generally, administration of anti-CTLA4 mAb and anti-CTLA4 + anti-PD1 mAb resulted in a significant decrease in tumor volume. Administration of anti-PD1 mAb resulted in a limited decrease in tumor volume that may be due to the injected 1×10 6 MC38 cells.

[0090] Intratumoral injection of the FoLD01 peptide significantly decreased tumor volume compared to the intratumoral DMSO / PBS control using a non-parametric Mann-Whitney t test. The p-value was 0.0499.

[0091] Example 4. Evaluation of peptides in the Pan02 tumor model 3×10 6 Pan02 (pancreatic adenocarcinoma) cells were subcutaneously injected into the posterior right flank of the mice. Tumor volume was measured at least twice a week in two dimensions using calipers. When the average tumor size reached approximately 80-120 mm 3 the mice were randomized and treatment was initiated. FoLD04 was administered intratumorally twice a week (50 μg dose). The monoclonal antibodies were administered intraperitoneally twice a week (5 mg / kg (anti-CTLA4) and 10 mg / kg (anti-PD1) doses).

[0092] It should be noted that some parts in the original text seem to be incomplete or have unclear notations (such as the "[parameter]" in the first paragraph and the "1×10" in the second paragraph without clear following content), which may affect the full understanding and accurate translation in a more comprehensive context. But the translation is done based on the provided text as much as possible.Tumor volume was measured on days 4, 7, 11, 14, 18, 21, 25, and 27 (end of study), and normalized for each animal relative to the day 0 value. Statistical test used: Unpaired, nonparametric two-sided Mann-Whitney U test.

[0093] Control used: Intratumoral injection of DMSO diluted in PBS (intratumoral injection for FoLD04). The results are shown in Figure 4.

[0094] Summary and interpretation of Pan02 model results Treatment with anti-CTLA4 mAbs and anti-CTLA4 + anti-PD1 mAbs resulted in a significant reduction in tumor volume, regardless of the time of evaluation and the statistical test used. Anti-PD1 mAbs resulted in sporadic, significant reductions in tumor volume, depending on the time of evaluation and the statistical test used.

[0095] When administered intratumorally, the FoLD04 peptide showed a significant reduction in tumor volume compared to the DMSO / PBS IT control, as measured by the non-parametric Mann-Whitney t test.

[0096] Based on the time of evaluation and the statistical tests used, subcutaneous administration of the peptide tended to lead to control of tumor volume, but this did not reach statistical significance. References [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The present invention provides, for example, the following items: (Item 1) A peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. (Item 2) An expression construct encoding a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. (Item 3) The expression constructs described in item 2, present in CAR-T cells or oncolytic viruses. (Item 4) (a) Below: (i) A peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4; (ii) nucleic acids encoding the peptide; (iii) CAR-T cells expressing the peptide; (iv) Oncolytic viruses expressing the peptide Active agents selected from the group consisting of; and (b) Pharmaceutically acceptable carriers, A pharmaceutical composition containing [a certain substance]. (Item 5) The active agent is a nucleic acid, wherein the nucleic acid is selected from the group consisting of DNA, cDNA, PNA, and RNA, as described in item 4 of the pharmaceutical composition. (Item 6) The nucleic acid is RNA, as described in item 5, for the pharmaceutical composition. (Item 7) The RNA comprises a modification selected from the group consisting of (i) modification of the ribose sugar, (ii) modification of the phosphate bond, and (iii) modification of the base, as described in item 6. (Item 8) The aforementioned modification is selected from the group consisting of ribo-difluorotoluyl nucleotide, 4'-thio-modified RNA, boranophosphate bond, phosphorothioate bond, 2'-O-methyl(2'-OMe) sugar substitution, 2'-fluoro(2'-F), 2'-O-methoxyethyl(2'-MOE) sugar substitution, locked nucleic acid (LNA), and L-RNA, as described in item 7. (Item 9) The pharmaceutically active agent is a peptide, wherein the peptide is provided together with a peptide carrier system selected from the group consisting of microparticles, polymer nanoparticles, liposomes, solid lipid nanoparticles, hydrophilic mucosal adhesive polymers, thiolated polymers, polymer matrices, nanoemulsions, and hydrogels, as described in item 4. (Item 10) A method for inhibiting the progression of hyperproliferative disorders, treating infectious diseases, enhancing the response to vaccination, treating sepsis, promoting hair repigmentation, or promoting the reduction of pigmented skin lesions, the method comprising the step of administering an effective amount of the pharmaceutical composition described in item 4 to an individual in need thereof. (Item 11) The method according to item 10, wherein the pharmaceutical composition is administered to inhibit the progression of the hyperproliferative disorder. (Item 12) The aforementioned hyperproliferative disorder is cancer, as described in item 11. (Item 13) The method according to item 12, wherein the cancer is pancreatic cancer or colon cancer. (Item 14) The method of item 10, further comprising the step of administering a second treatment to the patient. (Item 15) The second treatment is as follows: (i) Cancer vaccines; (ii) Chimeric antigen receptor (CAR) T cell therapy; (iii) PD1, PD-L1, lymphocyte activator gene-3 (LAG-3), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), V-domain immunoglobulin supplement for T cell activation Lesser (VISTA), T cell immunoglobulin domain and mucin domain 3 (TI M-3) Therapies comprising reducing or blocking the activity of molecules selected from the group consisting of killer immunoglobulin-like receptors (KIRs), indoleamine (2,3)-dioxygenase (IDO), B and T lymphocyte-reducing factors (BTLAs), and A2A adenosine receptors (A2ARs); (iv) cytokines; (v) Molecules selected from the group consisting of CD40, OX40, glucocorticoid-induced tumor necrosis factor-related protein (GITR), and inducible T cell costimulatory molecules (ICOS). Agonist of; (vi) oncolytic viruses; and (vii) A therapeutic agent selected from the group consisting of 4-1BB agonists, 4-1BB antagonists, anaplastic lymphoma kinase (ALK) inhibitors, histone deacetylase (HDAC) inhibitors, and VEGFR inhibitors. A method described in item 14, selected from the group consisting of the following. (Item 16) The method according to item 10, wherein the pharmaceutical composition is administered to treat an infectious disease or as a vaccine adjuvant to a vaccine against the infectious disease. (Item 17) The method according to item 16, wherein the infectious disease is malaria or hepatitis B. (Item 18) The method according to item 10, wherein at least one peptide is administered to treat sepsis. (Item 19) The method according to item 10, wherein at least one peptide is administered to promote hair re-pigmentation or to promote reduction of pigmented skin lesions.

Claims

[Claim 1] The invention described in the specification.

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