LAG3-binding peptide

Abstract

The present disclosure provides peptides that can be used to bind to LAG3 and block the interaction of LAG3 with other molecules (e.g., MHC-II, FGL1, and α-synuclein). These peptides can be used for various therapeutic purposes, such as inhibiting the progression of hyperproliferative disorders (including cancer), or synucleinopathies, inhibiting the progression of sepsis, inhibiting the progression of infectious diseases, and enhancing responses to vaccines.

Description

[Technical field]

[0001] This application incorporates by reference the contents of the 2.37kb of text submitted which is the Sequence Listing to this application, entitled "PCTsequencelisting.txt", filed on May 20, 2020.

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

[0003] Technical Field The present disclosure relates generally to immunomodulatory peptides. [Background technology]

[0004] background Lymphocyte activation gene 3 (LAG3 (also known as LAG-3, LAG 3, Lag3, CD223, FDC protein)) is a member of the immunoglobulin superfamily of receptors.

[0005] LAG3 is expressed on immune cells (activated T cells, Huard et al., 1994; natural killer cells, Triebel et al., 1990; B cells, Kisielow et al., 2005; plasmacytoid dendritic cells, Workman et al., 2009) where it binds to MHC class II (MHC-II) and acts as an immune checkpoint receptor. LAG3 also binds to fibrinogen-like protein (FGL1), and disruption of this binding can enhance antitumor immunity (Wang et al., 2019). There is a continuing need for useful regulators of immune checkpoint pathways. LAG3 is also expressed on neurons where it acts as a receptor for α-synuclein aggregates characteristic of synucleinopathies (Mao et al., 2016), disorders characterized by the abnormal accumulation of α-synuclein protein aggregates in neurons, nerve fibers, or glial cells. Synucleinopathies include: idiopathic and hereditary forms of Parkinson's disease (PD); diffuse Lewy body (DLB) disease (also known as Dementia with Lewy Bodies or Lewy body dementia); incident Lewy body disease; Lewy body variant of Alzheimer's disease (LBV); Combined Alzheimer's and Parkinson disease (CAPD); pure autonomic failure (PAF); multiple system atrophy (MSA) (e.g., olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome); pantothenate kinase-associated neurodegeneration; Down's syndrome; Gaucher-associated synucleinopathies; and neurodegeneration associated with brain iron accumulation. There is a continuing need for therapeutic agents to treat or manage the symptoms of synucleinopathies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Huard et al., "Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand," Immunogenetics 39 (3): 213-7, 1994 [Non-Patent Document 2] Triebel et al., “LAG3, a novel lymphocyte activation gene closely related to CD4,” J. Exp. Med. 171, 1393-405, 1990 [Non-Patent Document 3] Kisielow et al., "Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells". European Journal of Immunology 35 (7): 2081-8, 2005 [Non-Patent Document 4] Workman et al., "LAG-3 regulates plasmacytoid dendritic cell homeostasis," Journal of Immunology 182 (4): 1885-91, 2009 [Non-Patent Document 5] Wang et al., “Fibrinogen-like Protein 1 is a Major Immune Inhibitory Ligand of LAG-3,” Cell 176, 334-47, 2019 [Non-Patent Document 6] Mao et al., “Pathological α-synuclein transmission initiated by binding lymphocyte-activation gene 3,” Science 353, aah3374, 2016 [Brief description of the drawings]

[0007] [Figure 1A] 1A and 1B are graphs showing the results of the LAG3 blocking functional inhibition assay described in Example 2. [Figure 1B] 1A and 1B are graphs showing the results of the LAG3 blocking functional inhibition assay described in Example 2.

[0008] [Figure 2A] Figures 2A, 2B, 2C, and 2D are graphs showing the results of four independent homogeneous time-resolved fluorescence (HTRF) assays of peptide LAG3-11. Reference Ab-LAG3, anti-LAG3 antibody. OVA, ovalbumin (OVA) peptide (ISQAVHAAHAEINEAGR, SEQ ID NO: 8). "HTRF ratio" is the emission at 665 nm (acceptor) / emission at 620 nm (donor) multiplied by a factor, i.e., 104. [Figure 2B] Figures 2A, 2B, 2C, and 2D are graphs showing the results of four independent homogeneous time-resolved fluorescence (HTRF) assays of peptide LAG3-11. Reference Ab-LAG3, anti-LAG3 antibody. OVA, ovalbumin (OVA) peptide (ISQAVHAAHAEINEAGR, SEQ ID NO: 8). "HTRF ratio" is the emission at 665 nm (acceptor) / emission at 620 nm (donor) multiplied by a factor, i.e., 104. [Figure 2C] Figures 2A, 2B, 2C, and 2D are graphs showing the results of four independent homogeneous time-resolved fluorescence (HTRF) assays of peptide LAG3-11. Reference Ab-LAG3, anti-LAG3 antibody. OVA, ovalbumin (OVA) peptide (ISQAVHAAHAEINEAGR, SEQ ID NO: 8). "HTRF ratio" is the emission at 665 nm (acceptor) / emission at 620 nm (donor) multiplied by a factor, i.e., 104. [Figure 2D]Figures 2A, 2B, 2C, and 2D are graphs showing the results of four independent homogeneous time-resolved fluorescence (HTRF) assays of peptide LAG3-11. Reference Ab-LAG3, anti-LAG3 antibody. OVA, ovalbumin (OVA) peptide (ISQAVHAAHAEINEAGR, SEQ ID NO: 8). "HTRF ratio" is the emission at 665 nm (acceptor) / emission at 620 nm (donor) multiplied by a factor, i.e., 104.

[0009] [Diagram 3] Figure 3 is a graph showing the results of an HTRF assay of peptide LAG3-11. Anti-LAG-3 GMP-A092 is an anti-LAG3 antibody.

[0010] [Figure 4] FIG. 4 is a graph showing the results of an HTRF assay of peptide LAG3-42.

[0011] [Diagram 5] FIG. 5 is a graph showing the results of an HTRF assay of peptide LAG3-48.

[0012] [Figure 6] FIG. 6 is a graph showing the results of an HTRF assay of peptide LAG3-51.

[0013] [Figure 7] FIG. 7 is a graph showing the results of an HTRF assay of peptide LAG3-54.

[0014] [Figure 8] FIG. 8 is a graph showing the results of an HTRF assay of peptide LAG3-56.

[0015] [Figure 9] FIG. 9 is a graph showing the results of an HTRF assay of peptide LAG3-60.

[0016] [Figure 10] FIG. 10 is a graph showing the results of a peripheral blood mononuclear cell (PBMC) assay.

[0017] [Figure 11] FIG. 11 is a graph showing the results of a human LAG3 / FGL1 TR-FRET binding assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present disclosure provides peptides that bind to LAG3 and can be used to block its interactions with other molecules such as MHC-II, FGL1, and α-synuclein. [Table 1]

[0019] 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, replacing one 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, binding of myristic acid or palmitic acid, addition of N-terminal glycine, addition of lipophilic moieties (e.g., long fatty acid chains), and PEGylation.

[0020] Peptides can be produced by any method known in the art, including synthetic methods, recombinant methods, or both. Synthetic methods include solid-phase or liquid-phase methods and can include the use of protective groups. See, for example, Bodanszky et al. (1976), McOmie (1973), Merrifield (1963), Neurath et al. (1976), Stuart & Young (1984).

[0021] Recombinant production of peptides can be performed using any nucleotide sequence(s) encoding the peptide in any suitable expression system. A nucleic acid molecule encoding one or more of the disclosed peptides can be incorporated into an expression cassette that includes control elements operably linked to the coding sequence. Control elements include, but are not limited to, initiators, promoters (including inducible, repressible, and constitutive promoters), enhancers, and polyadenylation signals. A signal sequence can be included. The expression cassette can be provided in a vector that can be introduced into a suitable host cell for production of the peptide(s). Methods for constructing expression cassettes and expression vectors are well known. An expression vector can include one or more expression cassettes encoding one or more peptides (including, consisting essentially of, or consisting of any of SEQ ID NOs: 1-7).

[0022] In some embodiments, one or more peptides are expressed as components of a fusion protein. The other component of the fusion protein can be, for example, a cytokine or an engineered T cell receptor (TCR). The fusion protein can include one or more linkers between its components. In some embodiments, a linker between a peptide and another component of the fusion protein can include a proteolytic cleavage site to release the peptide after expression of the fusion protein. See, for example, US 2016 / 0138066; US 2018 / 0135060; US 2014 / 0343251; US ​​2012 / 0142891; Rodriguez et al., 2014.

[0023] In some embodiments, a component of the fusion protein is a moiety that can enhance the plasma half-life of the peptide (e.g., albumin or transthyretin). In other embodiments, a peptide or a modified version of a peptide is conjugated to the moiety. Methods for preparing such conjugates are well known in the art (e.g., Penchala et al., 2015; Kontermann, 2016; Zorzi et al., 2017).

[0024] In some embodiments, a component of the fusion protein is a partner molecule, such as 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). Alternatively, the peptide or modified version thereof may be conjugated to its partner molecule. Conjugation may be direct or through a linker. In some of these embodiments, the peptide or modified version thereof may be altered to replace one or more amino acids with the amino acid (e.g., lysine) used to attach to the partner molecule, or by N-terminally extending the peptide with, for example, one, two, three, or four glycine spacer molecules.

[0025] The present 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.

[0026] The present disclosure also provides an oncolytic virus comprising a nucleic acid molecule encoding one or more of the disclosed peptides.See US 2017 / 0157188; Lawler et al., 2017; US 2015 / 0250837. Oncolytic viruses include, but are not limited to, reovirus, Seneca Valley virus, vesicular stomatitis virus, Newcastle disease virus, herpes simplex virus, morbillivirus virus, retrovirus, influenza virus, Sindbis virus, poxvirus, and adenovirus.

[0027] Examples of oncolytic reoviruses include REOLYSIN® (pelareorep) and the reoviruses disclosed in US 2017 / 0049829.

[0028] An example of an oncolytic Seneca Valley virus is NTX-101 (Rudin et al., 2011).

[0029] Examples of oncolytic vesicular stomatitis viruses are disclosed in Stojdl et al., 2000; and Stojdl et al., 2003.

[0030] Examples of oncolytic Newcastle Disease Viruses include the 73-T PV701 and HDV-HUJ strains (Phuangsab et al., 2001; Lorence et al., 2007; and see also Freeman et al., 2006).

[0031] Examples of oncolytic herpes simplex viruses include NV1020 (Geevarghese et al., 2010) and T-VEC (Andtbacka et al., 2013).

[0032] Examples of oncolytic Morbillivirus viruses include oncolytic measles viruses (e.g., MV-Edm (McDonald et al., 2006) and HMWMAA (Kaufmann et al., 2013).

[0033] Examples of oncolytic retroviruses are disclosed in Lu et al., 2012.

[0034] Examples of oncolytic influenza viruses are disclosed, for example, in US 2018 / 0057594.

[0035] Examples of oncolytic Sindbis viruses are disclosed, for example, in Lundstrom, 2017.

[0036] Examples of oncolytic poxviruses are disclosed, for example, in Chan & McFadden, 2014.

[0037] Examples of oncolytic adenoviruses include ONYX-015 (Khuri et al., 2000) and H101 or Oncorine (Liang, 2018).

[0038] therapeutic use The peptides and modified versions thereof disclosed herein have many therapeutic applications, including treating hyperproliferative disorders, such as cancer. "Treat," as used herein, includes reducing or inhibiting the progression of one or more symptoms of the condition for which the peptide or modified version thereof is administered. The peptides and modified versions thereof may also be useful for reducing or treating one or more symptoms of synucleopathies, infectious diseases, and sepsis, as well as enhancing responses to vaccinations.

[0039] "Administer," as used herein, includes administration of the disclosed peptides or modified versions thereof per se, as well as administration by various vehicles as described below.

[0040] In some embodiments, one or more of the disclosed peptides and / or modified versions thereof are directly administered. In some of these embodiments, a peptide carrier system is used. Many peptide carrier systems are known in the art, including microparticles, polymer nanoparticles, liposomes, solid lipid nanoparticles, hydrophilic mucoadhesive polymers, thiolated polymers, polymer matrices, nanoemulsions, and hydrogels. See Patel et al. (2014), Bruno et al. (2013), Feridooni et al. (2016). Any suitable system can be used.

[0041] In some embodiments, engineered T cells expressing and secreting one or more of the disclosed peptides can be used to effect LAG3 inhibition at the site of T cell receptor and antigen engagement. The T cell-based therapy can be, for example, a CAR-T cell expressing one or more of the disclosed peptides. Either inducible or constitutive expression can be used.

[0042] In some embodiments, oncolytic viruses can be used to deliver one or more of the peptides disclosed above. Either inducible or constitutive expression can be used.

[0043] 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 combinations thereof) encoding the peptide(s); see, for example, US 2017 / 0165335. The nucleic acids encoding one or more peptides can be delivered using a variety of delivery systems known in the art. Nucleic acid delivery systems include, but are not limited to, gene guns; cationic lipids and cationic polymers; encapsulation in liposomes, microparticles, or microcapsules; electroporation; viral-based and bacterial-based delivery systems. Viral-based systems include, but are not limited to, modified viruses, such as adenoviruses, adeno-associated viruses, herpes viruses, retroviruses, vaccinia viruses, or hybrid viruses that contain elements of one or more viruses. US 2002 / 0111323 describes the use of "naked DNA," i.e., "non-infectious, non-immunogenic, non-integrated DNA sequences" that are free of "transfection-facilitating proteins, viral particles, liposomal formulations, charged lipids and calcium phosphate precipitants," to administer peptides. Bacteria-based delivery systems are disclosed, for example, in Van Dessel et al. (2015) and Yang et al. (2007).

[0044] In some embodiments, the peptide is administered via an RNA molecule that encodes the peptide, hi some embodiments, the RNA molecule is encapsulated in a nanoparticle. In some embodiments, the nanoparticles comprise a cationic polymer (e.g., poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan, poly(β-amino ester). In some embodiments, the nanoparticles comprise a cationic lipid or an ionizable lipid. In some embodiments, the RNA molecule is conjugated to a bioactive ligand (e.g., N-acetylgalactosamine (GalNAc), cholesterol, vitamin E, antibodies, cell penetrating peptides). See, e.g., Akinc et al. (2008), Akinc et al. (2009), 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. 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 & 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. See Xia et al. (2009), Yu et al. (2016).

[0045] In some embodiments, the RNA molecule may be modified to reduce its chance of degradation or recognition by the immune system. The ribose sugar, phosphate linkage, 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), Wittrup & Lieberman (2015). In some embodiments, the modifications are one or more of ribo-difluorotoluyl nucleotides, 4'-thio modified RNA, boranophosphate linkages, phosphorothioate linkages, 2'-O-methyl (2'-OMe) sugar substitutions, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE) sugar substitutions, locked nucleic acids (LNA), and L-RNA.

[0046] In some embodiments, administration is in conjunction with one or more other therapies. "In conjunction with" includes administration along with, prior to, or following administration of the one or more other therapies.

[0047] Pharmaceutical compositions, routes of administration, and devices One or more peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses (as discussed above) are typically administered in a pharmaceutical composition that includes a pharma- ceutically acceptable vehicle. A "pharma-ceutically acceptable vehicle" may include one or more substances that do not affect the biological activity of the peptide or modified versions thereof and do not cause adverse reactions when administered to a patient. The pharmaceutical composition may be liquid or lyophilized. Lyophilized compositions 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 pharmaceutical compositions include suspensions, emulsions, and tablets.

[0048] The pharmaceutical compositions may be administered by any suitable route, including, but not limited to, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, epidural, intratumoral, transdermal (e.g., US 2017 / 0281672), mucosal (e.g., intranasal or oral), pulmonary, and topical (e.g., US 2017 / 0274010). See, e.g., US 2017 / 0101474.

[0049] Administration can be systemic or local. In addition to local infusion and injection, implants can be used to achieve local administration. Examples of suitable materials include, but are not limited to, sialastic membranes, polymers, fibrous matrices, and collagen matrices.

[0050] Topical administration may be by creams, ointments, lotions, transdermal patches (eg, microneedle patches), or other suitable forms well known in the art.

[0051] Administration can also be by controlled release, for example, using microneedle patches, pumps and / or suitable polymeric materials. Examples of suitable materials 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), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.

[0052] Devices comprising any of the peptides, modified 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.

[0053] Treatment of hyperproliferative disorders (including cancer) In some embodiments, one or more of the peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above are administered to patients to inhibit the progression of hyperproliferative disorders (including cancer).Such inhibition can include, for example, reducing the proliferation of neoplastic or pre-neoplastic cells; destroying neoplastic or pre-neoplastic cells; and inhibiting tumor metastasis or reducing tumor size.

[0054] Examples of cancer include, but are not limited to, melanoma (including cutaneous or intraocular malignant melanoma), 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 area cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endometrial cancer, and ovarian cancer. Cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and lymphocytic lymphoma), bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, and T-cell lymphoma.

[0055] Cancer Combination Therapy In some embodiments, one or more of the peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above are administered together with one or more other cancer therapies or immunotherapies (e.g., those described below).

[0056] In some embodiments, the second treatment includes a second agent that reduces or blocks the activity of PD-1 (e.g., nivolumab, pembrolizumab, durvalumab) or reduces or blocks the activity of CTLA-4 (e.g., ipilimumab, tremelimumab).

[0057] In some embodiments, the second treatment includes an agent that reduces or blocks the activity of PD-L1 (e.g., atezolizumab).

[0058] In some embodiments, the second treatment comprises an agent that reduces or blocks the activity of LAG3 or other inhibitory checkpoint molecules and / or molecules that suppress the immune system. These molecules include, but are not limited to: 1. V-domain Immunoglobulin Suppressor of T cell Activation (VISTA (also known as c10orf54, PD-1H, DD1α, Gi24, Dies1, and SISP1); see US 2017 / 0334990, US 2017 / 0112929, Gao et al., 2017, Wang et al., 2011; Liu et al, 2015); 2. T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3; see US 2017 / 0198041, US 2017 / 0029485, US 2014 / 0348842, Sakuishi et al, 2010); 3. Killer immunoglobulin-like receptors (KIRs; see US 2015 / 0290316); 4. Drugs that inhibit indoleamine (2,3)-dioxygenase (IDO; see Mellemgaard et al., 2017); 5. B and T Lymphocyte Attenuator (BTLA; see US 2016 / 09222114); and 6. A2A adenosine receptor (A2AR; see Beavis et al, 2015; US 2013 / 0267515; US 2017 / 0166878; Leone et al, 2015; Mediavilla-Varela et al, 2017; Young et al, 2016).

[0059] Agents that reduce or block the activity of LAG3 include, but are not limited to, BMS-986016, IMP321, and GSK2831781 (He et al., 2016).

[0060] Agents that reduce or block the activity of VISTA include, but are not limited to, small molecules (e.g., CA-170) and antibodies (e.g., Le Mercier et al, 2014).

[0061] Agents that reduce or block the activity of TIM-3 include, but are not limited to, antibodies (e.g., MBG453 and TSR-022; see Dempke et al., 2017).

[0062] Agents that reduce or block the activity of KIR include, but are not limited to, monoclonal antibodies (e.g., IPH2101 and lirilumab (BMS-986015, formerly IPH2102); see Benson & Caligiuri, 2014).

[0063] Agents that reduce or block the activity of IDO include, but are not limited to, epacadostat and agents disclosed in US 2017 / 0037125.

[0064] Agents that reduce or block the activity of BTLA include, but are not limited to, peptides (e.g., Spodzieja et al., 2017).

[0065] Agents that reduce or block the activity of A2AR include, but are not limited to, small molecules (eg, CPI-444 and bipadenant).

[0066] In some embodiments, the second treatment comprises a cytokine (eg, interleukin 7).

[0067] In some embodiments, the second treatment comprises an agonist of a stimulatory 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 factor (ICOS).

[0068] CD40 agonists include, but are not limited to, CD40 agonist monoclonal antibodies (e.g., cp-870,893, ChiLob7 / 4, dacetuzumab, 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.

[0069] Agonists of OX40 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, e.g., Huseni et al., 2014;Linch et al., 2015;Messenheimer et al., 2017.See also, Shrimali et al., 2017.

[0070] Agonists of GITR include, but are not limited to, MEDI1873. See, e.g., Schaer et al., 2012; Tigue et al., 2017.

[0071] Agonists of ICOS include, but are not limited to, ICOS agonist antibodies JTX-2011 and GSK3359609.See, e.g., Harvey et al., 2015;Michaelson et al., 2016.

[0072] In other embodiments, the second therapy is a 4-1BB agonist (Shindo et al., 2015) (e.g., urelumab); a 4-1BB antagonist (see US 2017 / 0174773); an inhibitor of anaplastic lymphoma kinase (ALK; Wang et al., 2014; US 2017 / 0274074) (e.g., crizotinib, ceritinib, alectinib, PF-06463922, NVP-TAE684, AP26113, TSR-011, X-396, CEP-37440, RXDX-101); an inhibitor of histone deacetylase (HDAC; US See 2017 / 0327582); VEGFR inhibitors (e.g., axitinib, sunitinib, sorafenib, tivozanib, bevacizumab); and / or anti-CD27 antibodies (e.g., varlilumab).

[0073] In some embodiments, the second treatment comprises a cancer vaccine (e.g., Duraiswamy et al., 2013). A "cancer vaccine" is an immunogenic composition intended to elicit an immune response against a specific antigen in an individual to whom the cancer vaccine is administered. A cancer vaccine typically comprises a tumor antigen that can induce or stimulate an immune response against the tumor antigen. A "tumor antigen" is an antigen present on the surface of a target tumor. A tumor antigen may be a molecule that is not expressed by non-tumor cells, or may be, for example, an altered version of a molecule expressed by non-tumor cells (e.g., a misfolded, truncated, or otherwise mutated protein).

[0074] In some embodiments, the second treatment comprises chimeric antigen receptor (CAR) T cell therapy. See, e.g., John et al., 2013; Chong et al., 2016.

[0075] In some embodiments, one or more of the peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above are administered in conjunction with a CAR-T cell cancer therapy to increase the efficacy of the CAR-T cell cancer therapy.

[0076] In some embodiments, one or more of the peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above are administered with an oncolytic virus, e.g., as disclosed in US 2017 / 0143780. Non-limiting examples of oncolytic viruses are described above.

[0077] Further therapeutic uses Synucleinopathy In some embodiments, one or more of the peptides, modified peptides, nucleic acid molecules, CAR-T cells, and / or oncolytic viruses described above may be useful for reducing symptoms of synucleinopathies, either alone or in combination with other therapeutic interventions (e.g., L-DOPA, dopamine agonists (e.g., ropinirole, pramipexole), dopamine reuptake inhibitors (e.g., amantadine), and cholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine). Examples of synucleinopathies include: These include: idiopathic and hereditary forms of Parkinson's disease (PD); diffuse Lewy body (DLB) disease (also known as dementia with Lewy bodies or dementia with Lewy bodies); incidental Lewy body disease; Lewy body variant of Alzheimer's disease (LBV); combined Alzheimer's and Parkinson's disease (CAPD); pure autonomic failure (PAF); multiple system atrophy (MSA) (e.g., olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome); pantothenate kinase-associated neurodegeneration; Down's syndrome; Gaucher-related synucleinopathy; and neurodegeneration involving brain iron accumulation.

[0078] sepsis LAG3 expression is upregulated in sepsis (Patil et al., 2017). Thus, one or more of the peptides, modified peptides, or nucleic acids described above may be useful for treating sepsis, either alone or in combination with other therapeutic interventions (e.g., antibiotics, intravenous fluids, and vasopressors).

[0079] infectious disease In some embodiments, one or more of the disclosed peptides, modified peptides, or nucleic acids described above may be administered to treat infectious diseases, including chronic infections caused by viruses, fungi, bacteria, and protozoa, as well as helminths, either alone or in combination with other therapeutic interventions.

[0080] Examples of viral agents include human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) (including HSV1 and HSV2), human papilloma virus (HPV), varicella zoster virus (VSV), cytomegalovirus (CMV), hepatitis A virus, hepatitis B virus, and hepatitis C virus.

[0081] Examples of fungal agents include Aspergillus, Candida, Coccidioides, Cryptococcus, and Histoplasma capsulatum.

[0082] Examples of bacterial agents include Streptococcus (eg, pyogenes, agalactiae, pneumoniae), Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis.

[0083] Examples of protozoa include Sarcodina (eg, Entamoeba), Mastigophora (eg, Giardia), Ciliophora (eg, Balantidium), and Sporozoa (eg, Plasmodium falciparum, Cryptosporidium).

[0084] Examples of helminths include Platyhelminths (eg, flukes, cestodes), Acanthocephalins, and Nematodes.

[0085] Vaccine adjuvants In some embodiments, one or more of the disclosed peptides, modified peptides, or nucleic acids described above are used to ameliorate a response to vaccination (e.g., It can be administered as a vaccine adjuvant together with vaccine to enhance (by increasing effector T cells and / or reducing T cell exhaustion).The vaccine can 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 can be delivered, for example, using virus-like particles, as is well known in the art. In certain embodiments, for example, the following are provided: (Item 1) A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7. (Item 2) 2. The peptide according to item 1, which consists essentially of the amino acid sequence. (Item 3) 3. The peptide according to item 2, consisting of the amino acid sequence. (Item 4) A nucleic acid encoding the peptide according to item 1. (Item 5) 5. The nucleic acid according to item 4, which is an expression construct. (Item 6) The nucleic acid according to item 5, which is present in a CAR-T cell or an oncolytic virus. (Item 7) 5. The nucleic acid according to item 4, wherein the nucleic acid is selected from the group consisting of DNA, cDNA, PNA, and RNA. (Item 8) A host cell comprising the nucleic acid of item 4 or item 5. (Item 9) 2. A peptide composition consisting essentially of one or more peptides according to item 1. (Item 10) (a) an active agent selected from the group consisting of: (i) the peptide according to any one of items 1 to 3; (ii) a nucleic acid encoding the peptide; (iii) a CAR-T cell expressing the peptide; and (iv) an oncolytic virus expressing the peptide; and (b) a pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising: (Item 11) 11. The pharmaceutical composition of claim 10, wherein the active agent is a nucleic acid, the nucleic acid being selected from the group consisting of DNA, cDNA, PNA, and RNA. (Item 12) 11. The pharmaceutical composition of item 10, wherein the nucleic acid is RNA. (Item 13) 13. The pharmaceutical composition of claim 12, wherein the RNA comprises a modification selected from the group consisting of: (i) a ribose sugar modification, (ii) a phosphate linkage modification, and (iii) a base modification. (Item 14) 14. The pharmaceutical composition of claim 13, wherein the modification is selected from the group consisting of ribo-difluorotoluyl nucleotides, 4'-thio modified RNA, boranophosphate linkages, phosphorothioate linkages, 2'-O-methyl (2'-OMe) sugar substitutions, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE) sugar substitutions, locked nucleic acids (LNA), and L-RNA. (Item 15) 11. The pharmaceutical composition according to item 10, wherein the active agent is the peptide, and the peptide is provided with a peptide carrier system selected from the group consisting of microparticles, polymeric nanoparticles, liposomes, solid lipid nanoparticles, hydrophilic mucoadhesive polymers, thiolated polymers, polymer matrices, nanoemulsions, and hydrogels. (Item 16) 16. A method for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing response to a vaccine, the method comprising administering to an individual in need thereof an effective amount of the pharmaceutical composition according to any of items 10 to 15. (Item 17) 17. The method of claim 16, wherein the pharmaceutical composition is administered to inhibit progression of the hyperproliferative disorder. (Item 18) 18. The method of claim 17, wherein the hyperproliferative disorder is cancer. (Item 19) 20. The method of claim 18, wherein the cancer is melanoma. (Item 20) The second treatment may comprise: (i) Cancer vaccines; (ii) chimeric antigen receptor (CAR) T-cell therapy; (iii) a treatment comprising reducing or blocking the activity of a molecule selected from the group consisting of PD-1, PD-L1, lymphocyte activation gene-3 (LAG3), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), V-domain immunoglobulin inhibitor of T-cell activation (VISTA), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), killer immunoglobulin-like receptor (KIR), indoleamine (2,3)-dioxygenase (IDO), B- and T-lymphocyte attenuator (BTLA), and A2A adenosine receptor (A2AR); (iv) cytokines; (v) an agonist of a molecule selected from the group consisting of CD40, OX40, glucocorticoid-inducible tumor necrosis factor-related protein (GITR), and inducible T cell costimulator (ICOS); (vi) oncolytic viruses; and (vii) a therapeutic agent selected from the group consisting of a 4-1BB agonist, a 4-1BB antagonist, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of histone deacetylase (HDAC), and an inhibitor of VEGFR; 20. The method of claim 19, selected from the group consisting of: (Item 21) 17. The method of claim 16, wherein the pharmaceutical composition is administered to inhibit progression of a synucleinopathy. (Item 22) 22. The method of claim 21, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA). (Item 23) 17. The method of claim 16, wherein the pharmaceutical composition is administered to inhibit the progression of sepsis. (Item 24) 17. The method of claim 16, wherein the pharmaceutical composition is administered to inhibit the progression of an infectious disease. (Item 25) 17. The method of claim 16, wherein the pharmaceutical composition is administered to enhance a response to a vaccine. (Item 26) Use of the peptide according to any one of items 1 to 3 or the nucleic acid according to any one of items 4 to 7 in the manufacture of a medicament for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing the response to a vaccine. (Item 27) Use of the peptide according to any one of items 1 to 3 or the nucleic acid according to any one of items 4 to 7 for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing the response to a vaccine. (Item 28) 16. The composition according to any one of items 10 to 15, for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing response to a vaccine. EXAMPLES

[0086] Example 1. Peptide library screening TriCo-20 TM , TriCo-16 TM , Ph.D.-12 TM , and Ph.D. C7 TM Phage Display Peptide Libraries (Creative Biolabs, 45-1 Ramsey Road, Shirley, NY 11967) were screened by several rounds of biopanning to identify binders of soluble recombinant human LAG3.

[0087] After four rounds of screening, seven peptides showed clear enrichment of specific binders as defined by a >2-fold higher coated than uncoated signal in a clonal phage ELISA (Table 2). [Table 2]

[0088] Example 2. Peptide blockade of LAG3 signaling A cell-based reporter assay was used to assess whether binding of the seven peptides identified above was sufficient to block the interaction of LAG3 with its ligand MHC-II. The components of the assay included (1) a Jurkat T cell line stably expressing human LAG3 and a stable NFAT-luc2P luciferase reporter, (2) a Raji cell line expressing human MHC-II, and (3) a positive control anti-LAG3 antibody that blocks the interaction of LAG3 and MHC-II. Briefly, Jurkat cells expressing LAG3 are stimulated with a T cell receptor (TCR) activator molecule, resulting in the expression of luciferase. When Jurkat cells are co-cultured with a Raji cell line expressing the MHC-II branch, the interaction of LAG3 on the surface of Jurkat cells with MHC-II on the surface of Raji cells inhibits T cell activation and results in reduced luciferase expression. Addition of a neutralizing antibody against LAG3 blocks the inhibitory signal, allowing luciferase expression to proceed. TM Luciferase expression was measured using a ELISA kit (Promega). The seven LAG3 peptides were tested at concentrations of 0 μM, 0.64 μM, 1.6 μM, 4 μM, 10 μM, 25 μM and 100 μM.

[0089] The results of a positive control assay using an anti-LAG3 control antibody are shown in Figure 1 A. These results show that the control antibody restores luciferase expression in a dose-dependent manner, with a peak fold inhibition of approximately 5 at an antibody concentration of 25 μg / ml.

[0090] The results of an assay testing the seven LAG3 peptides identified above and a negative control peptide (SSYHHFKMPELHFGKNTFHQ; SEQ ID NO: 9) are shown in Figure IB. The data are expressed as fold increase in luciferase activity, where for each peptide, the relative light units (RLU) measured at 100 μM were compared to the RLU measured at 0.64 μM. The results show that two of the peptides, LAG3-11 and LAG3-56, restore luciferase expression at a concentration of 100 μM with approximately 2.8 and 2.2 fold inhibition, respectively.

[0091] Example 3. Peptide disruption of the LAG3-MHC-II interaction A homogeneous time-resolved fluorescence (HTRF) LAG3 / MHC-II binding assay (Cisbio US Inc.) was used to measure the interaction between MHC-II and LAG3 in the presence of peptide. In this assay, the interaction between Tag1-LAG3 and Tag2-MHC-II is detected by using anti-Tag1-Terbium (HTRF donor) and anti-Tag2-XL665 (HTRF acceptor). When the donor and acceptor antibodies are in close proximity due to LAG3 and MHC-II binding, excitation of the donor antibody induces fluorescence resonance energy transfer (FRET) to the acceptor antibody, which then emits specifically at 665 nm. This specific signal is directly proportional to the degree of LAG3 / MHC-II interaction. Thus, agents that block the interaction between LAG3 and MHC-II cause a reduction in the HTRF ratio.

[0092] Anti-human LAG3 antibody (Novoprotein #GMP-A092, Lot 0331158, 500nM) was tested in the above assay in an 11-point serially diluted semi-logarithmic dose-response curve starting at 100nM and served as a positive control. Ovalbumin peptide was used as a negative control. Peptides were reconstituted at a concentration of 20mM in 100μM DMSO and tested in an 11-point dose-response curve starting at 100μM, followed by 4-fold dilutions. Each dose was tested in triplicate.

[0093] 2A-D are graphs showing the results of four independent experiments testing peptide LAG3-11. These results show that peptide LAG3-11 disrupts the interaction of LAG3 and MHC-II.

[0094] Figures 3-9 are graphs showing the results of experiments testing peptides LAG3-11 (Figure 3), LAG3-42 (Figure 4), LAG3-48 (Figure 5), LAG3-51 (Figure 6), LAG3-54 (Figure 7), LAG3-56 (Figure 8), and LAG3-60 (Figure 9). The individual HTRF ratios used to construct these genes are shown in Tables 3A and 3B. [Table 3A] [Table 3B]

[0095] Example 5. Peptide enhancement of CD8+ T cell responses This example shows the effect of peptide LAG3-11 on IFN-γ secretion and proliferation in a human peripheral blood mononuclear cell (PBMC)-based recall assay. PBMCs were obtained from a human donor who had previously been identified as a positive responder to virus-specific peptides of Epstein-Barr virus (EBV) and cytomegalovirus (CMV). 5×10 5PBMCs were stimulated with virus-specific peptides of EBV and CMV in the presence or absence of LAG3-11. Seven days after stimulation, the percentage of virus-specific CD8+ T cells was identified via flow cytometry using MHC-I treatment. Supernatants were also collected and the amount of IFN-γ was determined via ELISA.

[0096] The results (shown in FIG. 11) show that LAG3-11 increases IFN-γ secretion by human PBMCs by 3-fold compared to the virus-specific peptide alone. + The approximately two-fold increase in the percentage of CD8 T cells indicates that the LAG3-11 peptide can enhance T cell proliferation.

[0097] Example 6. Interaction of peptides with FGL1 This example shows the ability of LAG-11 and LAG-56 to inhibit the interaction between human LAG3 and FGL1. The peptides were tested using the Human LAG3 / FGL1 TR-FRET Binding Assay (BPS Bioscience) performed according to the manufacturer's instructions.

[0098] Peptide stocks of LAG3-11, LAG3-42, LAG3-48, LAG3-51, LAG3-54, LAG3-56, LAG3-60 and OVA were prepared at 1 mM and subsequently diluted 1:5 in plate reactions. LAG3-11 and OVA were tested at 200 μM, 50 μM, 13 μM, 3 μM, 0.8 μM, 0.2 μM, 0.05 μM, and 0.01 μM. Other peptides were tested at 200 μM, 50 μM, 13 μM, and 3 μM. A neutralizing anti-human LAG3 antibody (BPS Bioscience Cat. #71219) was used as a positive control.

[0099] The reaction mixture was incubated for 1 hour at room temperature before development. After development, the plates were read on a Tecan M1000 TR-FRET instrument. Percent activity was calculated as follows: %Activity = 100 x [(Sample - Min) / (Max - Min)]

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Claims

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 6.

2. The peptide of claim 1 , consisting of the amino acid sequence.

3. A nucleic acid encoding the peptide of claim 1.

4. The nucleic acid of claim 3 which is an expression construct.

5. The nucleic acid of claim 4, which is present in a CAR-T cell or an oncolytic virus.

6. The nucleic acid of claim 3 , wherein the nucleic acid is selected from the group consisting of DNA, cDNA, PNA, and RNA.

7. A host cell comprising the nucleic acid of claim 3 or claim 4.

8. A peptide composition comprising one or more peptides according to claim 1.

9. (a) an active agent selected from the group consisting of: (i) a peptide according to any one of claims 1 to 2; (ii) a nucleic acid encoding the peptide; (iii) a CAR-T cell expressing the peptide; and (iv) an oncolytic virus expressing the peptide; and (b) a pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

10. The active agent is a nucleic acid, which can be derived from DNA, cDNA, PNA, and RNA.

10. The pharmaceutical composition of claim 9, selected from the group consisting of:

11. The pharmaceutical composition of claim 9 , wherein the nucleic acid is RNA.

12. 12. The pharmaceutical composition of claim 11, wherein the RNA comprises a modification selected from the group consisting of: (i) a ribose sugar modification, (ii) a phosphate linkage modification, and (iii) a base modification.

13. 13. The pharmaceutical composition of claim 12, wherein the modification is selected from the group consisting of ribo-difluorotoluyl nucleotides, 4'-thio modified RNA, boranophosphate linkages, phosphorothioate linkages, 2'-O-methyl (2'-OMe) sugar substitutions, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE) sugar substitutions, locked nucleic acids (LNA), and L-RNA.

14. 10. The pharmaceutical composition of claim 9, wherein the active agent is the peptide, and the peptide is provided with a peptide carrier system selected from the group consisting of microparticles, polymeric nanoparticles, liposomes, solid lipid nanoparticles, hydrophilic mucoadhesive polymers, thiolated polymers, polymer matrices, nanoemulsions, and hydrogels.

15. 15. The pharmaceutical composition of any one of claims 9 to 14 for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing the response to a vaccine.

16. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to inhibit progression of the hyperproliferative disorder.

17. 17. The pharmaceutical composition of claim 16, wherein the hyperproliferative disorder is cancer.

18. 18. The pharmaceutical composition of claim 17, wherein the cancer is melanoma.

19. The pharmaceutical composition, wherein the pharmaceutical composition is administered in combination with a second treatment, The second treatment comprises: (i) a cancer vaccine; (ii) chimeric antigen receptor (CAR) T cell therapy; (iii) a treatment comprising reducing or blocking the activity of a molecule selected from the group consisting of PD-1, PD-L1, lymphocyte activation gene-3 (LAG3), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), V-domain immunoglobulin inhibitor of T-cell activation (VISTA), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), killer immunoglobulin-like receptor (KIR), indoleamine (2,3)-dioxygenase (IDO), B- and T-lymphocyte attenuator (BTLA), and A2A adenosine receptor (A2AR); (iv) cytokines; (v) an agonist of a molecule selected from the group consisting of CD40, OX40, glucocorticoid-inducible tumor necrosis factor-related protein (GITR), and inducible T cell costimulator (ICOS); (vi) an oncolytic virus; and (vii) a therapeutic agent selected from the group consisting of 4-1BB agonists, 4-1BB antagonists, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of histone deacetylase (HDAC), and inhibitors of VEGFR; 20. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is selected from the group consisting of:

20. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to inhibit the progression of a synucleinopathy.

21. 21. The pharmaceutical composition of claim 20, wherein the synucleinopathy is selected from the group consisting of Parkinson's disease (PD), dementia with Lewy bodies (DLB), pure autonomic failure (PAF), and multiple system atrophy (MSA).

22. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to inhibit the progression of sepsis.

23. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to inhibit the progression of an infectious disease.

24. 16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is administered to enhance a response to a vaccine.

25. Use of a peptide according to any one of claims 1 to 2 or a nucleic acid according to any one of claims 3 to 6 in the manufacture of a medicament for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease or enhancing the response to a vaccine.

26. A composition for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing the response to a vaccine, the composition comprising a peptide according to any one of claims 1 to 2 or a nucleic acid according to any one of claims 3 to 6.

27. 15. The composition of any one of claims 9 to 14 for inhibiting the progression of a hyperproliferative disorder, inhibiting the progression of a synucleinopathy, inhibiting the progression of sepsis, inhibiting the progression of an infectious disease, or enhancing the response to a vaccine.

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