Viral peptides and uses thereof

Abstract

The present disclosure provides isolated peptides derived from human T-lymphotropic virus type 1 (HTLV-1), peptide-based molecules (e.g., peptide-MHC (pMHC) complexes), polynucleotides and vectors encoding the peptides or peptide-based molecules, pharmaceutical compositions (e.g., vaccine compositions), and their use for treating, preventing, and / or reducing the likelihood of HTLV-1 infection and / or HTLV-1-induced disease. The present disclosure also provides binding moieties that bind to the peptides or peptide-based molecules disclosed herein, and their use for treating, preventing, and / or reducing the likelihood of HTLV-1 infection and / or HTLV-1-induced disease. The present disclosure also provides methods and systems for identifying immunogenic virus-derived peptides.

Description

[0001] Cross-references to related applications

[0002] This patent application claims the benefit of U.S. Provisional Application No. 63 / 583,834, filed September 19, 2023, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0003] sequence list

[0004] This application includes a sequence list submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy was created on August 29, 2024, named 250298_000682_SL.xml, and is 126,068 bytes in size. Technical Field

[0005] This disclosure relates to methods and compositions involving isolated peptides derived from human T-lymphotropic virus type 1 (HTLV-1), and the use of such methods and compositions for treating, preventing HTLV-1 infection and / or HTLV-1-induced diseases or reducing their likelihood.

[0006] background

[0007] Human T-lymphotropic virus type-1 (HTLV-1) is a complex retrovirus belonging to the genus δ retrovirus. Deltaretrovirus HTLV-1, an estimated 5-10 million people worldwide, is the most pathogenic of the four known types of HTLV (HTLV-1, HTLV-2, HTLV-3, and HTLV-4). It is the primary cause of malignant lymphocytic proliferation known as adult T-cell leukemia / lymphoma (ATL) and a range of neuropathologies known as HTLV-1 myelopathy / tropical spastic paresis (HAM / TSP). HTLV-1 is prevalent in southwestern Japan, sub-Saharan Africa, South America, and the Caribbean, with localized foci in the Middle East and Australasia. HTLV-1 is primarily found in CD4+ cells. +HTLV-1 is a latent virus and can be transmitted through, for example, sexual contact, contaminated blood products, and mother-to-child transmission (parental / vertical). Furthermore, HTLV-1 is a latent virus and therefore cannot be cleared from the host's immune system; 4-5% of HTLV-1 carriers develop ATL during their lifetime, and another 0.25-4% develop HAM / TSP. Despite scientific advances in understanding the pathogenesis of HTLV-1 infection, the prognosis of ATL and the quality of life for those with HAM / TSP remain poor. There is currently no vaccine available to prevent HTLV-1 infection or HTLV-1-related disease. Therefore, identifying HTLV-1-related epitopes that may be suitable for targeting, such as therapeutic vaccines, immunotherapies, or other therapies targeting these HTLV-1-related antigens, will help combat HTLV-1-mediated infections and disease.

[0008] Overview

[0009] As described in the background section above, there is an urgent need in the art to develop methods for identifying HTLV-1 epitopes that may be useful to patients infected with HTLV-1. This application addresses these and other needs.

[0010] On the one hand, this article provides isolated peptides comprising an amino acid sequence having at least 90% identity with an amino acid sequence of any one of SEQ ID NO:1-89 and 143-146, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide is 5-20 amino acids in length.

[0011] In some embodiments, the isolated peptide comprises the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

[0012] In some embodiments, the isolated peptide consists essentially of the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

[0013] In some embodiments, the isolated peptide consists of the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

[0014] On the other hand, this article provides isolated peptides comprising two or more amino acid sequences selected from any one of SEQ ID NO:1-89 and 143-146, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof.

[0015] In some embodiments, the isolated peptide comprises one or more reverse peptide bonds, one or more non-peptide bonds, one or more D-isomers of amino acids, one or more chemical modifications, or any combination thereof.

[0016] In some implementations, the isolated peptide is generated by expression in a heterologous host cell.

[0017] In some implementations, the isolated peptide is synthesized.

[0018] In some embodiments, the isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof, when presented on the surface of antigen-presenting cells (APCs) as a complex with major histocompatibility complex (MHC) molecules, induces a human T-lymphotropic virus type 1 (HTLV-1)-specific immune response in subjects.

[0019] On the other hand, this article provides fusion proteins comprising one or more isolated peptides described herein fused with one or more heterologous molecules.

[0020] In some implementations, the one or more heterologous molecules enhance peptide-specific immune responses in the subject.

[0021] In some implementations, the one or more heterologous molecules mediate the delivery of the peptide to a specific site within the subject's body.

[0022] In some embodiments, the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

[0023] On the other hand, this article provides conjugates comprising one or more isolated peptides described herein conjugated to one or more heterologous molecules.

[0024] In some implementations, the one or more heterologous molecules enhance peptide-specific immune responses in the subject.

[0025] In some implementations, the one or more heterologous molecules mediate the delivery of the peptide to a specific site within the subject's body.

[0026] In some embodiments, the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

[0027] In some implementations, the one or more peptides are conjugated to particles.

[0028] On the other hand, this article provides oligomeric complexes comprising two or more peptides isolated herein.

[0029] On the other hand, this article provides non-covalent complexes comprising the isolated peptides and MHC molecules or fragments or derivatives thereof described herein.

[0030] In some embodiments, the MHC molecule or fragment thereof is a class I MHC molecule.

[0031] In some implementations, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0032] In some embodiments, the MHC molecule or fragment thereof is a class II MHC molecule.

[0033] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0034] On the other hand, this article provides fusion proteins comprising the isolated peptides and MHC molecules or fragments or derivatives thereof described herein.

[0035] In some embodiments, the MHC molecule or fragment thereof is a class I MHC molecule.

[0036] In some implementations, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0037] In some embodiments, the MHC molecule or fragment thereof is a class II MHC molecule.

[0038] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0039] On the other hand, this article provides conjugates comprising the isolated peptides and MHC molecules or fragments or derivatives thereof described herein.

[0040] In some embodiments, the MHC molecule or fragment thereof is a class I MHC molecule.

[0041] In some implementations, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

[0042] In some embodiments, the MHC molecule or fragment thereof is a class II MHC molecule.

[0043] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0044] On the other hand, this document provides pharmaceutical compositions comprising (i) one or more isolated peptides, one or more fusion proteins, one or more conjugates, one or more oligomeric complexes, or one or more non-covalent complexes, or any combination thereof, as described herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0045] In some embodiments, the pharmaceutical composition may also contain an adjuvant.

[0046] On the other hand, this document provides isolated molecules that combine with the isolated peptides, fusion proteins, conjugates, oligomeric complexes, or non-covalent complexes described herein.

[0047] In some embodiments, the molecule is an antibody or an antigen-binding fragment thereof.

[0048] In some implementations, the antibody is a bispecific antibody.

[0049] In some implementations, the molecule is an alternative scaffold.

[0050] In some implementations, the molecule is a chimeric antigen receptor (CAR).

[0051] In some implementations, the molecule is a T-cell receptor (TCR).

[0052] On the other hand, this article provides isolated cells containing the CAR described herein.

[0053] In some implementations, the isolated cells are immune cells.

[0054] In some implementations, the immune cells are T cells, NK cells, or macrophages.

[0055] On the other hand, this article provides isolated cells containing the TCR described herein.

[0056] In some implementations, the isolated cells are immune cells.

[0057] In some implementations, the immune cells are T cells, NK cells, or macrophages.

[0058] On the other hand, this article provides pharmaceutical compositions comprising (i) the isolated molecules or isolated cells described herein; and (ii) pharmaceutically acceptable carriers or excipients.

[0059] On the other hand, this document provides isolated polynucleotides containing nucleotide sequences encoding one or more isolated peptides or fusion proteins described herein.

[0060] In some implementations, the nucleotide sequence may be operatively linked to a promoter.

[0061] In some implementations, the isolated polynucleotide comprises DNA.

[0062] In some implementations, the isolated polynucleotide comprises RNA.

[0063] In some implementations, the RNA is mRNA.

[0064] In some implementations, the RNA is a self-replicating RNA.

[0065] On the other hand, this article provides vectors containing the isolated polynucleotides described herein.

[0066] In some implementations, the carrier is an expression carrier.

[0067] In some implementations, the vector is a viral vector.

[0068] On the other hand, this article provides host cells containing the isolated polynucleotides or vectors described herein.

[0069] In some implementations, the host cell is a prokaryotic cell.

[0070] In some implementations, the host cell is a eukaryotic cell.

[0071] In some implementations, the host cell is an APC.

[0072] On the other hand, this article provides pharmaceutical compositions comprising (i) the isolated polynucleotides described herein or the carriers described herein; and (ii) pharmaceutically acceptable carriers or excipients.

[0073] In some implementations, the pharmaceutically acceptable carrier is a lipid nanoparticle carrier.

[0074] On the other hand, this article provides a method for inducing an immune response against HTLV-1 infection in subjects in need, which includes administering a therapeutically effective dose to the subject:

[0075] a) One or more peptides isolated herein;

[0076] b) The fusion protein described herein;

[0077] c) The conjugates described herein;

[0078] d) The oligomeric complexes described herein;

[0079] e) The non-covalent complexes described herein;

[0080] f) The pharmaceutical compositions described herein;

[0081] g) The molecules described herein;

[0082] h) The isolated cells described herein;

[0083] i) The isolated polynucleotides described herein; or

[0084] j) The carrier described in this article.

[0085] On the other hand, this article provides a method for inducing an immune response against HTLV-1 infection in subjects in need, the method comprising administering to the subject a therapeutically effective amount of one or more isolated peptides described herein.

[0086] In another aspect, this article provides a method for inducing an immune response against HTLV-1 infection in subjects in need, comprising administering activated T cells to the subject, said activated T cells being generated by contacting the T cells with APCs, said APCs presenting the isolated peptide described herein in complex with MHC molecules.

[0087] On the other hand, this article provides a method for treating HTLV-1-induced diseases or conditions in subjects in need, which includes administering an effective amount of:

[0088] a) One or more peptides isolated herein;

[0089] b) The fusion protein described herein;

[0090] c) The conjugates described herein;

[0091] d) The oligomeric complexes described herein;

[0092] e) The non-covalent complexes described herein;

[0093] f) The pharmaceutical compositions described herein;

[0094] g) The molecules described herein;

[0095] h) The isolated cells described herein;

[0096] i) The isolated polynucleotides described herein; or

[0097] j) The carrier described in this article.

[0098] On another front, this article provides a method for preventing or reducing the likelihood of HTLV-1-induced diseases or conditions in subjects in need, which involves administering an effective dose to the subject:

[0099] a) One or more peptides isolated herein;

[0100] b) The fusion protein described herein;

[0101] c) The conjugates described herein;

[0102] d) The oligomeric complexes described herein;

[0103] e) The non-covalent complexes described herein;

[0104] f) The pharmaceutical compositions described herein;

[0105] g) The molecules described herein;

[0106] h) The isolated cells described herein;

[0107] i) The isolated polynucleotides described herein; or

[0108] j) The carrier described in this article.

[0109] On the other hand, this article provides a method for treating HTLV-1 induced diseases or conditions in subjects in need, the method comprising administering to the subject an effective amount of one or more isolated peptides described herein.

[0110] On the other hand, this article provides a method for preventing or reducing the likelihood of HTLV-1-induced diseases or conditions in subjects in need, the method comprising administering to the subject an effective amount of one or more isolated peptides described herein.

[0111] In some implementations, the HTLV-1 induced disease or condition is adult T-cell leukemia / lymphoma (ATL) or HTLV-1 myelopathy / tropical spastic paralysis (HAM / TSP).

[0112] On the other hand, this article provides a kit that includes:

[0113] (i) a) One or more isolated peptides described herein;

[0114] b) The fusion protein described herein;

[0115] c) The conjugates described herein;

[0116] d) The oligomeric complexes described herein;

[0117] e) The non-covalent complexes described herein;

[0118] f) The pharmaceutical compositions described herein;

[0119] g) The molecules described herein;

[0120] h) The isolated cells described herein;

[0121] i) The isolated polynucleotides described herein; or

[0122] j) The carrier described herein; and

[0123] (ii) Packaging and / or instructions for use thereof.

[0124] These and other aspects of this disclosure will be apparent to those skilled in the art from the following description, claims, and accompanying drawings. Brief description of the attached diagram

[0126] Figures 1A-1E This study demonstrates the affinity purification mass spectrometry of HTLV-transformed T lymphoblast cell lines. Figure 1A An example of the anti-HLA-I W6 / 32 affinity purification process and subsequent peptide identification by mass spectrometry is shown. Figures 1B-1E The results show the effects of mass spectrometry analysis on the cell line C8166 ( Figure 1B ), MT-2 ( Figure 1C ), MT-4 ( Figure 1D ) and C5 / MJ ( Figure 1E Description of the detected peptide length and count.

[0127] Figure 2 Eleven (11) HTLV peptides were detected in the C8166 cell line with good reproducibility, of which six (6) were predicted by NetMHCpan 4.0 to be HLA-binding agents of known C8166 HLA alleles (see, for example, Table 3). SEQ ID NOs 85, 63, 74, 83, 78, 81, 51, 24, 29, 70, and 31 are disclosed in column order in the figure.

[0128] Figure 3 Thirty-two (32) HTLV peptides were detected in the MT-2 cell line, of which twenty-one (21) were found in all three replicates, and most of them were predicted by NetMHCpan 4.0 to be conjugates of known MT-2 alleles (see, for example, Table 4). SEQ ID NOs 55, 46, 86, 80, 21 and 5 are disclosed in order of appearance, and SEQ ID NOs 84, 89, 88, 87, 72, 11, 9, 52, 67, 20, 44, 51, 57, 4, 45, 17, 60, 62, 54, 35, 77, 49, 76, 71, 82 and 73 are disclosed in order of column.

[0129] Figure 4Nineteen (19) HTLV peptides were detected in the MT-4 cell line, with sixteen (16) peptides overlapping between repeats. NetMHCpan 4.0 predicted that some of these peptides are conjugates of common HLA alleles (e.g., HLA-A02, HLA-A11, and HLA-A24) in the patient population (see, for example, Table 5). SEQ ID NOs 2, 1, 65, 66, 70, 68, 18, 56, 41, 64, 59, 47, 27, 31, 26, 42, 75, 15, and 53 are disclosed in column order in the figure.

[0130] Figure 5 The HLA-I immunopeptidomics from C5 / MJ cells treated with and without interferon (IFN) yielded nine (9) overlapping HTLV peptides, all of which were predicted by NetMHCpan 4.0 to be conjugates of the C5 / MJ HLA alleles (see, for example, Table 6). SEQ ID NOs 79, 50, 20, 58, 64, 36, 69, 9, 38, 8, 14, 89, and 37 are disclosed in column order in the figure.

[0131] Figures 6A-6E The results showed that sixteen (16) and twenty-one (21) HTLV peptides were detected in IFN-treated and untreated ED-41214(-) primary cells by mass spectrometry, respectively, and good reproducibility was observed among biological replicates (see, for example, see below). Figure 6B and Figure 6D Fifteen (15) HTLV peptides were found between untreated and IFN-treated ED-41214(-) cells. Figure 6E Only a few HTLV peptides were predicted by NetMHCpan 4.0 to be conjugates of common alleles (see, for example, Table 7). Figure 6A and Figure 6C The figures show descriptions of peptide lengths and counts detected by mass spectrometry analysis of ED-41214(-) cells that were not treated with IFN and were treated with IFN. The figures, in order of appearance, disclose SEQ ID NOs: 32, 7, 3, 25, 30, 52, 33, 61, 39, 19, 6, 28, 40, 23, 10, 43, 48, 32, 3, 7, 30, 34, 13, 52, 12, 33, 61, 39, 6, 19, 28, 40, 23, 10, 43, 16, and 22.

[0132] Figures 7A-7D The peptide distributions obtained for all HTLV primary samples are shown (see, for example, for ED41214(-), without IFN1). Figure 6A;ED40515(-), no IFN 1 ( Figure 7A ); ED40515(+), no IFN 1 ( Figure 7B ); ATL43T(+), no IFN 1 ( Figure 7C ); and ATL43Tb(-) IFN 1 (Figure 7E); however, HTLV peptides were detected only for ED-41214(-) (see, for example, Figure 6A (Joint search for human UniProt + HTLV sequences).

[0133] Figures 8A-8D Displayed via Thermo Orbitrap ( Figures 8A-8B ) and Bruker timeTOFSCP ( Figures 8C-8D An example of peptide distribution in HLA-I peptidomome analysis of ATL patient samples using mass spectrometry.

[0134] Figures 9A-9B An example of a procedure for reconstructing a patient-specific HTLV genome from RNA sequencing is shown. The RNA sequencing and RNA contig reconstruction steps are as follows: Figure 9A As shown. HTLV genome reconstruction includes BLAST analysis of the viral reference genome, viral genome reconstruction, and coding sequence extraction steps, such as... Figure 9B As shown.

[0135] Figure 10 This demonstrates the RNA sequencing read coverage of the patient-specific reconstructed genome.

[0136] Detailed description

[0137] This disclosure particularly provides isolated peptides and fragments or derivatives thereof derived from human T-lymphotropic virus type 1 (HTLV-1). Various peptide-based molecules are also provided, including peptide-containing complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, and conjugates. Polynucleotides and carriers encoding the peptides or peptide-based molecules described herein are also provided. Binding moieties (e.g., antibodies, alternative scaffolds, T-cell receptors (TCRs), or chimeric antigen receptors (CARs)) that bind to the peptides or peptide-based molecules are also provided. The compositions of this disclosure can be used to induce an immune response against HTLV infection (e.g., HTLV-1, HTLV-2, HTLV-3, and / or HTLV-4 infection), and / or to treat, prevent, and / or reduce the likelihood of HTLV-induced diseases or conditions (e.g., HTLV-1-induced, HTLV-2-induced, HTLV-3-induced, and / or HTLV-4-induced diseases or conditions). In one aspect, this disclosure also provides methods for identifying immunogenic viral-derived peptides.

[0138] Lymphocytes, such as T cells, play a crucial role in adaptive anti-infection, anti-tumor, autoimmune, and transplant rejection responses. Typically, T cell-mediated immune responses involve close contact between T cells and antigen-presenting cells (APCs), such as immune synapses. The pairing of several molecules is involved in the formation of immune synapses, including but not limited to: (a) the T-cell receptor (TCR) on T cells, which specifically binds to peptides in the peptide-binding groove of the major histocompatibility complex (MHC) molecule present on APCs; and (b) CD28 (on T cells), which pairs with the B7 molecule on APCs. The TCR, together with the CD3 molecule, forms the TCR complex, and when the TCR pairs with the peptide-MHC (pMHC) complex, signaling is emitted via CD3. Signaling via both the TCR complex and CD28 on T cells leads to T cell activation.

[0139] T cell receptors are composed of two types of chains ( (alpha) chain and (beta) chain, or (gamma) chain and The heterodimer structure is composed of (delta)). The chain is located in The nucleic acid sequence within a locus (located on human or mouse chromosome 14) encodes the entire δ-strand. loci, and The chain is located in The nucleic acid sequence within the locus (located on mouse chromosome 6 or human chromosome 7) encodes [the genome]. Most T cells possess [this genome]. TCR, while a small number of T cells have TCR. T cell receptor. and polypeptides (and similar) and The peptides are interconnected by disulfide bonds. Each of the two peptides that make up the TCR contains an extracellular domain, a transmembrane domain, and a cytoplasmic tail (which is also part of the constant region) that includes a constant region and a variable region.

[0140] Each TCR's variable region contains unique characteristic structures, namely unique sites or idiotypes that determine TCR specificity. Generally, a TCR will bind to a pMHC complex only if it contains an idiotype that recognizes peptides presented in the MHC context, such as a unique conformation of a specific pMHC complex.

[0141] Immunotherapy approaches for treating diseases focus on modulating T-cell activity in the body, for example, enhancing anti-infection and anti-tumor responses, or, for example, downregulating autoimmunity and transplant rejection. However, such approaches can lack specificity because immunotherapy can target signaling via the TCR complex by binding to CD3 and / or co-stimulatory molecule pairs. Such approaches can lead to undesirable side effects, such as overactive immune responses or systemic immunosuppression. Therefore, therapies utilizing the unique and specific interactions between the TCR and the pMHC complex can provide the ability to specifically modulate the activity of specific T cells in the body, offering T-cell-based therapy.

[0142] MHC molecules present virus-derived peptides on the surface of infected cells. CD8+ cytotoxic T cells recognize these pMHC complexes and are subsequently activated, providing an important mechanism for immune-based antiviral protection. Cells infected with HTLV-1, including those that develop into cancer cells, express various HTLV-1-associated antigens. Peptides derived from these antigens can be displayed on the cell surface in the form of complexes with MHC molecules. Detection of MHC-presented HTLV-1-derived peptides by T cells carrying the corresponding TCRs leads to targeted killing of infected cells. However, due to the selection process that occurs during T cell maturation in the thymus, the circulating pool typically lacks T cells that recognize HTLV-1-derived peptides with sufficiently high affinity. As a result, infected cells often evade clearance by the immune system.

[0143] Identification of HTLV-1-derived peptides present on infected cells (e.g., HTLV-1-induced cancer cells) can allow for the development of immunotherapeutic agents engineered to specifically target and destroy HTLV-1-infected cells (e.g., HTLV-1-induced cancer cells). Such agents can be portions and reagents that bind to HTLV-1-derived peptides and / or pMHC complexes, for example, portions that act by inducing a T-cell response. For example, such agents can be based on antibodies, TCRs, and / or CARs.

[0144] This disclosure is partly based on a proteogenomic approach that detects MHC-associated HTLV-1 peptides in HTLV-1-infected cells. The HLA-I-HTLV-1 peptide library characterized in this paper, expressed in HTLV-1-transformed cells and primary ATL cells, provides an accurate representation of HTLV-1 epitopes in the human population.

[0145] HLA-restricted viral peptides, as potential targets, can be used to deliver immunotherapeutic agents (such as antibodies, e.g., bispecific antibodies, and cell therapies based on engineered TCRs or CARs) to infected tissues. However, the combination of genomic variations between viral strains and differences in patient HLA alleles can pose challenges in developing therapeutics targeting these peptides. To address this challenge, in one aspect, this disclosure provides a proteogenomic approach for generating patient-specific databases that allows for the comprehensive identification of viral peptides, such as HTLV-1-derived peptides, based on viral transcriptome sequencing from individual patient samples. The HTLV-1-HLA-related peptides disclosed herein can be used to develop immunotherapeutic agents (such as antibodies, e.g., bispecific antibodies, and cell therapies based on engineered TCRs or CARs) to treat HTLV-1-related diseases or conditions, including T-cell leukemia / lymphoma (ATL), HTLV-1-spinal cord disease / tropical spastic paralysis (HAM / TSP), and hypersensitivity reactions (e.g., arthritis, uveitis, and HTLV-1-associated infectious dermatitis (IDH)). The protein genome discovery platform described in this article provides a method for identifying viral-derived peptides as targets for antiviral-related immunotherapies.

[0146] definition

[0147] 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 disclosure pertains.

[0148] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “a method” includes one or more methods, and / or steps of the type described herein, and / or steps that will become obvious to those skilled in the art upon reading this disclosure.

[0149] The term "about" or "approximately" includes a value that is within a statistically significant range. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and still more preferably within 5%. The permissible variations covered by the term "about" or "approximately" depend on the specific system being studied and are readily understood by those skilled in the art.

[0150] The term "antigen" includes any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, part thereof, or combination thereof) that, when introduced into an immune-capable host (directly or, as in a DNA or RNA vaccine), is recognized by the host's immune system and can elicit an immune response. As part of an immune synapse, the T-cell receptor (TCR) recognizes peptides presented in the context of the major histocompatibility complex (MHC). The peptide-MHC (pMHC) complex is recognized by the TCR, and the peptide (antigenic determinant) and the TCR idiotype provide specificity for the interaction. Therefore, the term "antigen" includes peptides presented in the context of the MHC, such as the pMHC complex. Peptides displayed on the MHC may also be referred to as "epitopes" or "antigenic determinants." The terms "peptide," "antigenic determinant," "epitope," etc., include not only peptides naturally presented by antigen-presenting cells (APCs) but can also be any desired peptide, provided it is recognized by immune cells, for example, when appropriately presented to cells of the immune system. For example, peptides with artificially prepared amino acid sequences can also be used as epitopes.

[0151] A single antigen (such as an antigenic polypeptide) may have more than one epitope. An epitope can be defined as a structural epitope or a functional epitope. A functional epitope is typically a subset of a structural epitope and is defined as those residues that directly contribute to the affinity between the MHC molecule and the antigen. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In some embodiments, epitopes may comprise determinant clusters that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in some embodiments, may have specific three-dimensional structural features and / or specific charge features. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from tertiary folds are generally lost upon treatment with denaturing solvents.

[0152] The terms “major histocompatibility complex” and “MHC” encompass the terms “human leukocyte antigen” or “HLA” (the latter two are generally used specifically for human MHC molecules), naturally occurring MHC molecules (e.g., MHC class I molecules containing MHC class I α (heavy) chains and β2 microglobulins; MHC class II molecules containing MHC class II α chains and MHC class II β chains), individual chains of MHC molecules (e.g., MHC class I α (heavy) chains, MHC class II α chains, and MHC class II β chains), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of MHC class I α chains, α1-α2 subunits of MHC class II α chains, and β1-β2 subunits of MHC class II β chains) and their portions (e.g., peptide-binding portions, such as peptide-binding grooves), mutants, and various derivatives (including fusion proteins), wherein such portions, mutants, and derivatives retain the ability to display antigenic peptides for recognition by TCRs (e.g., antigen-specific TCRs). MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy chain, which can accommodate peptides of about 8-10 amino acids. While both classes of MHC bind a core of about 9 amino acids (e.g., 5 to 17 amino acids) within the peptide, the open nature of the MHC class II peptide-binding groove (where the α1 domain of an MHC class II α peptide binds to the β1 domain of an MHC class II β peptide) allows for a wider range of peptide lengths. The length of MHC class II bound peptides typically varies between 13 and 17 amino acids, although shorter or longer lengths are not uncommon. Therefore, peptides can move within the MHC class II peptide-binding groove, thereby altering the 9-mer located directly within the groove at any given time. In some embodiments, the peptide-MHC complexes described herein may be peptide-MHC complexes derived from non-human animals. In other embodiments, the peptide-MHC complexes described herein may include peptide-HLA complexes, i.e., peptide-MHC complexes derived from humans. This document uses routine identification of specific MHC variants. For example, HLA-A11 refers to the human leukocyte antigen from the 11th gene position (called the locus) in the A gene group (therefore, a class I MHC); the gene HLA-DR11 refers to the human leukocyte antigen encoded by the gene from the 11th gene locus in the DR region (therefore, a class II MHC).

[0153] The terms “MHC-peptide complex,” “peptide-MHC complex,” “pMHC complex,” and “peptide in groove” include (i) MHC molecules, such as human and / or non-human animal MHC molecules, or portions thereof (e.g., their peptide-binding grooves and, for example, their extracellular portions), and (ii) antigenic peptides (e.g., HTLV-1-derived peptides), wherein the MHC molecule and the antigenic peptide are complexed in a manner in which the pMHC complex can specifically bind to T cell receptors. pMHC complexes include cell surface-expressed pMHC complexes and soluble pMHC complexes.

[0154] "HLA-peptide complex", "peptide-HLA complex", "pHLA complex", etc. refer to MHC-peptide complex, where MHC molecules are human leukocyte antigen (HLA) molecules.

[0155] The term "T cell" or "T lymphocyte" is used in its broadest sense herein to refer to all types of immune cells that express CD3, including but not limited to T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, regulatory T cells (Tregs), and NK-T cells. T cells can include thymocytes, naive T cells, memory T cells, immature T cells, mature T cells, resting T cells, or activated T cells. T cells may also include "gamma-delta T cells (γδ T cells)," which refers to a specialized cell population, a small subset of T cells that have a unique TCR on their surface and differ from most T cells, where the TCR consists of two glycoprotein chains called α-TCR chains and β-TCR chains, and in γδ T cells, the TCR consists of γ-chains and δ-chains.

[0156] The term “antigen-presenting cell” or “APC” refers to any cell that presents an antigen on its cell surface that is associated with a major histocompatibility complex molecule (MHC class I molecule or MHC class II molecule, or both).

[0157] The terms "antibody," "antibodies," and "immunoglobulin" refer to immunoglobulin molecules and their immunologically active portions, i.e., molecules containing antigen-binding sites that specifically bind to antigens, whether natural or partially or wholly synthetically produced. These terms include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fv (sdFv), intracellular antibodies, microantibodies, diabodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), as well as any of the epitope-binding fragments described above. The terms “antibody” and “antibodies” also refer to covalent biantibodies, such as those disclosed in U.S. Patent Application Publication 2007 / 0004909 (which is incorporated herein by reference in its entirety), and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910 (which is incorporated herein by reference in its entirety). Antibodies useful in this disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0158] The terms "specific binding," "binding in a specific manner," and "antigen specificity" indicate that molecules involved in specific binding can form relatively stable complexes with each other under physiological conditions and cannot nonspecifically form stable complexes with other molecules besides the designated binding pair. Therefore, a peptide-binding moiety (e.g., antibody, alternative scaffold, CAR, or TCR) that binds in a specific manner to an HTLV-1-derived peptide or peptide-based molecule (such as a complex containing said peptide (e.g., a pMHC complex), a fusion protein, or a conjugate) indicates that the peptide-binding moiety forms a stable intermolecular non-covalent bond with the HTLV-1-derived peptide or peptide-based molecule (such as a complex containing said peptide (e.g., a pMHC complex), a fusion protein, or a conjugate). Specific binding can be characterized by an equilibrium dissociation constant (Ki) in the low micromolar to picomolar range. D ) to characterize (i.e., smaller K) D(Indicating a tighter binding). High specificity can be found in the low nanomolar range, with very high specificity in the picomolar range. For example, the peptide-binding moiety may exhibit binding to HTLV-1-derived peptides or peptide-based molecules (such as complexes containing said peptides (e.g., pMHC complexes), fusion proteins, or conjugates), where K D The molecular weights are approximately 3000 nM or less, approximately 2000 nM or less, approximately 1000 nM or less, approximately 500 nM or less, approximately 300 nM or less, approximately 200 nM or less, approximately 100 nM or less, approximately 50 nM or less, approximately 1 nM or less, or approximately 0.5 nM or less. Methods for determining whether two molecules specifically bind to each other are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0159] The terms “protein” and “peptide”, used interchangeably herein, encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, as well as all other types of modified proteins (e.g., proteins produced by phosphorylation, acetylation, myristylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of fewer than 100 amino acids, preferably fewer than 50 amino acids, may be referred to as “peptides”.

[0160] The terms “polynucleotide” and “nucleic acid”, used interchangeably in this document, include polymers of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or their analogues or modified forms. These include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0161] The term "operably linked" or similar expression refers to a parallel relationship in which the components are positioned to allow them to function in their intended manner. For example, a "operably linked" control sequence to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include expression control sequences adjacent to the target gene and trans- or distantly acting expression control sequences to control the target gene (or target sequence). The term "expression control sequence" includes polynucleotide sequences that are essential to influencing the expression and processing of the coding sequence to which they are linked. "Expression control sequences" include: appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak concordant sequences); sequences that enhance polypeptide stability; and sequences that enhance polypeptide secretion when needed. The nature of such control sequences varies from host organism to host organism. For example, in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, they typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, but may also include additional components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.

[0162] The term "isolated" refers to a homogeneous population of molecules (such as polynucleotides or polypeptides) that has been substantially separated from and / or purified from other components in the system in which the molecule was produced (e.g., recombinant cells), and to proteins that have undergone at least one purification or isolation step. "Isolated" also refers to molecules that are substantially free of other cellular material and / or chemicals, including molecules isolated to higher purities (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity).

[0163] As used herein, the term "derivative" refers to a peptide, polypeptide, or polynucleotide, or a variant or analog thereof, that contains one or more mutations and / or chemical modifications compared to a reference peptide, polypeptide, or polynucleotide. Mutations and / or chemical modifications are further detailed below and may include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more positions in the amino acid or nucleotide sequence.

[0164] The term "treat" or "treatment" for a state, condition, disease, or ailment includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of developing at least one clinical or subclinical symptom of a state, condition, disease, or ailment in a subject who may have or be susceptible to the state, condition, disease, or ailment but has not yet experienced or exhibited clinical or subclinical symptoms of the state, condition, disease, or ailment; or (2) suppressing the state, condition, disease, or ailment, i.e., preventing, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom of the disease; or (3) alleviating the state, condition, disease, or ailment, i.e., causing the resolution of the state, condition, disease, or ailment or at least one clinical or subclinical symptom of the state, condition, disease, or ailment. The benefit to the subject treated is statistically significant, or at least perceptible to the patient or physician.

[0165] "Individual," "subject," or "animal" refers to a human, a veterinary animal (e.g., a cat, dog, cow, horse, sheep, pig, etc.), and an experimental animal model of a disease (e.g., a mouse, rat). In a preferred embodiment, the subject is a human.

[0166] When used in dosage or quantity terms, "effective" means an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered alone. The exact amount required varies from subject to subject to factors such as species, age and general condition, severity of the condition being treated, one or more specific medications used, method of administration, etc.

[0167] The phrase “pharmaceutical acceptable” used in conjunction with the compositions described herein means that the molecular entity and other components of such compositions are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). Preferably, the term “pharmaceutical acceptable” means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other recognized pharmacopoeia, for use in mammals, and more specifically in humans.

[0168] The term "administration," etc., refers to and includes administering the composition to a subject or system (e.g., to cells, organs, tissues, organisms, or related components or groups thereof). Those skilled in the art will understand that the route of administration may vary depending on, for example, the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, etc. For example, in some embodiments, administration to animal subjects (e.g., to humans or rodents) may be bronchial administration (including bronchial infusion), oral administration, enteral administration, intradermal administration, intradermal administration, gastric administration, intramedullary administration, intramuscular administration, intranasal administration, intraperitoneal administration, intrathecal administration, intravenous administration, intravenous administration, intraventricular administration, mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous administration, sublingual administration, local administration, tracheal administration (including tracheal infusion), transdermal administration, vaginal administration, and / or vitreous administration. In some embodiments, administration may include intermittent dosing. In some embodiments, administration may include continuous dosing (e.g., infusion) for at least a selected period of time.

[0169] Based on the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of this art can be employed. These techniques are well explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (in this paper, “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (edited by DNGlover, 1985); Oligonucleotide Synthesis (edited by MJGait, 1984); Nucleic Acid Hybridization [edited by BDHames & SJHiggins, 1985]; Transcription And Translation [edited by BDHames & SJHiggins, 1984]; Animal Cell Culture [edited by RIFreshney, 1986]; Immobilized Cells And Enzymes [IRL Press, 1986]; B. Perbal, A Practical Guide to Molecular Cloning (1984); Ausubel, FM et al., (edited). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site-directed mutagenesis as described in the following literature: Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Patent No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999); Kim and Maas, BioTech. 28:196-198 (2000); Parikh and Guengerich, BioTech. 24:428-431 (1998); Ray and Nickoloff, BioTech. 13:342-346 (1992); Wang et al., BioTech.19:556-559 (1995); Wang and Malcolm, BioTech. 26:680-682 (1999); Xu and Gong, BioTech. 26:639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419; Hogrefe, Strategies 14.3:74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270 and 6,242,222; Angag and Schutz, Biotech. 30:486-488 (2001); Wang and Wilkinson, Biotech. 29:976-978 (2000); Kang et al., Biotech. 20:44-46 (1996); Ogel and McPherson, ProteinEngineer. 5:467-468 (1992), Kirsch and Joly, Nucl. Acids. Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons and Meth. Molec. Biol. 67:209-218.

[0170] The peptides disclosed in this article

[0171] On the one hand, this disclosure provides isolated peptides containing an amino acid sequence derived from human T-lymphotropic virus type 1 (HTLV-1).

[0172] The HTLV genome follows the typical structure of a replicating retrovirus, containing GAG, POL, and ENV domains, flanked by two long terminal repeat (LTR) domains at either end of the provirus. Specifically, HTLV-1 has an 8.5 kb single-component, linear, dimer, single-stranded RNA (+) genome containing a 5' cap and a 3' poly-A tail. The two LTRs are approximately 600 nucleotide residues long and are located at the 5' and 3' ends of the genome. The LTRs contain U3, R, and U5 regions, which together serve as transcriptional units. The 5' end of the genome also contains a primer binding site, and the 3' end contains a polypurine segment. The integrated virus utilizes promoter elements in the 5' LTR to trigger and drive transcription, producing unsplit full-length mRNA, which can be used, for example, as genomic RNA for packaging into the virion. Although similar to other retroviruses, HTLV-1 differs from other retroviruses in its genomic RNA-coding structure and the enzyme proteins Gag (internal group-specific antigen), Pol (polymerases, reverse transcriptases, and integrase required for viral replication and maturation), and ENV (enveloped glycoprotein). This is because the pX region is located at the 3' end between the Env region and the 3' LTR. The pX region encodes several genes regulating alternative splicing, including but not limited to Tax and HTLV-I basic leucine zipper factor (HBZ), both of which are involved in viral pathogenesis.

[0173] The viral gene product Tax is a 40 kDa protein primarily located in the nucleus, but can also be found in the cytoplasm of infected cells. Tax interacts with a variety of host proteins and plays a key role in the transactivation of proviral transcription from the 5' LTR. Tax can functionally inactivate p53 and target pRB for degradation, thereby allowing infected cells to survive. Tax can also dysregulate several signaling pathways, such as, but not limited to, activator protein 1 (AP-1), nuclear factor κB (NF-κB), serum response factor (SRF), and cyclic AMP response element binding protein (CREB) pathways. Tax's pleiotropic function contributes to viral pathogenicity and the transformation of HTLV-1-infected cells.

[0174] HBZ is a nucleoprotein, but it can also be localized in the cytoplasm. HBZ contains three domains: an activation domain, a central domain, and a basic leucine zipper domain. HBZ antagonizes several Tax-mediated functions and is involved in viral persistence and immune evasion. The activation domain of HBZ contains two LXXLL-like motifs (where L is leucine and X is any amino acid), which can bind to the kinase-inducible domain interaction (KIX) domain of the p300-CBP transcriptional coactivator. The LXXLL-like motif and the KIX domain may be necessary for HBZ to activate TGF-β (transforming growth factor β) / Smad signaling.

[0175] After infecting the host, HTLV-1 infects cells via its glycoprotein gp62. HTLV-1 then achieves latency by integrating into the host genome and increasing proviral load through the proliferation of infected cells.

[0176] The HTLV-1 life cycle begins when the HTLV-1 virion attaches to a host cell via one or more surface receptors (e.g., GLUT1 / HSPG / NRP-1), through the HTLV-1 envelope surface and / or the transmembrane domain of the Env protein. After the outer membrane of the viral particle fuses with the host cell membrane, the contents of the virion can enter the cytoplasm of the host cell. The viral genomic RNA (gRNA) can be reverse transcribed using a virus-encoded reverse transcriptase (RNA-dependent / DNA-dependent polymerase) to convert the gRNA into double-stranded DNA (dsDNA). The viral dsDNA is transported to the nucleus and randomly integrated into the host genome using a virus-encoded integrase; at this point, the viral genome is now a provirus. The host cell machinery can then use host cell RNA polymerase II to drive the transcription of the proviral DNA, producing viral mRNA. The unspliced, full-length viral genomic RNA (~9kb) can be used to produce progeny virions and generate proteins such as Gag, Pol, and Pro proteins (proteases required for the maturation of structural and enzyme proteins). Single-splicing subgenomic mRNA (~4.3 kb) can be used to produce Env proteins, while double-splicing RNA (~2.1 kb) can be used to translate the regulatory proteins Tax (open reading frame (ORF) IV) and Rex (ORF III). Alternative splicing can also lead to the production of many accessory proteins from pX ORF I and II. Immature virions can assemble on the plasma membrane and are considered mature after viral budding.

[0177] The amino acid sequences derived from HTLV-1 disclosed herein may include naturally occurring proteinogenic amino acids as well as non-proteinogenic amino acids and non-naturally occurring amino acids, such as amino acid analogs. In some embodiments, the amino acids that can be used to implement this disclosure may include, for example, but not limited to, naturally occurring proteinogenic (L)-amino acids, their optically active (D)-isomers, chemically modified amino acids (including, for example, amino acid analogs, such as selenocysteine ​​(Sec), penicillamine (3-mercapto-D-valine), pyroglutamic acid (5-oxoproline), etc.), naturally occurring non-proteinogenic amino acids (such as ortholeucine), and chemically synthesized amino acids (having the amino acid characteristics known in the art), as well as amino acid equivalents.

[0178] In some embodiments, the isolated peptide of this disclosure comprises an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, the isolated peptide of this disclosure comprises the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146. In some embodiments, the isolated peptide of this disclosure comprises substantially the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146. In some embodiments, the isolated peptide comprises the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146. In some embodiments, the isolated peptide comprises two or more sequences selected from any one of SEQ ID NO:1-89 and 143-146, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof.

[0179] Table 1 below provides a list of non-limiting examples of HTLV-1 derived peptides.

[0180] Table 1. HTLV-1 Derivative Peptides

[0181]

[0182]

[0183]

[0184] The peptides disclosed herein can be synthesized or generated by hydrolysis. Synthetically generated peptides may include randomly generated peptides, specially designed peptides, and peptides in which at least some amino acid positions are conserved in several peptides while the remaining positions are random. Alternatively, the peptides disclosed herein can be generated by expression in a heterologous host cell.

[0185] In nature, peptides produced by hydrolysis undergo hydrolysis before the antigen binds to MHC molecules. Class I MHCs typically present peptides derived from proteins actively synthesized in the cytoplasm of the cell. In contrast, Class II MHCs typically present peptides derived from exogenous proteins that enter the endocytic pathway or proteins synthesized in the endoplasmic reticulum (ER). Intracellular transport allows peptides to associate with MHC molecules.

[0186] The binding of peptides to the MHC peptide-binding groove controls the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR. This spatial control is partly attributed to the hydrogen bonds formed between the peptide and the MHC molecule. Based on knowledge of how peptides bind to various MHC molecules, the major MHC-anchored amino acids and the different surface-exposed amino acids that exist between different peptides can be identified.

[0187] Preferably, the MHC-binding peptide has a length of about 5 to about 40 amino acid residues, more preferably about 6 to about 30 amino acid residues, even more preferably about 8 to about 20 amino acid residues, and even more preferably between about 9 and 11 amino acid residues, including peptides of any size with a length between 5 and 40 amino acids, in integer increments (i.e., 5, 6, 7, 8, 9...40). Although natural MHC class II binding peptides vary in length from about 9 to 40 amino acids, in almost all cases, the peptide can be truncated to a core of about 9 to 11 amino acids without losing MHC binding activity or T cell recognition.

[0188] In some embodiments, the isolated peptides of this disclosure may be about 8-12 amino acids in length. For example, the peptides disclosed herein may be 8, 9, 10, 11, or 12 amino acids in length.

[0189] The peptides disclosed herein may contain one or more reverse peptide bonds, one or more non-peptide bonds, one or more chemical modifications, one or more D-isomers of amino acids, or any combination thereof.

[0190] In some embodiments, the peptide may be modified to contain one or more reverse peptide bonds or non-peptide bonds. This modification can improve stability and / or the binding of the peptide to MHC molecules, thereby triggering a stronger immune response. In reverse peptide bonds, amino acid residues are not linked by peptide bonds (—CO—NH—), but the peptide bonds are reversed. Such reverse peptide mimics can be prepared using methods known in the art, such as those described in Meziere et al., 1997 (Meziere C., et al., J Immunol 1997). This method involves preparing pseudopeptides that involve changes in the main chain rather than changes in the side chain orientation. Such pseudopeptides can be used, for example, for desired MHC binding and / or T helper cell responses. Reverse-reverse peptides containing NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis. Other non-peptide bonds that can be used are, for example, —CH2—NH, —CH2S—, —CH2CH2—, —CH═CH—, —COCH2—, —CH(OH)CH2—, and —CH2SO—.

[0191] Amino acid residues comprising the peptides disclosed herein may be chemically modified. Non-limiting examples of chemical modifications include, for example, phosphorylation, acetylation, deamidation, amidation, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amidation of the carboxyl group, thiol modification by oxidation of cysteine ​​to sulfoalanine via performic acid, formation of mercury derivatives, formation of mixed disulfides with other thiols, reaction with maleimide, carboxymethylation using iodoacetic acid or iodoacetamide, and carbamylation using cyanate at alkaline pH. Chemical modifications may not correspond to chemical modifications that may be present in vivo.

[0192] For example, modifications to protein residues such as arginyl residues can be based on reactions that form adducts with ov-dicarbonyl compounds (such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione). Another example is the reaction of methylglyoxal with arginine residues. Cysteine ​​can be modified without the accompaniment of other nucleophilic sites such as lysine and histidine. Selective reduction of disulfide bonds in proteins is also possible. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Woodward reagent K can be used to modify specific glutamate residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine and glutamate residues. For example, diethyl pyrocarbonate and 4-hydroxy-2-nonenal can be used to modify histidine residues in proteins. Reactions of lysine residues with other α-amino groups can be used, for example, for peptide-surface binding or protein / peptide crosslinking. Lysine is an attachment site for polyethylene glycol and a major modification site for protein glycosylation. Methionine residues in proteins can be modified with, for example, iodoacetamide, bromoethylamine, and chloramine-T. Tetranitromethane and N-acetimidazole can be used to modify tyrosine residues. Cross-linking by forming dityrosine residues can be accomplished using hydrogen peroxide / copper ions. N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylthio)-3H-indole (BPNS-skatole) have been used for tryptophan modification in recent studies. Successful modification of therapeutic proteins and peptides with PEG can lead to an extended circulating half-life, while cross-linking proteins / peptides with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde can be used to prepare hydrogels. Chemical modification of allergens for immunotherapy can be achieved by carbamylation with potassium cyanate.

[0193] The peptides disclosed herein can also be synthesized with additional chemical groups at their N-terminus and / or C-terminus to enhance the peptide's stability, bioavailability, and / or affinity.

[0194] N-terminal modifications may include methylation (e.g., —NHCH3 or —N(CH3)2), acetylation (e.g., with acetic acid or its halogenated derivatives such as... chloroacetic acid, -bromoacetic acid or The N-terminus can be blocked with a benzyloxycarbonyl (Cbz) group, or with any blocking group containing a carboxylic acid ester functional group defined by RCOO- or a sulfonyl functional group defined by R-SO2-, where R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc., and similar groups. A deaminated amino acid can also be incorporated into the N-terminus (thus eliminating the N-terminal amino group) to reduce susceptibility to proteases or restrict peptide conformation. Furthermore, hydrophobic groups such as benzyloxycarbonyl, dansyl, or tert-butyloxycarbonyl can be added to the N-terminus. Similarly, an acetyl or 9-fluorenylmethoxycarbonyl group can be placed at the N-terminus.

[0195] C-terminal modification may include replacing a free acid with a carboxamide group or forming a cyclic lactam at the carboxyl terminus to introduce structural restriction. The peptides of this disclosure may also be cyclized, or deaminated or decarboxylated residues may be incorporated at the terminus of the peptide, resulting in the absence of a terminal amino or carboxyl group, thereby reducing susceptibility to proteases or restricting the conformation of the peptide. The C-terminal functional groups of the compounds of this disclosure include amides, lower alkyl amides, di(lower alkyl amides), lower alkoxy groups, hydroxyl groups, and carboxyl groups, as well as their lower ester derivatives and pharmaceutically acceptable salts thereof. Furthermore, hydrophobic groups, tert-butoxycarbonyl groups, or amide groups may be added to the C-terminus.

[0196] Other examples of non-natural modifications include incorporation of non-coding α-amino acids, photoreactive crosslinked amino acids, N-methylated and β-amino acids, skeleton reduction, retroinversion using D-amino acids, and C-terminal amidation and polyethylene glycol modification.

[0197] The peptides described herein may contain one or more (e.g., 1, 2, 3, or 4) amino acid substitutions and / or insertions and / or deletions. An amino acid substitution means that an amino acid residue is replaced at the same position. Inserted amino acid residues may be inserted at any position and may be inserted in such a way that some or all of the inserted amino acid residues are directly adjacent to each other, or in such a way that no inserted amino acid residue is directly adjacent to another inserted amino acid residue. One or more (e.g., 1, 2, 3, or 4) amino acids may be substituted and / or inserted and / or deleted from the sequence of any one of SEQ ID NO: 1-89 and 143-146. Each substitution and / or insertion and / or deletion may occur at any position in any one of SEQ ID NO: 1-89 and 143-146.

[0198] In some embodiments, the peptides of this disclosure may contain additional amino acids (e.g., 1, 2, 3, or 4) at the C-terminus and / or N-terminus of any one of SEQ ID NO:1-89 and 143-146. The peptides of this disclosure may contain the amino acid sequence of any one of SEQ ID NO:1-89 and 143-146, except for one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions, or deletions.

[0199] The inserted and substituted amino acids can be naturally occurring or non-naturally occurring, for example, they may contain non-natural side chains and / or be linked together by non-natural peptide bonds. Such modified peptide ligands are further discussed in Douat-Casassus et al., J. Med. Chem, 2007; 50(7):1598-609 and Hoppes et al., J. Immunol 2014; 193(10):4803-13 and their references. If more than one amino acid residue is substituted and / or inserted, the substituted / inserted amino acid residues may be the same as or different from each other. Each substituted amino acid may have a different side chain than the substituted amino acid.

[0200] In the antigenic peptides of the present invention, D-amino acids may replace L-amino acids. Furthermore, non-standard amino acids (i.e., amino acids other than common naturally occurring protein-derived amino acids, such as β-γ-δ-amino acids and many derivatives of L-α-amino acids) may also be used for substitution or addition to produce the peptides of this disclosure.

[0201] Amino acid substitutions can be conserved, meaning that the substituted amino acid has similar chemical properties to the original amino acid. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: Group 1 - Ala, Ser, Thr, Pro, Gly; Group 2 - Asp, Asn, Glu, Gln; Group 3 - His, Arg, Lys; Group 4 - Met, Leu, Ile, Val, Cys; Group 5 - Phe, Thy, Trp.

[0202] Significant changes in function (e.g., affinity for MHC molecules and / or TCRs) can be achieved by selecting substitutions with lower conservation than those described above (in other words, selecting residues that are more significantly different in terms of their effect on the maintenance of the peptide backbone structure (e.g., as a folded sheet or helical conformation) in the substituted region, the body portion of the side chain, or the charge or hydrophobicity of the peptide at the site involving MHC or TCR binding). Substitutions that are generally expected to produce the greatest changes in peptide properties are those in which (a) a hydrophilic residue (e.g., Ser) replaces a hydrophobic residue (e.g., Leu, Ile, Phe, Val, or Ala) (or is replaced by it); (b) a residue with an electronegative side chain (e.g., Lys, Arg, or His) replaces an electronegative residue (e.g., Glu or Asp) (or is replaced by it); or (c) a residue with a large side chain (e.g., Phe) replaces a residue without a side chain (e.g., Gly) (or is replaced by it).

[0203] Other side chains can also be used, such as those alkyl, lower alkyl, cyclic 4-, 5-, 6-, and 7-membered alkyl groups, amides, amide lower alkyl groups, amide di(lower alkyl), lower alkoxy groups, hydroxyl groups, carboxyl groups, and their lower ester derivatives, as well as those substituted with 4-, 5-, 6-, and 7-membered heterocyclic groups, replacing the natural side chains of the 20 genetically encoded amino acids (or stereoisomers of D-amino acids). For example, proline analogs in which the ring size of the proline residue is changed from 5-membered to 4-, 6-, or 7-membered can be used. The cyclic group can be saturated or unsaturated, and if unsaturated, it can be aromatic or non-aromatic. The heterocyclic group preferably contains one or more nitrogen, oxygen, and / or sulfur heteroatoms. Examples of such groups include furazanyl, furanyl, imidazoalkyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidinyl (e.g., 1-piperidinyl, piperidino), pyranyl, pyrazinyl, pyrazolyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolidinyl, thiadiazolyl, thiazolyl, thiophenyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups can be substituted or unsubstituted. When a group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or a substituted or unsubstituted phenyl group.

[0204] Other examples of amino acid substitutions include stereoisomers (e.g., D-amino acids) and non-natural amino acids such as L-ornithine, L-homocysteine, L-homoserine, L-citrulline, 3-sulfinyl-L-alanine, N-(L-arginino)succinic acid, 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, triiodothyronine, L-thyroxine, L-selenocysteine, N-(L-arginino)taurine, 4-aminobutyrate, (R,S)-3-amino-2-methylpropionate, α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, β-alanine, 3-pyridylalanine, 4-hydroxyproline, o-phosphoserine, N-methylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ortholeucine, and other similar amino acids and imino acids.

[0205] Amino acid residues that do not significantly contribute to the interaction with T cell receptors can be modified by replacing them with other amino acids. The incorporation of these other amino acids will not significantly affect the responsiveness of T cells, nor will it eliminate the binding to the relevant MHC.

[0206] Peptides may also comprise isosteres of two or more residues. As used herein, “isostere” refers to a sequence that can substitute two or more residues of a second sequence because the spatial conformation of the first sequence is adapted to a binding site specific to the second sequence. This term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include modifications to the amide nitrogen, α-carbon, and amide carbonyl groups, as well as complete substitution, extension, deletion, or crosslinking of the amide bond.

[0207] Combinations of substitutions / additions / deletions at more than one position can be developed and tested to determine whether the combination has an additive or synergistic effect on the immunogenicity of the peptide. In some embodiments, no more than four positions within the peptide are altered simultaneously.

[0208] Preferably, the peptide of this disclosure binds to an MHC molecule in the peptide-binding groove of the MHC molecule. Typically, the above-described amino acid modifications do not weaken the peptide's ability to bind to an MHC molecule. In some embodiments, the amino acid modifications enhance the peptide's ability to bind to an MHC molecule. For example, a mutation can be made at the site that anchors the peptide to the MHC molecule. This is particularly important for binding (especially binding to HLA-A). For peptides of type 02), such anchoring sites and preferred residues at these sites may include, for example, amino acid residues at position 2 and / or the C-terminus of the peptide, which are generally considered to be the primary anchoring sites. Preferred anchoring residues may differ for each HLA type. As a non-limiting example, HLA-A The preferred amino acid at position 2 of the peptide 02 is Leu, Lie, Val, or Met, and the preferred amino acid at the C-terminus is Val or Leu. Multiple positions (including positions 2, 3, 5-7, and 9) are also relevant for the peptide's interaction with HLA-A. Stable binding at position 02 may be important. Anchor residues at positions 2 and 9 are likely crucial for peptide binding to HLA-A2. However, other peptide side chains (e.g., at position 3) may contribute to the stability of the interaction. In some cases, the optimal peptide binding length can exceed 9 residues.

[0209] The immunological properties of peptides can be described as their association with MHC molecules (K+). on and K off The binding function of the MHC-peptide complex (MHC-MHC) and the TCR (the affinity of the TCR for the interaction between the TCR and the MHC-peptide complex) is investigated. Modification of major MHC anchor residues exhibits a significant degree of predictability in its overall effect on binding to MHC molecules. Modification of minor MHC anchor residues can affect the affinity of the MHC-peptide complex for the TCR interaction, as well as the K-axis associated with peptide-MHC interactions. on and K off .

[0210] When the HTLV-1 peptide is a mutant peptide, T cell lines targeting the native (non-mutant) epitope are generated, employing an immunization strategy potent enough to elicit an effective response in transgenic mice carrying human MHC (such as the A2 allele). The HTLV-1 peptide is studied in vitro in the presence of competent APCs, and its functional effects on native (non-mutant) epitope-specific T cells are measured. Evaluation is performed at different concentrations of the HTLV-1 peptide because, in the case of cross-reactive peptides, the expected effect is biphasic (activation at limited concentrations, and inhibition at higher concentrations due to antigen-induced cell death [AICD]). Measurements of the following three parameters are used to characterize the disclosed HTLV-1 peptide:

[0211] 1. The minimum concentration of HTLV-1 marker required to induce effects indicating T cell activation (e.g., the production of cytokines [e.g., IFN-γ]);

[0212] 2. Maximum (peak) effect at any HTLV-1 peptide concentration (e.g., cytokine production [e.g., IFN-γ]); and

[0213] 3. HTLV-1 peptide concentration at peak activation effect (e.g., cytokine concentration [e.g., IFN-γ]).

[0214] As a non-limiting example, HTLV-1 peptides that result in decreased values ​​associated with parameters 1 and 3 but increased values ​​associated with parameter 2 can be useful. Using native epitopes and / or unrelated non-cross-reactive peptides as references is valuable for identifying peptide classes with potential values. Peptides with properties quantitatively comparable to or even moderately weakened by native epitopes are still considered useful because they may exhibit different immunological properties from native peptides while retaining cross-reactivity, such as a reduced ability to break tolerance or reactivate reactivity in vivo or a reduced tendency to induce AICD.

[0215] In addition to its practicality and speed, other advantages of this screening method include, but are not limited to: using more relevant polyclonal T cell lines rather than potentially biased T cell clones as readouts, and integrating features such as K... on K off The combined values ​​of parameters such as TCR affinity can be translated into cross-reactivity and functional affinity of the peptide-MHC complex relative to the TCR. These parameters can predict in vivo immunological properties, thereby defining a useful group of peptides eligible for further evaluation, optimization, and practical application. Peptides predicted to bind to MHC and retain cross-reactivity with a nominal wild-type peptide-specific TCR will elicit measurable effects in this assay.

[0216] The peptides disclosed herein, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof, may be used to induce an immune response. If this is the case, it is important that the immune response be specific to the intended target (e.g., HTLV-1, HTLV-2, HTLV-3, and / or HTLV-4) to avoid the risk of unwanted side effects that may be associated with an “off-target” immune response. Therefore, preferably, the amino acid sequence of the peptides disclosed herein does not match the amino acid sequence of peptides from any other one or more endogenous proteins (particularly the amino acid sequence of a peptide from another human protein). Furthermore, the amino acid modifications described herein should not impair the ability of the peptide to induce an antigen-specific immune response when presented on the surface of antigen-presenting cells (APCs) in a complex with MHC molecules.

[0217] The half-life and / or bioavailability can also be extended by, for example, PEGylation, glycosylation, polysialylation, hydroxyethyl starchization, recombinant PEG mimicry, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, or acylation of the peptide.

[0218] The peptides disclosed herein can also serve as structural models for non-peptide compounds with similar biological activities. Various techniques can be used to construct compounds possessing the same or similar desired biological activities as the lead peptide compounds, but exhibiting more favorable activities in terms of solubility, stability, and sensitivity to hydrolysis and proteolysis. These techniques include replacing the peptide backbone with a backbone composed of amide esters, phosphonates, carbamates, sulfonamides, secondary amines, and N-methyl amino acids.

[0219] The multiple peptides described herein can be operatively linked together. Therefore, in one aspect, this disclosure provides isolated peptides or polypeptides comprising two or more amino acid sequences selected from SEQ ID NO:1-89 and 143-146, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof. For example, such multi-epitope peptides or polypeptides may comprise 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 monoepitope peptides described herein (e.g., SEQ ID NO:1-89 and 143-146). Monoepitope peptides (e.g., SEQ ID NO:1-89 and 143-146) may be arranged in any order and may be identical or different.

[0220] In some embodiments, this disclosure provides isolated peptides or polypeptides comprising the numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, ... An isolated peptide or polypeptide, or a pharmaceutically acceptable salt thereof, or a fragment thereof, or a derivative thereof, of a sequence of 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, or 93 amino acids.

[0221] Monoepitope peptides can be linked by linkers. Linkers may contain relatively small neutral molecules, such as amino acids or amino acid mimics, which are essentially uncharged under physiological conditions. Linkers can be selected from, for example, those shown in Table 2, or neutral spacer regions of other nonpolar or neutral polar amino acids. It should be understood that the optionally present linkers do not need to consist of identical residues and can therefore be heterooligomers or homooligomers. When present, linkers are typically composed of at least one or two residues, and more commonly three to six residues.

[0222] The peptides disclosed herein can be synthesized, for example, by solid-phase synthesis. This allows the peptides to be immobilized, for example, onto a solid support (such as beads). The peptides disclosed herein can be synthesized using the Fmoc-polyamide mode of solid-phase peptide synthesis. Temporary N-amino protection is provided by a 9-fluorenylmethoxycarbonyl (Fmoc) group. This protecting group, which is highly unstable to bases, is repeatedly cleaved using 20% ​​piperidine in N,N-dimethylformamide. Side-chain functional groups can be protected as their butyl ethers (in the case of serine, threonine, and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butoxycarbonyl derivatives (in the case of lysine and histidine), triphenylmethyl derivatives (in the case of cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of arginine). When glutamine or asparagine is a C-terminal residue, 4,4′-dimethoxydiphenylmethyl is used to protect the side-chain amide functional group. The solid-phase support is based on a polydimethacrylamide polymer, which consists of three monomers: dimethacrylamide (main chain monomer), bisacrylamide ethylenediamine (crosslinking agent), and methyl acryloylsarcosinate (functionalizing agent). The peptide-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. Except for asparagine and glutamine, all amino acid derivatives are added in their pre-formed symmetrical anhydride derivative forms. Asparagine and glutamine are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or indigo assays. After synthesis, the peptide is cleaved from the resin support by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture, simultaneously removing the side-chain protecting groups. Commonly used scavengers include ethylenedithiol, phenol, anisole, and water, the specific choice depending on the constituent amino acids of the synthesized peptide. Furthermore, combinations of solid-phase and solution-phase methods for peptide synthesis are also possible.

[0223] Trifluoroacetic acid was removed by vacuum evaporation, followed by grinding with diethyl ether to obtain crude peptides. Any scavenging agents present were removed by a simple extraction procedure that yielded scavenging-free crude peptides upon aqueous freeze-drying.

[0224] Purification can be achieved by techniques such as recrystallization, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography or combinations thereof using, for example, acetonitrile / water gradient separation.

[0225] Peptides can be analyzed using thin-layer chromatography, electrophoresis (especially capillary electrophoresis), solid-phase extraction (CSPE), reversed-phase high-performance liquid chromatography, amino acid analysis after acid hydrolysis, and analysis by rapid atomic bombardment (FAB) mass spectrometry, as well as MALDI and ESI-Q-TOF mass spectrometry.

[0226] Alternatively, peptides can be generated through recombinant expression in heterologous host cells. Such methods typically involve expressing the peptide in vivo using a vector containing a nucleic acid sequence encoding the peptide to be expressed; for example, in bacterial, yeast, insect, or mammalian cells.

[0227] In other embodiments, an in vitro cell-free system may be used. The peptides may be isolated and / or may be provided in a substantially pure form. For example, they may be provided in a form that is substantially free of other peptides or proteins.

[0228] The peptide-MHC (pMHC) complex disclosed in this article

[0229] On the other hand, this disclosure provides a peptide-MHC molecule complex. Preferably, the peptide binds to the peptide-binding groove of the MHC molecule. In some embodiments, the peptide forms a non-covalent complex with the MHC molecule. In other embodiments, the peptide and MHC molecule may be covalently linked, for example, through a linker.

[0230] MHC molecules are generally classified into two classes: class I and class II. Class I MHC molecules are complete membrane proteins containing a glycoprotein heavy chain (also referred to herein as the α chain) with three extracellular domains (i.e., α1, α2, and α3) and two intracellular domains (i.e., the transmembrane domain (TM) and the cytoplasmic domain (CYT)). The heavy chain is non-covalently associated with a soluble subunit called β2-microglobulin (β2m or β2M). Class II MHC molecules, or MHC class II proteins, are heterodimeric integrated membrane proteins containing a non-covalently associated α chain and a β chain. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (TM and CYT domains). The β chain contains two extracellular domains (β1 and β2) and two intracellular domains (TM and CYT domains).

[0231] Class I and Class II MHC molecules organize their domains to form antigenic determinant binding sites, such as peptide-binding moieties or peptide-binding grooves. A peptide-binding groove is a cavity-forming portion of an MHC molecule in which a peptide (e.g., an antigenic determinant) can bind. The conformation of the peptide-binding groove can change upon peptide binding, allowing the correct alignment of amino acid residues crucial for the binding of the TCR to the peptide-MHC (pMHC) complex.

[0232] In some embodiments, the MHC molecule includes a segment of the MHC chain sufficient to form a peptide-binding groove. For example, the peptide-binding groove of a class I protein may include portions of α1 and α2 domains capable of forming two β-sheets and two α-helices in a heavy chain. Including a portion of the β2 microglobulin chain can stabilize class I MHC molecules. While for most types of class II MHC molecules, the interaction between the α and β chains can occur in the absence of a peptide, double-stranded class II MHC molecules are unstable until the binding groove is filled with a peptide. The peptide-binding groove of a class II protein may include portions of α1 and β1 domains capable of forming two β-sheets and two α-helices. A first portion of the α1 domain forms a first β-sheet, and a second portion of the α1 domain forms a first α-helix. A first portion of the β1 domain forms a second β-sheet, and a second portion of the β1 domain forms a second α-helix. X-ray crystal structures of class II proteins with peptides bound in their binding grooves show that one or both ends of the bound peptide can extend beyond the MHC protein. Therefore, the ends of the α1 and β1 α-helices of class II proteins form open cavities, preventing the ends of peptides bound to the binding groove from being buried within these cavities. Furthermore, X-ray crystal structures of class II proteins show that the N-terminus of the MHC β chain extends distinctly from the side of the MHC protein in a non-structural manner, as the first four amino acid residues of the β chain cannot be localized by X-ray crystallography.

[0233] The peptides disclosed herein can bind to MHC molecules in a specific manner, enabling the pMHC complex to bind to the TCR in a preferred manner. In some embodiments, the binding of the pMHC complex to the TCR can induce a T cell response.

[0234] Whether a given peptide will form a complex with an MHC molecule can be determined by assessing whether MHC can refold in the presence of the peptide using methods described, for example, PCT application WO2018 / 083505 (which is incorporated herein by reference in its entirety for all purposes). If the peptide does not form a complex with MHC, then MHC will not refold. Refolding can be confirmed using antibodies that recognize only the folded state of MHC. Alternatively, the ability of the peptide to stabilize MHC on the surface of antigen-processing-associated transporter (TAP) deficient cell lines (such as T2 cells, which lack the TAP-mediated ability to transport cytoplasmic peptides into the endoplasmic reticulum (ER) to load peptides onto MHC class I molecules) can be measured, or other biophysical methods can be used to determine interaction parameters.

[0235] The peptides disclosed herein can be provided as MHC groove-binding peptides. In some embodiments, MHC groove-binding peptides can be engineered such that the peptide can be modified at some or all of the sites involved in MHC binding. For example, MHCBN is a comprehensive database of MHC-binding and non-binding peptides compiled from published literature and existing databases. The latest version of the database has 25,860 entries, including 20,717 MHC-binding peptides and 4,022 inactive MHC peptides from over 450 MHCs. The database contains (a) sequence and structural data of peptide-derived proteins and (b) MHCs. MHCBN has various web tools, including: (i) peptide localization on the query sequence; (ii) arbitrary field retrieval; (iii) dataset creation; and (iv) online data submission.

[0236] In some cases, peptide binding tools used to predict binding to MHC-I or MHC-II can be, for example, Antibody Epitope Prediction, ANTIGENIC, BepiPred, CTLPred, DiscoTope, EPIPREDICT, Epitope Cluster Analysis, Epitope Conservancy Analysis, EUiPro, HLA Peptide Binding Predictions, HLA Binding, MAPPP, MHCBench, MHC-I Processing Predictions, Mosaic Vaccine Tool Suite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, and Prediction of Antigenic Determinants. Determinants, ProPred, ProPred-1, RankPep, SMM, SVMHC, TAPPred, VaxiJen, or combinations thereof. Use other example programs such as BIMAS, SYFPEITHI, or Rankpep.

[0237] In one specific implementation, an altered peptide library is generated by genetically engineering the library using polymerase chain reaction (PCR) or any other suitable technique to construct DNA fragments encoding peptides. Using PCR, the resulting fragment library encodes all possible codon combinations at specific triplet codons by employing oligonucleotides with random mutations within those triplet codons. Preferably, certain amino acid positions remain constant; these are conserved amino acids required for binding to the MHC peptide-binding groove and do not contact the T cell receptor (TCR).

[0238] In some embodiments, when a library is genetically engineered to construct a DNA fragment encoding a peptide to generate a library of altered peptides using polymerase chain reaction (PCR) or any other suitable technique, the target TCR is a TCR for which it is desired to identify a peptide epitope recognized by a receptor. In some embodiments, the target TCR is derived from a patient with HTLV-1 infection and / or HTLV-1-induced disease or condition. In some embodiments, the TCR comprises an α-chain and a β-chain.

[0239] The MHC molecules used in the pMHC complex described herein include naturally occurring full-length MHC molecules and single chains of MHC molecules (e.g., MHC class I α (heavy) chains, β2-microglobulin, MHC class II α chains, and MHC class II β chains), single subunits of such chains of MHC (e.g., α1, α2, and / or α3 subunits of MHC class I α chains, α1 and / or α2 subunits of MHC class II α chains, and β1 and / or β2 subunits of MHC class II β chains), as well as fragments, mutants, and various derivatives (including fusion proteins, e.g., fusions with viral envelope proteins or fusogens), wherein such fragments, mutants, and derivatives retain the ability to display antigenic determinants for recognition by antigen-specific TCRs. In one specific embodiment, the MHC comprises transmembrane domains embedded in the lipid envelope of liposomes, recombinant viral particles, or virus-like particles (VLPs).

[0240] Naturally occurring MHC molecules are encoded by gene clusters on human chromosome 6 or mouse chromosome 17. MHC is also known as H-2 in mice and as human leukocyte antigen (HLA) in humans. MHC class I molecules specifically bind to CD8 molecules expressed on cytotoxic T lymphocytes (CD8+ T cells), while MHC class II molecules specifically bind to CD4 molecules expressed on helper T lymphocytes (CD4+ T cells). MHC includes, but is not limited to, HLA-specific molecules such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), E, ​​G, DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9), and DP (e.g., DP1-DP6). More preferably, HLA specificity includes A1, A2, A3, A11, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8 and DR-11.

[0241] In some embodiments, the MHC molecule in the pMHC complex of this disclosure is a human leukocyte antigen (HLA) molecule. The MHC molecule may be a human HLA molecule selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In some embodiments, the MHC class I or MHC II polypeptide may be derived from any functional human HLA-A, B, C, DR, or DQ molecule. Non-limiting examples of HLA-A alleles include, but are not limited to, A. 0101, A 0201, A 0202, A 0301, A 1101, A 2301, A 2402, A 2501, A 2601, A 2901, A 2902, A 3101, A 3201, A 3301, A 3401, A 3601, A 4301, A 6601, A 6801, A 6901, A 7401 and A 8001. Non-restrictive examples of the HLA-B allele include, but are not limited to, B. 0702, B 0801, B 1301, B 1401, B 1402, B 1501, B 1801, B 1802, B 2701, B 2702, B 3501, B 3502, B 3701, B 3801, B 3901, B 4001, B 4101, B 4201, B 4402, B 4501, B 4601, B 4701, B 4801, B 4901, B 5001, B 5101, B 5201, B 5301, B 5401, B 5501, B 5502, B 5601, B 5701, B 5801, B 5901, B 6701, B 7301, B 1517, B 8101, B 8201 and B 8301. Non-restrictive examples of HLA-C alleles include, but are not limited to, Cw. 0101, Cw 0202, Cw 0303, Cw 0401, Cw 0501, Cw 0602, Cw 0701, Cw 0702, Cw 0802, Cw 1203, Cw 1401, Cw 1502, Cw 1601, Cw 1701 and Cw 1801. Non-restrictive examples of HLA-DR alleles include, but are not limited to, DRB1. 0101, DRB1 0103, DRB1 1501, DRB1 1502, DRB1 1601, DRB1 1602, DRB1 0301, DRB1 0401, DRB1 0404, DRB1 1101, DRB1 1201, DRB1 1301, DRB1 1302, DRB1 1401, DRB1 1402, DRB1 0701, DRB1 0801, DRB1 0802, DRB1 0803, DRB1 0901 and DRB1 1001.

[0242] In some embodiments, the MHC class I molecule may be selected from HLA-A. 02, HLA-A 01. HLA-A 03. HLA-A 11. HLA-A 23. HLA-A 24. HLA-B 07. HLA-B 08. HLA-B 40. HLA-B 44. HLA-B 15. HLA-C 04. HLA C 03 HLA-C 07. Allele variants of the above-mentioned HLA types also exist, all of which are covered in this disclosure. In some embodiments, the MHC molecule may be HLA-A. 02 or HLA-A 11.

[0243] The MHC molecules used in this article can also be derived from any other mammal or bird species, such as non-human primates, rodents (e.g., mice), rabbits, horses, cattle, dogs, cats, pigs, etc.

[0244] Naturally occurring MHC class I molecules bind to peptides derived from proteolytically degraded proteins, particularly endogenously synthesized proteins, via cell-mediated binding. The resulting small peptides are transported to the endoplasmic reticulum, where they associate with nascent MHC class I molecules, then are transported via the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0245] Naturally occurring MHC class I molecules consist of an α (heavy) chain associated with β2-microglobulin. The heavy chain is composed of subunits α1-α3. The β2-microglobulin protein associates with the α3 subunit of the heavy chain. In some embodiments, the β2-microglobulin and α3 subunit are covalently bound. In some embodiments, the β2-microglobulin and α3 subunit are non-covalently bound. The α1 and α2 subunits of the heavy chain fold to form grooves that allow peptides (e.g., antigenic determinants) to be displayed and recognized by TCRs.

[0246] Class I molecules can bind peptides that are approximately 8-10 amino acids in length. Each person has three to six different types of Class I molecules, each of which can bind many different types of peptides.

[0247] In some embodiments, the MHC contained in the pMHC complex of this disclosure comprises (i) a class I MHC polypeptide or a fragment, mutant, or derivative thereof, and optionally (ii) a β2-microglobulin polypeptide or a fragment, mutant, or derivative thereof. In one specific embodiment, the class I MHC polypeptide is linked to the β2-microglobulin polypeptide via a peptide linker.

[0248] In one specific embodiment, the class I MHC peptide is a human class I MHC peptide selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC peptide is a mouse class I MHC peptide selected from H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0249] In some embodiments, the peptide disclosed herein forms a complex with one or more MHC class I α heavy chains. In some embodiments, the MHC class I α heavy chain is entirely human. In some embodiments, the MHC class I α heavy chain is humanized. Humanized MHC class I α heavy chains are described, for example, in U.S. Patent Publications 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467. In some embodiments, the MHC class I α heavy chain comprises a human extracellular domain (human α1, α2, and / or α3 domains) and a cytoplasmic domain of another species. In some embodiments, the class I α heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence may be HLA-A The 0201 sequence. In various respects, peptide MHC can include all domains of the MHC class I heavy chain.

[0250] In some embodiments, the MHC molecule contains β2-microglobulin. In some embodiments, the β2-microglobulin is entirely human. In some embodiments, the β2-microglobulin is humanized.

[0251] In some implementations, MHC class I molecules contain mutations in the β2-microglobulin (β2m or β2M) polypeptide and heavy chain sequences to establish a disulfide bond between B2M and the heavy chain. In some cases, the heavy chain is HLA, and the disulfide bond connects one of the following residue pairs: B2M residue 12, HLA residue 236; B2M residue 12, HLA residue 237; B2M residue 8, HLA residue 234; B2M residue 10, HLA residue 235; B2M residue 24, HLA residue 236; B2M residue 28, HLA residue 232; B2M residue 98, HLA residue 192; B2M residue 9... 9. HLA residue 234; B2M residue 3, HLA residue 120; B2M residue 31, HLA residue 96; B2M residue 53, HLA residue 35; B2M residue 60, HLA residue 96; B2M residue 60, HLA residue 122; B2M residue 63, HLA residue 27; B2M residue Arg3, HLA residue Gly120; B2M residue His31, HLA residue Gln96; B2M residue B2M residues: Asp53, HLA residue Arg35; B2M residue Trp60, HLA residue Gln96; B2M residue Trp60, HLA residue Asp122; B2M residue Tyr63, HLA residue Tyr27; B2M residue Lys6, HLA residue Glu232; B2M residue Gln8, HLA residue Arg234; B2M residue Tyr10, HLA residue Pro235; B2M residues... The residues are Ser11, Gln242 (HLA); Asn24 (B2M), Ala236 (HLA); Ser28 (B2M), Glu232 (HLA); Asp98 (B2M), His192 (HLA); Met99 (B2M), Arg234 (HLA), Gly2 (first linker), Tyr84 (heavy chain (HLA)); Arg12 (light chain (B2M)), Ala236 (HLA); and / or Arg12 (B2M), Gly237 (HLA).

[0252] In some embodiments, the antigenic determinant amino acid sequence may be the amino acid sequence of the peptide described herein, which may be presented by an MHC class I molecule. In some embodiments, the sequence may contain about 8 to about 15 consecutive amino acids. In some embodiments, the sequence may contain about 8 to about 12 consecutive amino acids.

[0253] In some embodiments, at least one chain of the MHC and the peptide are included in the fusion protein. In one specific embodiment, the MHC and the peptide are separated by a linker sequence. For example, the single-chain molecule may contain an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence from the amino to the carboxyl terminus. Alternatively, the single-chain molecule may contain an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence from the amino to the carboxyl terminus. The single-chain molecule may also contain a signal peptide sequence at the amino terminus. In some embodiments, a linker sequence may be present between the peptide sequence and the β2-microglobulin sequence. In some embodiments, a linker sequence may be present between the β2-microglobulin sequence and the class I α (heavy) chain sequence. The single-chain molecule may also contain a signal peptide sequence at the amino terminus, a first linker sequence extending between the peptide sequence and the β2-microglobulin sequence, and / or a second linker sequence extending between the β2-microglobulin sequence and the class I heavy chain sequence. In some embodiments, the β2-microglobulin and class I α (heavy) chain sequences may be human, mouse, or porcine.

[0254] In some embodiments, the single-chain molecule may include a first flexible linker between the peptide ligand segment and the β2-microglobulin segment. For example, the linker may extend from the carboxyl terminus of the peptide ligand segment to and connect to the amino terminus of the β2-microglobulin segment. Preferably, the structure of the linker allows the connected peptide ligand to fold into the binding groove, thereby generating a functional MHC-antigenic peptide. In some embodiments, the linker may contain at least about 10 amino acids and at most about 15 amino acids. In some embodiments, the single-chain molecule may include a second flexible linker inserted between the β2-microglobulin and heavy chain segments. For example, the linker may extend from the carboxyl terminus of the β2-microglobulin segment to and connect to the amino terminus of the heavy chain segment. In some embodiments, the β2-microglobulin and the heavy chain may fold into the binding groove, thereby generating a molecule that can function in promoting T cell proliferation.

[0255] Suitable linkers used in MHC can have any suitable length, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Non-limiting examples of linkers include, for example, glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 92), and (GGGS)n (SEQ ID NO: 93), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers can be used; glycine and serine are both relatively unstructured and can therefore serve as neutral linkers between components. Glycine polymers can be used; glycine enters significantly more phi-psi spaces than alanine and is much less restricted than residues with longer side chains. Exemplary linkers may comprise amino acid sequences, including but not limited to those listed in Table 2. In some embodiments, the linker peptide includes cysteine ​​residues that can form disulfide bonds with cysteine ​​residues present in the second polypeptide.

[0256] Table 2. Examples of connector sequences

[0257]

[0258]

[0259] In some embodiments, the single-chain molecule may comprise a peptide covalently linked to an MHC class I α (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cysteine ​​residues). In some embodiments, the disulfide bond comprises a first cysteine ​​and a second cysteine ​​residue, the first cysteine ​​comprising a linker extending from the carboxyl terminus of the antigenic peptide, and the second cysteine ​​comprising an MHC class I heavy chain (e.g., an MHC class I α (heavy) chain having a non-covalent binding site of the antigenic peptide). In some embodiments, the second cysteine ​​residue may be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In some embodiments, the single-chain molecule may comprise a continuous polypeptide chain along with a disulfide bond. In some embodiments, the single-chain molecule may comprise two continuous polypeptide chains linked by a disulfide bridge (as the sole covalent bond). In some embodiments, in addition to the Cys residue, the linking sequence may also comprise at least one amino acid, including one or more Gly residues, one or more Ala residues, and / or one or more Ser residues.

[0260] In some embodiments, if the pMHC complex contains a first cysteine ​​residue at the Gly-Ser linker extending between the C-terminus of the peptide and β2-microglobulin and a second cysteine ​​residue at the proximal heavy chain position, then a disulfide bridge can link the antigenic peptide described herein to the class I groove of the pMHC complex.

[0261] Linking peptides to MHC class I or MHC class II molecules via flexible linkers can help ensure that the peptide occupies and remains associated with the MHC molecule during biosynthesis, transport, and display. However, there are cases where such linkers can interfere with the binding of the peptide to the MHC molecule or interfere with the TCR recognition of the complex. As an alternative, in some embodiments, the MHC molecule and the peptide are expressed separately.

[0262] In some embodiments, the β2-microglobulin sequence may comprise the full-length β2-microglobulin sequence. In some embodiments, the β2-microglobulin sequence lacks the leader peptide sequence. Therefore, in some conformations, the β2-microglobulin sequence may comprise approximately 99 amino acids and may be mouse-derived. β2-microglobulin sequence (e.g., GenBank accession number X01838). In some other conformations, the β2-microglobulin sequence may contain approximately 99 amino acids and can be human. 2-Microglobulin sequence (e.g., GenBank accession number AF072097.1).

[0263] In some embodiments, the pMHC complex may comprise an MHC sequence disclosed in the following documents: U.S. Patents 4,478,823, 6,011,146, 518,697, 8,895,020, 8,992,937, and WO 96 / 04314; Mottez et al., J. Exp. Med. 181:493-502, 1995; Madden et al., Cell 70:1035-1048, 1992; Matsumura et al., Science 257:927-934, 1992; Mage et al., Proc. Natl. Acad. Sci. USA 89:10658-10662, 1992; Toshitani et al., Proc. Natl. Acad. Sci. 93:236-240. 1996; Chung et al., J. Immunol. 163:3699-3708, 1999; Uger and Barber, J. Immunol. 160:1598-1605, 1998; Uger et al., J. Immunol. 162, pp. 6024-6028, 1999; White et al., J. Immunol. 162:2671-2676, 1999; Yu et al., J. Immunol. 168:3145-3149, 2002; Truscott et al., J. Immunol. 178:6280–6289, 2007. All these references are incorporated in their entirety by citation.

[0264] In some embodiments, the MHC comprises a class II MHC polypeptide or a fragment, mutant, or derivative thereof. In one specific embodiment, the MHC comprises α and β polypeptides of a class II MHC complex or a fragment, mutant, or derivative thereof. In one specific embodiment, the α and β polypeptides are linked by a peptide linker. In one specific embodiment, the MHC comprises α and β polypeptides of a human class II MHC complex selected from HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another specific embodiment, the MHC comprises α and β polypeptides of a mouse H-2A or H-2E class II MHC complex.

[0265] Naturally occurring class II molecules can contain two polypeptide chains: an α chain and a β chain. These chains can originate from the DP, DQ, or DR genome. There are approximately 40 known different human MHC class II molecules. All of them share the same basic structure but may have subtle differences in their molecular structure. MHC class II molecules can bind peptides of 13–18 amino acids in length.

[0266] In some embodiments, the MHC class II α chain is fully human. In some embodiments, the MHC class II α chain is humanized. Humanized MHC class II α chains are described, for example, in U.S. Patent Nos. 8,847,005, 9,043,996, and 10,154,658 (which are incorporated herein by reference in their entirety). In some embodiments, the humanized MHC class II α chain polypeptide comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II α chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, the class II α chain polypeptide is humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA.

[0267] In some embodiments, the peptide of this disclosure forms a complex with one or more MHC class II β chains. In some embodiments, the MHC class II β chains are entirely human. In some embodiments, the MHC class II β chain polypeptide is humanized. Humanized MHC class II β chain polypeptides are described, for example, in U.S. Patent Nos. 8,847,005, 9,043,996, and 10,154,658 (which are incorporated herein by reference in their entirety). In some embodiments, the humanized MHC class II β chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the class II β chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB.

[0268] The pMHC complexes disclosed herein may be isolated and / or in substantially pure form. For example, the complexes may be provided in a form substantially free of other peptides or proteins. The MHC molecules disclosed herein may include recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC, including their derivatives or variants, as long as peptide binding is retained. For example, MHC molecules may be fused to therapeutic portions, attached to solid carriers, in soluble forms, attached to tags, biotinylated, and / or in polymeric forms. The peptides disclosed herein may be covalently linked to MHC.

[0269] Methods for generating soluble recombinant MHC molecules that can form complexes with the peptides disclosed herein include, but are not limited to, those derived from *Escherichia coli* (…). E. coli MHC molecules can be expressed and purified in cells or insect cells. Alternatively, they can be synthesized or produced using cell-free systems.

[0270] The peptides disclosed herein can be present on the cell surface in complex with MHC. Therefore, this disclosure also provides cells that present the pMHC complex disclosed herein on their surface. Such cells can be mammalian cells, preferably cells of the Y1 immune system, and specialized antigen-presenting cells (APCs), such as dendritic cells or B cells. Other preferred cells include T2 cells. Cells presenting the peptides or pMHC complexes disclosed herein can be isolated, preferably in a homogeneous population, or provided in a substantially pure form. Such cells may not naturally present the complexes disclosed herein, or optionally, the cells may present the complex at a level higher than their native level. Such cells can be obtained by pulsed cells with one or more peptides disclosed herein (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides), or by genetically modified cells (via DNA or RNA transfer) to express one or more peptides disclosed herein (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides). The pulse involves incubating cells with peptides for several hours, typically using peptide concentrations of 10. -5 M to 10 -12 Within the range of M. These cells can also be used with HLA molecules (such as HLA-A). 02) Transduction to further induce the presentation of one or more peptides. Cells can be recombinantly generated. Cells presenting the peptides disclosed herein can be used to isolate T cells and TCRs activated or bound to said cells.

[0271] The fusion proteins, conjugates, and oligomeric complexes disclosed in this article

[0272] The peptides or pMHC complexes disclosed herein can be fused or conjugated to one or more heterologous molecules. The peptides or pMHC complexes disclosed herein can also be in multimeric form. Therefore, this disclosure also provides fusion proteins, conjugates, and oligomeric complexes comprising the peptides or pMHC complexes of this disclosure.

[0273] In some embodiments, the peptide is fused or conjugated with one or more heterologous molecules, including MHC molecules (or fragments thereof).

[0274] Heterologous molecules suitable for gene fusion and / or chemical conjugation with the peptides or pMHC complexes disclosed herein include, but are not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, etc. One or more heterologous molecules may be fused to the N-terminus and / or C-terminus of another polypeptide chain in the peptide and / or pMHC complex.

[0275] Heteropeptides and polypeptides include, but are not limited to: epitopes (e.g., FLAG) or tag sequences (e.g., His6 (SEQ ID NO: 1)). IDNO: 147, etc. (to allow for the detection and / or isolation of fusion proteins); transmembrane receptor proteins or portions thereof, such as extracellular domains or transmembrane and intracellular domains; ligands or portions thereof that bind to transmembrane receptor proteins; enzymes or portions thereof that have catalytic activity; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; polypeptides or peptides that enhance stability, such as immunoglobulin constant regions (e.g., Fc domains); half-life-extending sequences comprising combinations of two or more (e.g., 2, 5, 10, 15, 20, 25, etc.) naturally occurring or non-naturally occurring charged and / or uncharged amino acids (e.g., Ser, Gly, Glu, or Asp), designed to form a major hydrophilic or major hydrophobic fusion partner of the fusion protein; functional or non-functional antibodies (e.g., antibodies specific to dendritic cells), or their heavy or light chains; and polypeptides having activities different from those of the fusion proteins disclosed herein, such as therapeutic activity. In some embodiments, the one or more heterologous molecules enhance peptide-specific immune responses in a subject. In some implementations, the one or more heterologous molecules mediate the delivery of the peptide to a specific site within the subject's body.

[0276] In some embodiments, the fusion protein of this disclosure may include one or more affinity tags, for example, to allow affinity purification or conjugation to another molecule. Examples of affinity tags include, but are not limited to, His6 tags (SEQ ID NO:147), Avi tags, biotin, hemagglutinin (HA) tags, FLAG tags, Myc tags, GST tags, MBP tags, chitin-binding protein tags, calmodulin tags, V5 tags, streptavidin-binding protein tags, green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, SUMO tags, and ubiquitous protein tags.

[0277] In some embodiments, the fusion protein of this disclosure may comprise one or more epitopes not present in the antigen. One such example is the use of a fusion peptide in which a heterozygous T helper cell epitope is covalently linked to a peptide sequence (e.g., via a peptide linker or spacer region). Non-limiting examples of heterozygous T helper cell epitopes include the PADRE peptide, tetanus toxoid peptides (830-843), or influenza hemagglutinin HA (307-319).

[0278] The peptides or pMHC complexes disclosed herein can be conjugated with additional components such as carrier molecules or adjuvants for use as vaccines. Examples of adjuvants used in vaccines include microorganisms, such as bacterial BCG and / or substances produced by bacteria, such as Detox B (a monophosphoryl lipid A and mycobacterial cell wall skeleton oil droplet emulsion). KLH (keyhole hemocyanin), bovine serum albumin (BSA), and the E2 core protein of the pyruvate dehydrogenase complex are examples of suitable carrier proteins for use in vaccine compositions. Other examples of carrier proteins suitable for the compositions of this disclosure include, but are not limited to, ovalbumin (OVA), blue carrier protein (BCP), thyroglobulin (THY), soybean trypsin inhibitor (STI) and multiple attachment peptide (MAP), albumin, serum albumin, C-reactive protein, conalbumin, whey protein, ionocarrier protein, acyl carrier protein, signal transduction adaptor protein, androgen-binding protein, calcium-binding protein, calmodulin-binding protein, ceruloplasmin, cholesterol ester transfer protein, f-box protein, fatty acid-binding protein, follicle-staphylin, follicle-staphylin-related protein, GTP-binding protein, insulin-like growth factor-binding protein, iron-binding protein, potential TGFβ-binding protein, light-harvesting protein complex, lymphocyte antigen, membrane transport protein, neurohypophysin, periplasmic-binding protein, phosphate-binding protein, phosphatidylethanolamine-binding protein, phospholipid transport protein, retinol-binding protein, RNA-binding protein, S-phase kinase-related protein, sex hormone-binding globulin, thyroxine-binding protein, cobalamin transporter, cortisol transporter, transferrin-binding protein, and / or vitamin D-binding protein.

[0279] As another example, the peptide or pMHC complex disclosed herein can be fused to the N-terminal 80 amino acids of, for example, an HLA-DR antigen-associated invariant chain (p33 or Ii) derived from NCBI GenBank accession number X00497. The Ii fragment can facilitate the efficient introduction of the peptide or pMHC complex into cells.

[0280] The peptides or pMHC complexes of this disclosure may also be covalently (e.g., via a linker) or non-covalently linked to portions capable of eliciting therapeutic effects (such as antibodies or cytokines, such as interleukin-2, interferon-α, and granulocyte-macrophage colony-stimulating factors). Optionally or additionally, the peptides or pMHC complexes may be encapsulated in liposomes.

[0281] Other suitable heterologous molecules include, but are not limited to, fluorescent or luminescent markers, radioactive markers, nucleic acid probes, and contrast agents, antibodies, or enzymes that produce detectable products. Methods for detecting heterologous molecules may include flow cytometry, microscopy, electrophoresis, or scintillation counting.

[0282] In some embodiments, the peptides or pMHC complexes of this disclosure can be conjugated with fluorocarbons to enhance cellular immunogenicity. When a peptide or another polypeptide chain of a pMHC complex is linked to a fluorocarbon, the ends of the peptide or polypeptide chain, such as those not conjugated to the fluorocarbon, or other linkages, can be altered, for example, to promote the solubility of the fluorocarbon-peptide / polypeptide construct by forming micelles. To facilitate the large-scale synthesis of the construct, the N-terminal or C-terminal amino acid residues of the peptide or another polypeptide chain of the pMHC complex can be modified. When the desired peptide or another polypeptide chain of the pMHC complex is particularly sensitive to cleavage by peptidases, cleavable peptide mimics can be used to replace normal peptide bonds. Such bonds and synthetic methods are well known in the art.

[0283] The peptides or pMHC complexes disclosed herein may be provided in a soluble form or immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, beads, membranes, agarose gels, magnetic beads, plates, tubes, and columns. pMHC complexes may be attached to ELISA plates, magnetic beads, or surface plasmon resonance biosensor chips. Methods for attaching peptides or pMHC complexes to solid supports are known to those skilled in the art, including, for example, using affinity pairs such as biotin and streptavidin, or antibodies and antigens. In some embodiments, the peptides or pMHC complexes are biotin-labeled and attached to a surface coated with streptavidin.

[0284] The peptide or pMHC complexes disclosed herein may be in polymeric form, such as dimers, tetramers, pentamers, octamers, or larger polymers. Therefore, in some aspects, this disclosure provides oligomeric complexes comprising the peptide or pMHC complexes of this disclosure. As used herein, the terms “oligomer,” “oligomeric,” “oligomer,” and “oligomerization,” etc., encompass dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, or more advanced types of polymeric monomers comprising peptide or pMHC complexes. Having multiple copies of the peptide or pMHC complex in a large complex can enhance its biological activity, such as immunogenic activity.

[0285] For example, the peptides of this disclosure can be oligomerized using a biotin / streptoavidin system. Biotinylated analogs of the peptide monomers can be synthesized using standard techniques. For example, the peptides can be C-terminally biotinylated. These biotinylated peptide monomers are then oligomerized by incubation with streptoavidin [e.g., incubation for 1 hour at a 4:1 molar ratio in phosphate-buffered saline (PBS) or HEPES-buffered RPMI medium at room temperature]. In a variation of this embodiment, the biotinylated peptide monomers can be oligomerized by incubation with an anti-biotin antibody [e.g., goat anti-biotin IgG].

[0286] Generally, oligopMHC complexes can be generated using biotin-labeled pMHC and complexed with fluorescently labeled streptavidin. Biotinylation sites can be introduced into the pMHC complex, allowing biotin to be added to these sites, for example, using the BirA enzyme. Alternatively, oligopMHC complexes can be formed using immunoglobulins as a molecular scaffold. In this system, the extracellular domains of the MHC molecule are fused to the constant region of the immunoglobulin heavy chain, separated by short amino acid linkers. OligopMHC complexes have also been generated using carrier molecules such as dextran. Due to affinity effects, oligopMHC complexes can be used to improve the detection of binding sites (such as T-cell receptors) of said complexes.

[0287] In other embodiments, the peptides or pMHC complexes of this disclosure can be oligomerized by covalently linking to at least one linker. The linker moiety can be a peptide linker, such as those described herein (e.g., in Table 2). In some embodiments, polyethylene glycol (PEG) can be used as a linker for oligomerizing the peptide monomer. For example, a single PEG moiety can be simultaneously linked to the N-terminus of both peptide chains of the peptide dimer.

[0288] Alternatively, the oligopeptide or pMHC complex may also contain one or more intramolecular disulfide bonds between cysteine ​​residues of the peptide or pMHC monomers. Preferably, both monomers contain at least one intramolecular disulfide bond. Most preferably, both monomers contain intramolecular disulfide bonds, such that each monomer contains a cyclic group. Such disulfide bonds can be formed by oxidizing cysteine ​​residues in the core sequence of the peptide. In one embodiment, the formation of cysteine ​​bonds is controlled by selecting the type and concentration of the oxidant that effectively optimizes the formation of the desired isomer. For example, when the oxidant is DMSO, oxidation of the peptide dimer is preferentially achieved (to form two intramolecular disulfide bonds (one on each peptide chain)) (rather than the formation of intermolecular disulfide bonds). The formation of cysteine ​​bonds can be controlled by selectively using thiol protecting groups during peptide synthesis.

[0289] In some embodiments, the peptide or pMHC complex described herein may be fused or conjugated to a dimerizing moiety. The dimerizing moiety may include, for example, an immunoglobulin domain (e.g., derived from an IgG antibody, such as human IgG) that links two monomers to produce a homodimer or heterodimer molecule. As a non-limiting example, the dimerizing motif in a protein according to this disclosure may be constructed to include a hinge region and an immunoglobulin domain (e.g., a Cy3 domain), such as a carboxyl-terminal C-domain (CH3 domain), or a sequence substantially identical to the C-domain. The hinge region may be derived from Ig and facilitates dimerization by forming one or more interchain covalent bonds (e.g., one or more disulfide bridges). Furthermore, such homodimers or heterodimers may also include one or more targeting moieties that bind to target molecules present on, for example, antigen-presenting cells (APCs), such as dendritic cells or B cells. In this case, the hinge region may serve as a flexible spacer between domains, allowing two targeting units to simultaneously bind to two target molecules on an APC expressed at variable distances. Immunoglobulin domains facilitate dimerization through non-covalent interactions (e.g., hydrophobic interactions). In a preferred embodiment, the CH3 domain is derived from IgG. These dimerized motifs may exchange with other multimerized motifs from, for example, other Ig isotypes / subtypes. Preferably, the dimerizing motif is derived from a natural human protein, such as human IgG. Examples of such homodimeric protein constructs are described in US 10,590,195 (which is incorporated herein by reference in its entirety).

[0290] Nucleic acid and vector

[0291] On the other hand, this disclosure provides isolated polynucleotides comprising nucleic acid sequences encoding one or more peptides of this disclosure and / or peptide-based molecules (such as complexes (e.g., pMHC complexes), fusion proteins, or conjugates containing said peptides). The polynucleotides may be, for example, DNA, cDNA, PNA, RNA, or combinations thereof (single-stranded and / or double-stranded), or polynucleotides in natural or stable forms (e.g., polynucleotides having a phosphate thioester backbone), and may or may not contain introns, provided that they encode a peptide.

[0292] In some embodiments, the polynucleotide described herein encodes a peptide comprising an amino acid sequence or fragment thereof having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity with any of the amino acid sequences of SEQ ID NO: 1-89 and 143-146. In some embodiments, the polynucleotide described herein encodes a peptide or fragment thereof comprising an amino acid sequence of any of SEQ ID NO: 1-89 and 143-146.

[0293] In some embodiments, the polynucleotides described herein encode more than one peptide selected from any one of SEQ ID NO:1-89 and 143-146, or fragments or derivatives thereof. For example, the polynucleotides described herein may encode 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides (e.g., SEQ ID NO:1-89 and 143-146), or fragments or derivatives thereof. The peptides may be arranged in any order and may be the same or different.

[0294] In some implementations, the polynucleotides described herein encode numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28 from SEQ ID NO:1-89 and 143-146. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 amino acid sequences or fragments or derivatives thereof.

[0295] In some implementations, the polynucleotides described herein are DNA molecules.

[0296] Methods for delivering DNA to subjects include, for example, direct delivery as naked DNA. Delivery can also be achieved via nanoparticles, gene guns, microneedle arrays, and in-situ electroporation. Ballistic delivery can also be used to administer nucleic acids. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles (such as gold particles).

[0297] In some embodiments, the polynucleotide described herein is an RNA molecule. For example, the RNA molecule may be mRNA or self-replicating RNA.

[0298] The RNA-encoding polynucleotides disclosed herein can be used to prepare vaccines. RNA cannot integrate into the genome and therefore has little carcinogenic potential; therefore, RNA can be used to manufacture vaccines. Furthermore, RNA only needs to enter the cytoplasm, unlike DNA which needs to enter the cell nucleus. The RNA molecules disclosed herein can be chemically modified and / or incorporated with modified nucleosides to overcome susceptibility to degradation. RNA vaccines may contain mRNA and / or self-replicating RNA (also known as RNA replicons). Delivery techniques for RNA vaccines may also include, for example, condensation with protamine and encapsulation in liposomes or nanoparticles.

[0299] Nucleic acids (DNA or RNA) can also be delivered after being complexed with cationic compounds (such as cationic lipids). Lipid-mediated gene delivery methods are described, for example, in WO 91 / 06309, WO 93 / 24640, WO 96 / 18372, and U.S. Patent No. 5,279,833 (which are incorporated herein by reference in their entirety).

[0300] The nucleic acid molecules described herein can be synthesized. One method is the phosphoramide method. Not wishing to be bound by theory, in this chemical method, phosphoramide (a nucleoside with a side-chain protecting group that maintains the integrity of the sugar, phosphodiester bond, and base during the chain extension step) is coupled to the 5' hydroxyl group of the nucleoside immobilized on a solid support column via its reactive 3' phosphorus group. The steps of oligonucleotide synthesis may include: (1) detriphenylmethylation, wherein the dimethoxytriphenylmethyl (DMT or triphenylmethyl) group on the 5' hydroxyl group of the supporting nucleoside is removed by treatment with trichloroacetic acid (TCA). (2) In the coupling step, the phosphoramide, made reactive by tetrazolium (a weak acid), is chemically coupled to the base finally added to the column support material. (3) In the capping step, any free 5' hydroxyl group of the unreacted column nucleotide is acetylated by treatment with acetic anhydride and N-methylimidazole. (4) In the oxidation step, the unstable internucleotide phosphate bonds between previously coupled bases and the recently added bases are oxidized to more stable phosphotriester bonds by treatment with iodine and water. After all bases in the oligonucleotide sequence are coupled, the complete nucleic acid chain can be cleaved from the column by treatment with ammonium hydroxide, and the base protecting groups are removed by heating in an ammonium hydroxide solution.

[0301] As a non-limiting example, the synthetic cycle may include growing a nucleotide chain from a starting protected nucleotide, the protected nucleotide being derivatized to a solid support via its terminal 3' hydroxyl group. Reagents and solvents may be pumped through the support to induce the sequential removal and addition of sugar protecting groups, thereby isolating the reactivity of specific chemical moieties on the monomer and allowing them to be added stepwise to the growing oligonucleotide chain. Assembly of the protected oligonucleotide chain may be performed in chemical steps (e.g., but not limited to: unblocking, activation / coupling, oxidation, and capping). Cleavage and deprotection then expose the single-stranded nucleic acid.

[0302] The nucleic acid synthesis methods disclosed herein may include, for example, oligonucleotide synthesis, column-based oligonucleotide synthesis, microarray-based oligonucleotide synthesis, gene synthesis from oligonucleotides, gene synthesis from an array-derived oligonucleotide pool, and any of various error correction and sequence verification steps, or any combination thereof.

[0303] The chemical synthesis of RNA can be similar to that used for DNA. In some embodiments, RNA chemical synthesis methods may include an additional protecting group at the 2' hydroxyl group of the ribose. The 2' hydroxyl group at the ribose position can be protected with tert-butyldimethylsilyl, which can be stable throughout the synthesis and can be removed in the final deprotection step by adding a basic fluoride ion such as tetrabutylammonium fluoride (TBAF). The remaining positions on the sugar and base can be protected in the same manner as in DNA. Stepwise coupling efficiencies of up to 99% can be obtained by adjusting several parameters in the DNA synthesis protocol, such as, but not limited to, coupling time, monomer delivery rate, frequency of washing steps, and type of blocking reagent.

[0304] Viral nucleic acid synthesis can be catalyzed by both viral and host enzymes, with their relative contributions determined by viral type and specific molecules. Except for retroviruses, viruses with RNA genomes utilize virus-encoded RNA-dependent RNA polymerases to synthesize mRNA and replicate their genome. Conversely, retroviruses use virion-encoded RNA-dependent DNA polymerases (reverse transcriptases) to synthesize double-stranded complementary DNA (cDNA) copies of their single-stranded RNA genome. In subsequent steps, the retroviral cDNA can be integrated into the host chromosome and transcribed by host-encoded DNA-dependent RNA polymerase II (pol II), producing viral information and genomic RNA. Besides poxviruses, DNA viruses also use host-encoded pol II to transcribe their information. Poxviruses, because they replicate in the cytoplasm and cannot access pol II, assemble novel transcriptases composed of multiple poxvirus-specific (and possibly one or more host-derived) subunits. Most DNA virus families (e.g., poxviruses) assemble novel transcriptases composed of multiple poxvirus-specific (and possibly one or more host-derived) subunits. Poxviridae ), Iridoviridae ( Iridoviridae Herpesviridae ( Herpesviridae ), Adenoviridae ( Adenoviridae The virus synthesizes DNA-dependent DNA polymerases encoded by the virus. However, two families (i.e., parvoviruses) Parvoviridae ) and the family Polycystic Papillomaviruses ( Papovaviridae )) utilizes the host's DNA polymerase, while hepatotropic DNA viruses ( Hepadnaviridae Virus DNA is replicated via RNA intermediates using a virus-encoded reverse transcriptase.

[0305] Due to the degeneracy of the genetic code, nucleic acid molecules with different nucleotide sequences can encode the same amino acid sequence. To enable expression in various hosts, polynucleotides can be codon-optimized.

[0306] On the other hand, this disclosure provides vectors comprising nucleic acid sequences according to a third aspect of this disclosure. In addition to nucleic acid sequences encoding only the peptides of this disclosure, the vectors may also comprise one or more additional nucleic acid sequences encoding one or more other peptides. Once expressed, such additional peptides may be fused to the N-terminus or C-terminus of the peptide of this disclosure. Examples of such additional peptides are described in detail in the preceding sections. In one embodiment, the vector comprises a nucleic acid sequence encoding a peptide or protein tag (e.g., a biotinylation site, a FLAG tag, a MYC tag, a HA tag, a GST tag, a Strep tag, or a polyhistidine tag).

[0307] The vectors used in the context of this disclosure ideally contain sequences suitable for introduction into cells. For example, the vector may be an expression vector, a vector in which the coding sequence of a polypeptide is under the control of its own cis-regulatory elements, a vector designed to promote gene integration or gene replacement in host cells, etc.

[0308] In the context of this disclosure, the term "vector" encompasses DNA molecules, such as plasmids, bacteriophages, phage particles, viruses, or other vectors, which contain one or more heterologous or recombinant nucleotide sequences (e.g., the aforementioned nucleic acid molecules of this disclosure under the control of a functional promoter and possibly an enhancer), and are capable of functioning as a vector in the sense understood by one of ordinary skill in the art.

[0309] For example, the following vectors are provided: bacteriophages, such as λ(X) phage and EMBL phage; bacterial vectors, such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a, pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5; eukaryotic vectors, such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG, and pSVL; and transposons, such as Sleeping Beauty transposons and PiggyBac transposons.

[0310] In some embodiments, the vector is a viral vector. Viral vectors can be derived from naturally occurring viral genomes and are typically modified to be non-replicating, for example, non-replicating. Non-replicating viruses require trans-donation of proteins for replication. Typically, these proteins are stably or transiently expressed in virus-producing cell lines, thereby allowing viral replication. Therefore, viral vectors are generally infectious and non-replicating. Viral vectors can be adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV types 5 and 2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindebes virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimera), herpesvirus vectors (e.g., vectors derived from cytomegaloviruses (such as rhesus macaque cytomegalovirus (RhCMV)), arenavirus vectors (e.g., lymphocytic choriomeningitis virus (LCMV) vectors), measlesvirus vectors, poxvirus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara strain (MVA), NYVAC (derived from vaccinia virus r Copenhagen strain), and fowlpoxvirus vectors (canarypox virus (ALVAC) and fowlpox virus (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retroviral vectors, lentiviral vectors, and simian virus 40. (SV40), bovine papillomavirus, Epstein-Barr virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, Rous sarcoma virus, poxvirus virus-like particles, baculovirus vectors and bacterial spores.

[0311] As further examples, adenovirus vectors can be derived from human adenovirus (Ad), adenoviruses infecting other species such as bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), canine adenovirus (e.g., CAdV2), porcine adenovirus (e.g., PAdV3 or 5), or from great apes such as chimpanzees (Pan), gorillas (Gorillas), orangutans (Pongo), bonobos (Pan paniscus), and common chimpanzees (Pan troglodytes). Poxvirus (Poxviridae) vectors can be derived from variola, vaccinia virus, cowpox virus, or monkeypox virus. Exemplary vaccinia viruses are Copenhagen vaccinia virus (W), New York attenuated vaccinia virus (NYVAC), ALVAC, TROVAC, and modified vaccinia virus Ankara strain (MVA).

[0312] Many expression systems are known in the art, including bacteria (e.g., Escherichia coli and Bacillus subtilis). Bacillus subtilis )), yeast (e.g., brewer's yeast ( Saccharomyces cerevisiae ), filamentous fungi (e.g., a certain species of Aspergillus genus) Aspergillus spec. ( ), plant cells, animal cells (e.g., mammalian cells), and insect cells.

[0313] In another aspect, this disclosure provides host cells comprising the vectors of this disclosure. The host cells may be prokaryotic or eukaryotic. In some cases, bacterial cells may be preferred prokaryotic host cells, typically strains of *Escherichia coli*, such as *E. coli* strains DH5 and RR1. Non-limiting examples of eukaryotic host cells include yeast, insect, and mammalian cells (e.g., derived from mouse, rat, monkey, or human cell lines). Non-limiting examples of yeast host cells include, for example, YPH499, YPH500, and YPH501. Non-limiting examples of mammalian host cells include Chinese hamster ovary (CHO) cells, NIH Swiss mouse embryonic cells NIH / 3T3, monkey kidney-derived COS-1 cells, and human embryonic kidney cells 293 cells. Examples of insect cells include Sf9 cells, which can be transfected with baculovirus expression vectors.

[0314] Transformation of suitable cell hosts using the DNA constructs of this disclosure is accomplished by known methods, which typically depend on the type of vector used. Successfully transformed cells, i.e., cells containing the DNA constructs of this disclosure, can be identified, for example, by PCR. Alternatively, the presence of proteins in the supernatant can be detected using antibodies.

[0315] It should be understood that certain host cells of this disclosure (e.g., bacterial, yeast, and insect cells) can be used to prepare the peptides or peptide-based molecules of this disclosure. However, other host cells may be useful in certain therapeutic applications. For example, antigen-presenting cells (APCs), such as dendritic cells or B cells, can be used to express the peptides of this disclosure, allowing the peptides to be loaded into suitable MHC molecules.

[0316] Another aspect of this disclosure provides a method for producing the peptides or peptide-based molecules of this disclosure, the method comprising culturing host cells and isolating the peptides or peptide-based molecules from the host cells or their culture medium.

[0317] Peptide and pMHC binding site

[0318] The peptides, pMHC complexes, or other peptide-based molecules disclosed herein (such as complexes, fusion proteins, or conjugates containing the peptides disclosed herein) can be used to identify and / or isolate binding moieties that specifically bind to the peptides, pMHC complexes, or other peptide-based molecules disclosed herein. Such binding moieties can be used as immunotherapeutic agents and may include, for example, antibodies (or antigen-binding fragments thereof), alternative scaffolds, TCRs, and CARs.

[0319] On one hand, this disclosure provides a peptide-binding moiety that binds to the peptide of this disclosure. Preferably, the peptide-binding moiety binds the peptide when the peptide is complexed with MHC. In the latter case, the peptide-binding moiety may partially bind MHC, provided that the peptide-binding moiety also binds the peptide. The peptide-binding moiety may bind only the peptide, and this binding may be specific. The peptide-binding moiety may bind only the pMHC complex, and this binding may be specific.

[0320] This disclosure also provides a method for identifying peptide-binding moieties that bind to the pMHC complex of this disclosure, the method comprising contacting a candidate peptide-binding moieties with the pMHC complex and determining whether the candidate peptide-binding moieties bind to the complex. Methods for determining binding to the pMHC complex include, for example, surface plasmon resonance or any other biosensor technique, ELISA, flow cytometry, chromatography, microscopy. Optionally, or additionally, binding may be determined by a functional assay, wherein a biological response, such as cytokine release or apoptosis, is detected after binding.

[0321] The candidate peptide-binding moiety can be a peptide-binding moiety of the type already described, such as an antibody or a TCR.

[0322] For example, antibodies and TCRs can be obtained from display libraries, where the pMHC complex disclosed herein is used for library panning. TCRs can be displayed on surfaces such as phage particles and yeast particles, and such libraries have been used to isolate high-affinity variants of TCRs derived from T cell clones. TCR phage libraries can be used to isolate TCRs with novel antigen specificity. Such libraries can be constructed using α- and β-chain sequences corresponding to those found in naturally occurring libraries. However, random combinations of these α- and β-chain sequences during library creation can produce TCR libraries that may not be naturally occurring.

[0323] In some embodiments, the pMHC complex of this disclosure can be used to screen libraries of different TCRs displayed on the surface of phage particles. The TCRs displayed in the libraries may not correspond to those contained in the natural library; for example, they may contain α- and β-chain pairings not present in vivo, and / or the TCRs may contain non-natural mutations and / or the TCRs may be in a soluble form. Screening may include panning the phage library with the pMHC complex of this disclosure, followed by isolation of the bound phage particles. For this purpose, the pMHC complex may be attached to a solid support (such as magnetic beads or a column matrix), and then the phage-bound pMHC complex may be isolated using a magnet or chromatography, respectively. The panning step may be repeated several times. The isolated phages may be further amplified in *E. coli* cells. The specific binding of the isolated phage particles to the pMHC complex of this disclosure can be tested. Binding may be detected using techniques including, but not limited to, ELISA or SPR (e.g., using a BiaCore instrument). The DNA sequence of the T-cell receptor displayed by the pMHC-bound phage may also be further identified by PCR methods.

[0324] Alternatively, antigen-binding T cells and TCRs can be isolated from fresh blood obtained from a patient or healthy donor. This method involves using autologous dendritic cells (DC-stimulated T cells), followed by stimulation with autologous B cells, and then pulsed with the peptides disclosed herein. Several rounds (e.g., three or four rounds) of stimulation can be performed. The specificity of activated T cells can then be tested by measuring cytokine release in the presence of T2 cells pulsed with the peptides disclosed herein (e.g., using an IFNγ ELISpot assay). Activated cells can then be sorted by fluorescence-activated cell sorting (FACS) using labeled antibodies that detect the expression of intracellular cytokines (e.g., IFNγ) or cell surface markers (such as CD137). The sorted cells can be amplified and further validated, for example by an ELISpot assay and / or by cytotoxicity against target cells and / or by peptide-MHC tetramer staining. The TCR strand from a validated clone can then be amplified by rapid amplification of cDNA ends (RACE) and sequenced.

[0325] The peptide-binding moiety disclosed herein may include, for example, but not limited to, antibodies, TCRs, or CARs.

[0326] In some embodiments, the peptide-binding portion of this disclosure may be an antibody or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof encompass antibodies, derivatives of humanized antibodies, functional equivalents, and homologs, including any polypeptide containing an immunoglobulin-binding domain, whether natural or wholly or partially synthetic, and any polypeptide or protein having a binding domain that is an antibody-binding domain or homologous to an antibody-binding domain. Therefore, chimeric molecules comprising an immunoglobulin-binding domain or equivalent fused to another polypeptide are included. Humanized antibodies may be modified antibodies having a variable region of a non-human (e.g., mouse) antibody and a constant region of a human antibody. Examples of antibodies are immunoglobulin isotypes (e.g.,...

[0327] Antibodies can be polyclonal or monoclonal. Antibodies can be IgG, IgE, IgM, IgD, and IgA, and their isotype subclasses; or fragments containing antigen-binding domains, such as Fab, scFv, Fv, dAb, Fd; and biclonal antibodies. Monoclonal antibodies may be referred to as "mAb" in this document.

[0328] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. A bispecific antibody may include a second targeting portion that targets desired cells or tissues or another desired antigen associated with the same or similar disease or condition.

[0329] Antibodies, such as monoclonal antibodies, can be used, and recombinant DNA technology can be employed to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may include introducing DNA encoding the variable region or complementarity-determining region (CDR) of an immunoglobulin into the constant region, or the constant region plus the framework region, of a different immunoglobulin. Hybridomas (or other antibody-producing cells) may undergo genetic mutations or other changes that may or may not alter the binding specificity of the produced antibody.

[0330] It has been shown that fragments of complete antibodies can perform the function of binding antigens. Examples of binding fragments are (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of the VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two linked Fab fragments; (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site; (viii) bispecific single-chain Fv dimers; (ix) “diabody”, a multivalent or multispecific fragment constructed by gene fusion; and (x) VHH or VNAR antibodies, also known as single-domain antibodies or nanobodies (Nb), which may be derived from heavy-chain antibodies of, for example, dromedary camels, alpacas, llamas, or sharks.

[0331] Biantibodies are multimers of polypeptides, each containing a first domain with a binding region of the immunoglobulin light chain and a second domain with a binding region of the immunoglobulin heavy chain. These two domains are linked (e.g., via peptide linkers) but cannot associate with each other to form an antigen-binding site: the antigen-binding site is formed by association between the first domain of one polypeptide in the multimer and the second domain of another polypeptide in the multimer (WO94 / 13804). When using bispecific antibodies, these antibodies can be conventional bispecific antibodies, which can be prepared in a variety of ways, such as chemical preparation or hybridoma preparation, or can be any bispecific antibody fragment mentioned above. Using scFv dimers or biantibodies may be preferred over using intact antibodies. Biantibodies and scFvs can be constructed using only the variable region, without the Fc region, which may reduce the impact of anti-idiotype reactions. Other forms of bispecific antibodies include single-chain "Janusins". In contrast to intact bispecific antibodies, bispecific biantibodies can also be useful because they are easy to construct and express in E. coli. Phage display can be used to easily select biantibodies (and many other peptides such as antibody fragments) with appropriate binding specificity from a library. If one arm of the biantibody remains constant, for example, having specificity against a target antigen, then the library can be constructed with the other arm varying, and antibodies with appropriate specificity can be selected. An "antigen-binding domain" is a part of an antibody that contains a region that specifically binds to and is complementary to a portion of the antigen. When the antigen is large, the antibody may bind only to a specific portion of the antigen, called an epitope. Antigen-binding domains can be provided by one or more antibody variable domains. Antigen-binding domains may contain antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH).

[0332] In some embodiments, the peptide-binding moiety may be an antibody-like molecule programmed to specifically bind to the peptide or peptide-MHC complex disclosed herein. In some embodiments, the peptide-binding moiety may comprise a TCR mimic antibody. In some embodiments, such a TCR mimic antibody may comprise a high-affinity soluble antibody molecule conferred with TCR-like specificity against tumor or viral epitopes, which can target tumor and / or virus-infected cells and mediate their specific killing.

[0333] This disclosure also covers binding portions based on engineered protein scaffolds or “alternative scaffolds.” Alternative scaffolds are derived from stable, soluble, native protein structures that have been modified to provide binding sites for target molecules. Examples of alternative scaffolds include, but are not limited to, affinities based on the Z domain of staphylococcal protein A, which provides a binding interface on its two α-helices; anti-carrier proteins derived from lipid carriers, incorporating small ligand binding sites at the open end of a β-barrel fold; monobodies designed to incorporate the fibronectin type III domain (FN3) of fibronectin or tenosynovin as a protein scaffold or synthetic Fn3 domains (e.g., tencon); nanobodies; and DARPins. Other alternative scaffolds include Adnectin™, iMab, EETI-II / AGRP, Kunitz domains, thioredoxin aptamers, Affilin, Tetranectin, Fynomer, and Avimer. Alternative scaffolds typically target and bind to the same antigen protein as antibodies and are potential therapeutic agents. They can function as inhibitors or antagonists, or as delivery vehicles to target molecules (such as toxins) to specific tissues in the body. 。 Short peptides can also be used to bind target proteins. Phylomers are naturally occurring structural peptides derived from bacterial genomes. These peptides represent a range of different protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo.

[0334] Alternative scaffolds are typically single-chain polypeptide frameworks containing a highly structured core associated with variable domains exhibiting high conformational tolerance, allowing for insertions, deletions, or other substitutions within the variable domains. Libraries can be generated using known protocols, introducing diversity into one or more variable domains and, in some cases, into the structured core. The resulting libraries can then be screened for binding to the peptides and / or pMHC complexes disclosed herein, and the specificity of the identified conjugates can be further characterized using known methods. Alternative scaffolds can be derived from protein A, particularly its Z-domain (affinity), ImmE7 (immunoprotein), BPTI / APPI (Kunitz domain), CTLA-4, charybdotoxin (scorpion venom), Min-23 (knottins), lipid carriers (anticarriers), Ras-binding protein AF-6 (PDZ domain), neokarzinostatin, fibronectin domains, ankyrin concordant repeat domains, or thioredoxin.

[0335] In some embodiments, the antibodies or alternative scaffolds described herein can be immobilized on viral vectors. Such modified recombinant viral vectors can be used to target the delivery of genetic material encoded by the viral vector into cells and / or tissues. Various methods can be used to transfer the antibodies or alternative scaffolds onto the viral vectors, for example, by using affinity binding pairs (such as c-Myc / anti-Myc antibodies, streptavidin / biotin) or via a spy-tag / spy-catcher system. Exemplary vectors that can be modified with the antibodies or alternative scaffolds described herein include, but are not limited to, adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV6, AAV9, or AAV9.PHP), retroviral vectors, lentiviral vectors, and targeted oncolytic viruses (e.g., herpes simplex virus (HSV)).

[0336] In some implementations, the peptide-binding moiety may be a TCR. TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and the IMGT TCR sequence public database.

[0337] The TCR disclosed herein can be in any form. For example, the TCR can be an αβ heterodimer, or an αα or ββ homodimer.

[0338] α / β heterodimer TCRs have α and β chains. Broadly speaking, each chain contains a variable region, a linker region, and a constant region. The β chain typically also contains a short, diverse region between the variable and linker regions, but this diverse region is usually considered part of the linker region. Each variable region contains three hypervariable CDRs (complementarity-determining regions) embedded in the frame sequence; CDR3 is considered the primary mediator of antigen recognition. Several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions exist, distinguished by their frame regions, CDR1 and CDR2 sequences, and partially defined CDR3 sequences.

[0339] The TCRs disclosed herein may not correspond to TCRs as they exist in nature. For example, they may contain α-chain and β-chain combinations not present in the natural library. Optionally or additionally, the TCRs described herein may be soluble, and / or the α-chain constant region and / or β-chain constant region may be truncated relative to the natural / natured TRAC / TRBC sequence, such that, for example, the C-terminal transmembrane domain and intracellular region are absent. This truncation may result in the removal of cysteine ​​residues that form natural interchain disulfide bonds from the TRAC / TRBC.

[0340] Furthermore, the TRAC / TRBC domain may contain modifications. For example, the α-chain extracellular sequence may include modifications relative to the native / naturally occurring TRAC, whereby, referring to the IMGT number, amino acid T48 of TRAC is replaced by C48. Similarly, the β-chain extracellular sequence may include modifications relative to the native / naturally occurring TRBC1 or TRBC2, where S57 (referring to the IMGT number) of TRBC1 or TRBC2 is replaced by C57. These cysteine ​​substitutions relative to the native α- and β-chain extracellular sequences enable the formation of non-natural interchain disulfide bonds that stabilize refolded soluble TCRs, i.e., TCRs formed by refolding the extracellular α- and β-chains. Such non-natural disulfide bonds facilitate the display of correctly folded TCRs on bacteriophages. Furthermore, the use of soluble TCRs linked by stable disulfide bonds makes it easier to assess binding affinity and binding half-life. Alternative sites for forming non-natural disulfides include, for example, TRAC Thr 45 and TRBC1 of exon 1 of 01 01 or TRBC2 Ser77 of exon 1 of 01; TRAC Tyr 10 and TRBC1 in exon 1 of 01 01 or TRBC2 Ser 17 of exon 1 of 01; TRAC Thr 45 and TRBC1 of exon 1 of 01 01 or TRBC2 Asp 59 of exon 1 of 01; and TRAC Ser15 and TRBC1 of exon 1 of 01 01 or TRBC2 Glu 15 of exon 1 of 01. TCRs with non-natural disulfide bonds can be full-length or truncated.

[0341] The TCR disclosed herein may be in single-chain form. Single-chain TCRs include the following types of αβ TCR polypeptides: optionally Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ, wherein Vα and Vβ are the α and β variable regions of the TCR, respectively, Cα and Cβ are the α and β constant regions of the TCR, respectively, and L is a linker sequence. Single-chain TCRs may contain non-natural disulfide bonds. TCRs may be in a soluble form (i.e., without transmembrane or cytoplasmic domains) or may comprise full-length α and β chains. TCRs may be provided on the surface of cells such as T cells.

[0342] The TCR disclosed herein can be engineered to include mutations. Methods for generating high-affinity TCR variants with mutations include phage display and site-directed mutagenesis. Preferably, affinity-enhancing mutations are generated within the variable regions of the α and / or β chains. More preferably, affinity-enhancing mutations are generated within the CDR. The α and / or β chain variable regions may contain 1 to 15 mutations.

[0343] The TCRs disclosed herein can also be labeled with imaging compounds (e.g., markers suitable for diagnostic purposes). Such labeled high-affinity TCRs can be used in methods for detecting TCR ligands selected from CD1-antigen complexes, bacterial superantigens, and MHC-peptide / superantigen complexes, the methods comprising contacting the TCR ligand with a high-affinity TCR (or a multimeric high-affinity TCR complex) specific to the TCR ligand; and detecting binding to the TCR ligand. In multimeric high-affinity TCR complexes (e.g., formed using biotinylated heterodimers), fluorescent streptavidin can be used to provide a detectable marker. Fluorescently labeled multimers are suitable for FACS analysis, for example, to detect antigen-presenting cells carrying peptides specifically targeted by the high-affinity TCR.

[0344] The TCR (or its multivalent complex) disclosed herein may alternatively or additionally be conjugated (e.g., covalently linked or otherwise linked) to a therapeutic agent, which may be, for example, a toxic fraction for cell killing, or an immunostimulant (such as an interleukin or cytokine). The multivalent high-affinity TCR complexes of this disclosure may have enhanced TCR ligand binding capacity compared to non-multimeric wild-type or high-affinity T-cell receptor heterodimers. Therefore, the multivalent high-affinity TCR complexes according to this disclosure are particularly suitable for tracking or targeting cells presenting specific antigens in vitro or in vivo, and are also suitable as intermediates for the production of other multivalent high-affinity TCR complexes with such uses. Thus, high-affinity TCRs or multivalent high-affinity TCR complexes can be provided in pharmaceutically acceptable formulations for in vivo application.

[0345] The high-affinity TCR disclosed herein can be used to produce soluble bispecific reagents. A preferred embodiment is a reagent comprising a soluble TCR fused to an anti-CD3 specific antibody fragment via a linker.

[0346] On the other hand, this disclosure provides nucleic acids encoding the TCR of this disclosure, TCR expression vectors containing nucleic acids encoding the TCR of this disclosure, and cells carrying such vectors. The TCR can be encoded in a single open reading frame or in two different open reading frames. The scope of this disclosure also includes cells containing a first expression vector and a second expression vector, the first expression vector containing nucleic acid encoding the α-chain of the TCR of this disclosure, and the second expression vector containing nucleic acid encoding the β-chain of the TCR of this disclosure. Alternatively, a vector can simultaneously encode both the α-chain and β-chain of the TCR of this disclosure.

[0347] Another aspect of this disclosure provides cells displaying the TCR of this disclosure on their surface. These cells may be T cells or other immune cells. T cells may be modified so that they do not correspond to naturally occurring T cells. For example, cells may be transfected with a vector encoding the TCR of this disclosure, so that the T cells express additional TCRs besides the natural TCR. Alternatively or additionally, T cells may be modified so that they cannot present the natural TCR. Numerous methods are suitable for transfecting T cells with DNA or RNA encoding the TCR of this disclosure. As a non-limiting example, transfection methods may include rapid RNA-based transfection systems. T cells expressing the TCR of this disclosure are suitable for adoptive therapy-based treatment of diseases such as cancer. Numerous suitable methods exist for performing adoptive therapy. For example, adoptive cell therapy (ACT) may include the use of autologous tumor-infiltrating lymphocytes and may include a lymphocyte clearance pretreatment protocol prior to ACT. In some embodiments, viruses encoding the TCR, such as retroviruses, may be used for the genetic modification of lymphocytes to convert normal lymphocytes into lymphocytes with anti-cancer activity. Adoptive transfer of lymphocytes with anti-cancer activity into patients requiring treatment, such as metastatic melanoma, can mediate tumor regression. In some implementations, ACT may include treatment of cancer patients expressing viruses or alloantigens, treatment of cancer patients expressing viral antigens, and / or ACT using genetically modified lymphocytes. In some embodiments, the ACT method may include, for example, genetically modifying lymphocytes to introduce new recognition specificity using, for example, one or more αβTCRs and / or one or more chimeric TCRs; genetically modifying lymphocytes to alter T cell function using, for example, co-stimulatory molecules (e.g., CD28, 41BB), cytokines (e.g., IL2, IL15), homing molecules (e.g., CD62L, CCR7), and / or apoptosis-preventing molecules (BCL2); modifying host lymphocyte clearance using, for example, selectively clearing CD4+ cells or regulatory T cells; blocking inhibitory signals on reactive lymphocytes using, for example, antibodies against CTLA4 and / or PD-1; stimulating metastatic cells with a vaccine using, for example, a recombinant virus encoding one or more antigens; administering alternative cytokines to support cell growth using, for example, IL15 and / or IL21; stimulating APCs using, for example, a Toll-like receptor agonist; using, for example, alternative culture conditions and in vitro growth to promote the production of poorly differentiated lymphocytes using cytokines; and using, for example, natural killer cells to overcome antigen escape variants.

[0348] When expressed on transfected T cells, the TCRs of this disclosure used for adoptive therapy are typically glycosylated. The glycosylation pattern of the transfected TCRs can be modified by mutations in the transfected gene.

[0349] In some embodiments, the peptide-binding portion may be a chimeric antigen receptor (CAR). A CAR is a genetically engineered receptor. CARs that bind to the peptides or pMHC complexes disclosed herein can be generated by incorporating the antigen-binding domain of a specific binding peptide or pMHC complex into the extracellular domain of the CAR. The CAR can be introduced into and expressed by immune cells (such as T cells, NK cells, or macrophages). The CAR can be programmed to recognize specific antigens and, upon binding to those antigens, activate immune cells to attack and destroy the cells presenting those antigens. When these antigens are present on tumor cells, CAR-expressing immune cells can target and kill the tumor cells.

[0350] The general structure of a CAR typically includes an extracellular domain for binding antigens (e.g., peptides or pMHC complexes disclosed herein), a hinge, a transmembrane domain, and an intracellular domain comprising a signal transduction domain and optionally one or more co-stimulatory domains.

[0351] The extracellular domain of a CAR may contain any polypeptide that specifically binds to a desired antigen (e.g., a peptide or pMHC complex disclosed herein). For example, the extracellular domain may contain an antibody fragment, such as scFv or VHH. A CAR may also be engineered to bind two or more desired antigens, which may be tandemly arranged and separated by a linker sequence. For example, one or more domain antibodies, scFv, alpaca VHH antibodies, or other VH-only antibody fragments may be tandemly organized via a linker to provide bispecificity or multispecificity for the CAR.

[0352] The hinge domain may be located between the extracellular domain and the transmembrane domain of the CAR, for example, to provide flexibility that allows the extracellular domain to bind efficiently to its intended target. The hinge region may be a polypeptide of about 2 to 100 amino acids in length. The hinge may contain or consist of flexible residues (such as Gly and Ser) that allow adjacent protein domains to move freely relative to each other. Longer hinges may be used when it is desirable to ensure that two adjacent domains do not spatially interfere with each other. The hinge may be derived from the hinge region or a portion thereof of any immunoglobulin. Non-limiting examples of linkers include a portion of the human CD8α chain, the extracellular domain of CD28, an Ig hinge from IgG, IgM, IgA, IgD, or IgE, the FcyRllla receptor, or a functional fragment thereof.

[0353] The transmembrane domain of a CAR can be derived from transmembrane proteins, such as the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD2, CD4, CD5, CD8, CD9, CD16, CD18, CD19, CD22, CD27, CD29, CD33, CD37, CD40, CD45, CD49a, CD64, CD80, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134, CD154, CD160 (BY55), KIRDS2, OX40, LFA-1 (CD11a, CD18), CD11b, CD11c, CD11d, ICOS (CD278), 4-1 BB (CD137), 4-1 BBL, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, ITGB2, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp30, NKp44, NKp46, NKG2D and NKG2C or their functional fragments.

[0354] The intracellular signaling domains of CARs are involved in transducing signals for effective CAR binding to target antigens into the interior of immune effector cells to trigger effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the release of cytotoxic factors into CAR-bound target cells, or other cellular responses triggered after antigen binding to the extracellular CAR domain. Non-restricted examples of intracellular signaling domains of CARs include those derived from CD3ζ, CD3... Those intracellular signal transduction domains of CD3δ, CD3γ, CD5, CD22, CD39, CD79A, CD79B, CD66d, CD226, DAP10, DAP12, Fcε receptor Iγ chain (FCER1G), or FcRβ.

[0355] The intracellular co-stimulatory domains of CARs can provide a second signal required for the efficient activation and function of T lymphocytes after binding to antigens. Such co-stimulatory domains can be derived from one or more co-stimulatory molecules, such as, but not limited to, 4-1BB, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, BTLA, GITR, CD226, HVEM, and ZAP70.

[0356] CARs can be generated using standard molecular biology techniques. The extracellular domain that binds to the desired antigen can be derived from the antibody or its antigen-binding fragment described herein.

[0357] On the other hand, this disclosure also provides cells comprising the peptide-binding portions of this disclosure (e.g., TCRs and CARs). In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell, an NK cell, or a macrophage. For the subject receiving the host cell (as a treatment), it can be autologous or allogeneic.

[0358] In some embodiments, the TCR of this disclosure is provided as TCR-T cells. In some embodiments, the CAR of this disclosure is provided as CAR-T cells. Any method known in the art for modifying T cells to express TCR or CAR can be used to generate the TCR-T or CAR-T cells of this disclosure.

[0359] Cells expressing the peptide-binding moieties of this disclosure (e.g., TCRs and CARs) may also contain one or more additional genes. These additional genes can be used to enhance effector function in cells expressing peptide-binding moieties (e.g., TCRs and CARs). Non-limiting examples of additional gene classes include (a) second targeting moieties, such as antibodies, including fragments thereof, and bispecific antibodies (e.g., bispecific T-cell connectives (BiTEs)); (b) secretory cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18); (c) membrane-bound cytokines (e.g., IL-15); (d) chimeric cytokine receptors (e.g., IL-2 / IL-7, IL-4 / IL-7); and (e) constitutively active cytokine receptors (e.g., C7R, TGFRII DNR), dominant-negative receptors (DNRs; e.g., TGFβRII). (g) ligands of co-stimulatory molecules (e.g., CD80, 4-1BBL), (h) nuclear factors (NFAT) of activated T cells (e.g., NFATC1, NFATC2, NFATC3, NFATC4, and NFAT5), or (j) suicide genes (e.g., CD20, truncated EGFR or HER2, inducible cysteine ​​9 molecules). In some embodiments, cells expressing the peptide-binding moieties of this disclosure (e.g., TCR and CAR) may express a second targeting moieties that target specific tissues and / or cell types or known cancer antigens.

[0360] Pharmaceutical compositions, dosage forms and administration

[0361] On the other hand, this disclosure provides pharmaceutical compositions comprising peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates containing the peptide), nucleic acid molecules, carriers, cells, or peptide-binding moieties of this disclosure, and pharmaceutically acceptable carriers and / or excipients. The pharmaceutical compositions of this disclosure may be in any suitable form (depending on the desired method of administration to a patient). Suitable compositions and methods of administration are known to those skilled in the art, for example, see Johnson et al., Blood. 2009; 114(3):535-46.

[0362] Pharmaceutical compositions may comprise the disclosed peptide or peptide-based molecules in free form or in pharmaceutically acceptable salt form. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the disclosed peptide, wherein the peptide is modified by forming an acidic or basic salt of the drug. For example, an acidic salt is prepared by reacting a free base (typically in which the neutral form of the drug has a neutral -NH2 group) with a suitable acid. Suitable acids for preparing acidic salts include organic acids (e.g., acetic acid, benzoic acid, citric acid, propionic acid, glycolic acid, trifluoroacetic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, maleic acid, succinic acid, fumaric acid, tartaric acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.) and inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.). Conversely, the preparation of basic salts of the acidic portions that may be present on peptides is done using pharmaceutically acceptable bases (such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc.).

[0363] The compositions disclosed herein may comprise a variety of peptides, for example, 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides as described herein (e.g., SEQ ID NO: 1-89 and 143-146). In some embodiments, the compositions disclosed herein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ... The sequence of 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, or 93 amino acids, or a pharmaceutically acceptable salt thereof, or a fragment thereof or a derivative thereof.

[0364] In some embodiments, the peptide or peptide-based molecule can be from about 1 μg / mL to 50 mg / mL, for example, about 0.1 mg / mL to 10 mg / mL, about 0.2 mg / mL to 5 mg / mL, about 0.5 mg / mL to 8 mg / mL, about 0.8 mg / mL to 12 mg / mL, about 1 mg / mL to 15 mg / mL, about 2 mg / mL to 20 mg / mL, or about 5 mg / mL to 25 mg / mL, or about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, 2 mg / mL, 2.25 mg / mL, 2.5 mg / mL, 2.75 mg / mL, 3 mg / mL, 3.25 mg / mL. Concentrations of 3.5 mg / mL, 3.75 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, or 20 mg / mL are present in solution.

[0365] The pharmaceutical composition may be suitable for administration via any suitable route (e.g., parenteral (including subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation or intranasal route).

[0366] Such compositions can be prepared by any method known in the pharmaceutical field, for example by mixing the active ingredient with one or more carriers or excipients under sterile conditions.

[0367] In addition, this document discloses pharmaceutical formulations comprising peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes containing one or more peptides), fusion proteins or conjugates), nucleic acid molecules, carriers, cells or binding portions of the present disclosure.

[0368] Pharmaceutical compositions based on the peptides, peptide-based molecules (e.g., complexes (e.g., peptide-MHC (pMHC) complexes containing one or more peptides), fusion proteins, or conjugates), nucleic acid molecules, carriers, cells, or binding moieties disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. Peptides, peptide-based molecules (e.g., complexes containing one or more peptides (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates), nucleic acid molecules, carriers, cells, or binding moieties can be formulated for administration by, for example, injection, inhalation, or isolation (oral or nasal), or by oral, oral, parenteral, or rectal administration, or by direct administration to an organ or tissue.

[0369] Pharmaceutical compositions can be formulated for various routes of administration, including systemic, local, or regional administration. Techniques and formulations are available, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injection. For injection purposes, pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologically compatible buffers (such as Hank's solution or Ringer's solution). Furthermore, pharmaceutical compositions can be formulated in solid form and reconstituted or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0370] In some embodiments, the pharmaceutical compositions of this disclosure may be lyophilized. As a non-limiting example, the obtained lyophilized material can be reconstituted into an aqueous composition by adding an aqueous solvent. In some embodiments, the aqueous composition may be able to be administered directly to a patient parenterally. Therefore, another embodiment of this disclosure is an aqueous pharmaceutical composition that can be obtained by reconstituted a lyophilized material with an aqueous solvent.

[0371] In some embodiments, the pharmaceutical compositions disclosed herein may comprise a lyophilized formulation. As a non-limiting example, the lyophilized formulation may comprise the peptides, mannitol, and / or TWEEN 80® disclosed herein. As another non-limiting example, the lyophilized formulation may comprise the peptides, mannitol, and poloxamer 188 disclosed herein. In some embodiments, the pharmaceutical composition may comprise a lyophilized formulation containing a reconstituted liquid composition.

[0372] In some embodiments, the pharmaceutical compositions of this disclosure can provide formulations with improved solubility and / or wettability compared to lyophilized compositions previously known. As a non-limiting example, suitable excipient compositions can be used to improve the solubility and / or wettability of the lyophilized material. Thus, the pharmaceutical compositions of this disclosure (comprising peptides of SEQ ID NO: 1-89 and 143-146, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof) can be developed to exhibit desired shelf stability at (e.g., at -20°C, +5°C, or +25°C) and be readily redissolved, such that the lyophilized material can be completely dissolved within seconds to two minutes or longer using a buffer or other excipient, with or without the use of an ultrasonic homogenizer. Furthermore, the composition can be readily administered to patients in need of treatment via any suitable delivery route disclosed herein, such as parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes. As a non-limiting example, the resulting solution may have a pH between pH 2.7 and pH 9.

[0373] For oral administration, pharmaceutical compositions may be in the form of tablets or capsules, for example, prepared by conventional methods using pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or humectants (e.g., sodium lauryl sulfate). Tablets may also be coated using methods well known in the art. Liquid formulations for oral administration may be in the form of, for example, solutions, syrups, or suspensions, or they may be provided as an anhydrous product for reconstitution with water or other suitable carriers prior to use. Such liquid formulations can be prepared using conventional methods with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or gum arabic), non-aqueous carriers (e.g., almond oil, oily esters, ethanol, or fractionated vegetable oils), and preservatives (e.g., methylparaben, propylparaben, or sorbic acid). The formulation may also contain buffer salts, flavoring agents, coloring agents, and sweeteners, as appropriate.

[0374] Pharmaceutical compositions can be formulated for parenteral administration by injection (e.g., by rapid concentration or continuous infusion). Injectable formulations can be provided in unit dosage forms (e.g., in ampoules or multi-dose containers, optionally with preservatives). Pharmaceutical compositions can also be formulated as suspensions, solutions, or emulsions in oily or aqueous media and may contain other agents, including suspending agents, stabilizers, and / or dispersants.

[0375] In addition, pharmaceutical compositions can also be formulated as reservoir formulations. These long-acting formulations can be administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, compounds can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or formulated as slightly soluble derivatives, such as slightly soluble salts. Other suitable delivery systems include microspheres, which offer the possibility of local, non-invasive drug delivery over an extended period of time. This technique may include microspheres with pre-capillary dimensions, which can be injected via a coronary catheter into any selected site of an organ without causing inflammation or local ischemia. The administered therapeutic agent is then slowly released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0376] Systemic application can also be performed transmucosal or transdermal. For transmucosal or transdermal application, a penetrant suitable for the permeability barrier is used in the formulation. Such penetrants are generally known in the art and, for example, for transmucosal application, include bile salts and fusidic acid derivatives. Additionally, detergents can be used to enhance penetration. Transmucosal application can be performed using nasal sprays or suppositories. For topical application, the carrier particles described herein can be formulated into ointments, creams, gels, or lotions generally known in the art. Lotions can also be used topically to treat injuries or inflammation to accelerate healing.

[0377] Suitable injectable drug forms may include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be a fluid. It must be stable under manufacturing conditions and certain storage parameters (such as refrigeration and freezing), and must be protected against contamination by microorganisms such as bacteria and fungi.

[0378] If the formulations disclosed herein are used as therapeutic agents to enhance the immune response of a subject, the therapeutic agents may be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino group of a protein) formed from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with a free carboxyl group may also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0379] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Suitable flowability can be maintained, for example, by using coatings (such as lecithin), maintaining the desired particle size in the case of dispersions, and using surfactants. Microbial action can be prevented by a variety of antibacterial and antifungal agents known in the art. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Extended absorption of the injectable composition can be achieved by using agents with delayed absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0380] Sterile injection solutions can be prepared by mixing the required amount of the active compound or construct with various other ingredients listed above (if needed) in a suitable solvent, followed by filtration and sterilization.

[0381] After formulation, the solvent can be prepared in a manner compatible with the dosage form and to a therapeutically effective amount. The formulation is readily applicable in various dosage forms, such as injectable solutions of the type described above, but sustained-release capsules, microparticles, and microspheres may also be used.

[0382] For example, for parenteral administration in aqueous solutions, the solution should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. In this context, those skilled in the art will recognize the sterile aqueous media that can be used according to this disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous infusion solution, or injected into the intended infusion site.

[0383] In any case, the person responsible for administration will determine the appropriate dose for the individual subject. For example, the peptides, peptide-based molecules (such as complexes containing one or more peptides (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates), nucleic acid molecules, carriers, cells, or binding moieties described herein may be administered to the subject on a daily or weekly basis, or on a monthly, semi-annual, or annual basis, depending on need, exposure to a pathogenic organism (e.g., HTLV-1), or the subject's condition (e.g., cancer).

[0384] In addition to formulations intended for parenteral administration (such as intravenous, intratumoral, intradermal, or intramuscular injection), other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomes; sustained-release capsules; biodegradable forms; and any other forms currently in use.

[0385] Nasal solutions or sprays, aerosols, or inhalers can also be used. Nasal solutions can be aqueous solutions designed to be applied to the nasal cavity in the form of drops or sprays. Nasal solutions can be prepared such that they resemble nasal secretions in many respects. Therefore, aqueous nasal solutions are generally isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. Furthermore, if desired, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers may be included in the formulation. Various commercial nasal preparations are known and may include, for example, antibiotics and antihistamines, and are used for asthma prevention.

[0386] Oral formulations may include excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are available in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In some specific embodiments, the oral pharmaceutical compositions will contain inert diluents or assimilateable edible carriers, or they may be encapsulated in hard or soft-shell gelatin capsules, or they may be compressed into tablets, or they may be mixed directly with food in the diet. For oral therapeutic administration, the active compound may be mixed with excipients and used in the form of ingestible tablets, lozenges, tablets, capsules, elixirs, suspensions, syrups, wafers, etc.

[0387] Tablets, lozenges, pills, capsules, etc., may also contain the following substances: binders, such as gum arabic, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin, or flavorings, such as peppermint, wintergreen oil, or cherry flavorings. When the dosage unit is in capsule form, it may also contain a liquid carrier in addition to the substances of the above types. Various other materials may be present as coatings or otherwise alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Elixir syrups may contain the active compound sucrose (as a sweetener), methylparaben and propylparaben (as preservatives), dyes, and flavorings (such as cherry or orange flavorings).

[0388] Other embodiments disclosed herein may relate to kits for use with the methods and compositions. Kits may also include suitable containers, such as vials, tubes, micro or microcentrifuge tubes, test tubes, flasks, bottles, syringes, or other containers. If additional components or reagents are provided, the kit may include one or more additional containers into which such reagents or components can be placed. Kits described herein typically also include means for containing peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes containing one or more peptides), fusion proteins, or conjugates), nucleic acid molecules, carriers, cells, or binding moieties, and any other sealed reagent containers for commercial sale. Such containers may include injectable or blow-molded plastic containers that retain the desired vial within. Optionally, the compositions may require one or more additional active agents.

[0389] Dosage ranges and administration frequencies can vary depending on the nature of the composition and the medical condition, as well as the specific patient's parameters and the route of administration. The dosage also depends on the subject receiving the medication. For example, a lower dosage may be required if the subject is an adolescent, and a higher dosage may be required if the subject is an adult. In some embodiments, a more precise dosage may depend on the subject's weight. Suitable, non-limiting examples of dosages of pharmaceutical compositions containing the active agents disclosed herein may vary depending on the age and body size of the subject to be administered, the target disease, the therapeutic purpose, the condition, the route of administration, etc. Suitable, non-limiting examples of dosages include, for example, 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment may be adjusted according to the severity of the patient's condition. In some implementations, a second dose or multiple subsequent doses may be administered after the initial dose, the amount of which may be substantially the same as or less than the initial dose, wherein the interval between subsequent doses is at least 1 to 3 days, at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0390] The composition may be administered to the subject via: intravenous, intratumoral, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, local, intratumoral, intramuscular, intrasheathal, subcutaneous, subconjunctival, intravesical, mucosal, intraperitoneal, intraumbilical, intraocular, oral, local, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion, via catheter, by irrigation, in cream form, or in lipid composition form.

[0391] Certain additional medications used in combination therapies may be formulated and administered in any manner known in the art.

[0392] The compositions disclosed herein may also include adjuvants, such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, mediators, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy.

[0393] The peptides or peptide-based molecules disclosed herein may be provided in the form of a vaccine composition. This vaccine composition may be used to treat, prevent, or reduce the likelihood of HTLV-1 infection and / or HTLV-1-induced disease or condition. It is understood that vaccines may take several forms (see, for example, Schlom, J Natl Cancer Inst. 2012; 104(8):599-613; Salgaller, Cancer Res. 1996; 56(20):4749-57 and Marchand, Int J Cancer. 1999; 80(2):219-30). The vaccine composition may contain additional peptides or peptide-based molecules such that the peptides or peptide-based molecules disclosed herein are one of a mixture of peptides or peptide-based molecules. Adjuvants may be added to the vaccine composition to enhance the immune response. Specifically, for the peptide-containing vaccine compositions disclosed herein, pharmaceutically acceptable adjuvants include, but are not limited to, aluminum salts, Amplivax, AS15, Aquila's QS21 stimulon, AsA404 (DMXAA), β-glucan, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact EV1P321, IS Patch, ISS, 1018 ISS, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, poly-ICLC, PepTel®, Pam3Cys, PLGA microparticles, ricinole, SRL172, virions and other virus-like particles, YF-17D, VEGF traps, R848 and / or vadimezan.

[0394] Alternatively, the vaccine composition may take the form of an APC displaying a peptide of this disclosure that is complexed with MHC. Preferably, the APC is an immune cell, more preferably a dendritic cell or a B cell. The peptide may be pulsed onto the cell surface (Thurner, J ExpMed. 1999; 190(11): 1669-78), or a nucleic acid encoding the peptide of this disclosure may be introduced into a dendritic cell or a B cell (e.g., by electroporation. Van Tendeloo, Blood. 2001; 98(1): 49-56).

[0395] The pharmaceutical compositions disclosed herein can be administered directly to a patient, directly to an affected organ, or systemically (intradermal (id), intramuscular (im), intraperitoneal (ip), and intravenous (iv)), or ex vivo to cells derived from the patient or human cell lines, subsequently administered to the patient, or used to select, in vitro, a subset of immune cells derived from the patient, and then re-administered to the patient. If nucleic acids are administered to cells in vitro... ， Transfecting cells to co-express immunostimulatory cytokines (such as interleukin-2) may be useful. Peptides or peptide-based molecules can be essentially pure, or used in combination with immunostimulatory adjuvants, in conjunction with immunostimulatory cytokines, or administered with a suitable delivery system (e.g., liposomes, viral particles, VLPs). Peptides or peptide-based molecules can also be conjugated with suitable carriers such as keyforaminifera hemocyanin (KLH) or mannan (see, e.g., WO 95 / 18145 and Longecker et al., 1993).

[0396] In some embodiments, the peptide-containing compositions described herein also include an accessory molecule capable of regulating the survival or activity of TCR-expressing cells.

[0397] Non-limiting examples of useful accessory molecules include, for example, anti-CD28 antibodies, anti-CD80 (B7.1) antibodies, anti-CD86 (B7.2) antibodies, anti-anti-CD3 antibodies, anti-CD2 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD47 antibodies, and their functional derivatives, mutants, and fragments.

[0398] The accessory molecules used in the peptide compositions described herein include signaling molecules that, in addition to the primary signal provided by, for example, the binding of the TCR / CD3 complex to the pMHC complex, mediate T cell responses, including but not limited to proliferation, activation, and differentiation.

[0399] The auxiliary molecules can be, for example, inhibitory or stimulating antibodies, peptide ligands, co-stimulatory peptides, cytokines, etc. Non-limiting examples of auxiliary molecules that can be used in the peptide-containing compositions described herein include, for example, CD7, B7.1 (CD80), B7.2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, FIVEM, lymphotoxin β receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies binding to Toll ligand receptors and ligands specifically binding to B7-H3, and ligands binding to CD27, CD28, B7.1 (CD80), B7.2 (CD86), 4-1BB, OX40, CD30, CD40, PD-1, ICOS, and lymphocyte function-associated antigen-1. Antibodies that specifically bind to (LFA-1), CD2, CD3, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0400] Other non-limiting examples of helper molecules include, for example, members of the TNF / TNF family (e.g., OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD7); members of the immunoglobulin superfamily (e.g., VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chain, ICOS, ICOS ligand, HHLA2, butyrophilm, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF). CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILR), cytotoxic cell immunoglobulin-like receptors (KIR), lectin superfamily members, selectins, cytokines / chemokines and cytokine / chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrin, fibronectin, cadherin), or extracellular domains of multi-span integrated membrane proteins, or antibodies against any of these molecules.

[0401] In some embodiments, the peptide-containing compositions described herein further comprise a cytotoxic agent. In one specific embodiment, the cytotoxic agent is a toxin or a radioactive isotope (e.g., a radioconjugate) or a suicide gene. Non-limiting examples of toxins that can be used in the peptide-containing compositions described herein include, for example, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments, mutants, or derivatives thereof. Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), etc. Pseudomonas aeruginosa Suicide genes include, for example, ricin A chain, abrin A chain, modeccin A chain, α-arcinoxin, tung oil protein, caryophyllin protein, American pokeweed protein (PAPI, PAPII, and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, sapaonaria officinalis inhibitor, gelonin, mitogellin, localized arcinoxin, phenolmycin, enoxacin, and trichothecene compounds. Non-restrictive examples of suicide genes include, for example, thymidine kinase, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, β-galactosidase, hepatocyte pigment P450-2B1, linamarinase, horseradish peroxidase, and carboxypeptidase.

[0402] Methods for introducing the polypeptides or polynucleotides of this disclosure into cells or subjects may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implantable device-mediated delivery. In some embodiments, nucleic acids or proteins may be introduced into cells or subjects in a vector, such as polylactic acid (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, or lipid microtubules.

[0403] This document envisions the use of nanoparticles to deliver the peptide or polynucleotide compositions of this disclosure. Exemplary nanoparticles include, but are not limited to, polymer nanoparticles, inorganic nanoparticles, liposomes, lipid nanoparticles (LNPs), immunostimulatory complexes (ISCOMs), virus-like particles (VLPs), or self-assembled proteins. Nanoparticles may be calcium phosphate nanoparticles, silicon nanoparticles, or gold nanoparticles. For example, polymer nanoparticles may contain one or more synthetic polymers, such as poly(d,L-lactide-co-glycolic acid) (PLG), poly(d,L-lactic acid-co-glycolic acid) (PLGA), poly(γ-glutamic acid) (γ-PGA), poly(ethylene glycol) (PEG), or polystyrene, or one or more natural polymers, such as polysaccharides, such as pullulan, alginate, inulin, and chitosan. The use of polymer nanoparticles may be advantageous due to the properties of the polymers that can be contained within them. For example, the natural and synthetic polymers listed above may have good biocompatibility and biodegradability, non-toxicity, and / or the ability to be molded into desired shapes and sizes. Polymer nanoparticles can also form hydrogel nanoparticles and hydrophilic three-dimensional polymer networks, which possess advantageous physical properties, including flexible mesh sizes, large surface areas for multivalent conjugation, high water content, and high antigen loading capacity. Polymers such as poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for forming hydrogel nanoparticles. Inorganic nanoparticles typically have a rigid structure and contain a shell encapsulating antigens or a core in which antigens can be covalently linked. The core may contain one or more atoms, such as gold (Au), silver (Ag), copper (Cu) atoms, Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd, or Au / Ag / Cu / Pd or calcium phosphate (CaP).

[0404] Other molecules suitable for complexing with the polypeptides or polynucleotides of this disclosure include cationic molecules such as polyamide amine, dendritic polylysine, polyethyleneimine or polypropyleneimine, polylysine, chitosan, DNA-gelatin aggregates, DEAE dextran, dendritic polymers or polyethyleneimine (PEI).

[0405] In some embodiments, the antibodies of this disclosure may be conjugated to nanoparticles. Nanoparticles that can be conjugated to the antibodies of this disclosure include, but are not limited to: polyethylene glycol-modified liposomes, poly(d,l-lactide-co-glycolic acid) / montmorillonite nanoparticles (PLGA / MMT NP), poly(lactide-co-glycolic acid) (PLGA) nanoparticles, poly(malic acid) nanoparticles, chitosan-shelled nanoparticles, carbon nanotubes, and other inorganic nanoparticles (such as nanoparticles made from magnesium-aluminum layered double hydroxides and disuccinimide carbonate (DSC) and TiO2 nanoparticles). Nanoparticles can be developed and conjugated to antibodies included in pharmaceutical compositions for targeting virus-infected cells.

[0406] Treatment

[0407] The compositions disclosed herein, including peptides, peptide-based molecules (such as complexes comprising one or more peptides (e.g., peptide-MHC (pMHC) complexes), fusion proteins or conjugates), nucleic acid molecules, carriers, cells or binding moieties, may be used to prevent and / or treat viral infections (e.g., HTLV-1 infection) and / or diseases or conditions caused by viral infections (e.g., HTLV-1 infection).

[0408] On the one hand, this document discloses methods for regulating the activity, proliferation, or survival of cells containing TCRs, including contacting the cells with compositions disclosed herein (e.g., peptides, complexes (e.g., pMHC complexes), fusion proteins, or conjugates).

[0409] In some embodiments, the cells are lymphocytes, such as T cells (e.g., CD4+ T cells or CD8+ T cells). In some embodiments, the target T cells are CD4+ T cells, such as helper T cells (e.g., Th1, Th2, or Th17 cells) or CD4+ / CD25+ / FOXP3+ regulatory T (Treg) cells. In some cases, the target T cells are CD8+ T cells, such as cytotoxic T cells. In some cases, the target T cells are memory T cells, which can be CD4+ T cells or CD8+ T cells, wherein memory T cells are typically CD45RO+. In some cases, the target T cells are NK-T cells.

[0410] In some implementations, the contact is external. In some implementations, the contact takes place inside the subject (e.g., a human).

[0411] In some implementations, the cell is a mammalian cell (e.g., a human cell).

[0412] In some implementations, for example, when the target T cell is a CD8+ T cell, the peptide is presented by a class I MHC peptide. In some implementations, for example, when the target T cell is a CD4+ T cell, the peptide is presented by a class II MHC peptide.

[0413] When the T cell's TCR is bound by a TCR-binding molecule (e.g., the pMHC complex), the interaction of the T cell with the peptide described herein can lead to, for example, T cell activation, induction of unresponsiveness, or death. "T cell activation" refers to the induction of signal transduction pathways in a T cell, resulting in the production of cellular products (e.g., interleukin-2). "Unresponsiveness" refers to a reduced responsiveness of T cells to an antigen. Activation and unresponsiveness can be measured, for example, by measuring the amount of IL-2 produced by T cells after the pMHC complex has bound to the TCR. Unresponsive cells will have reduced IL-2 production compared to stimulated T cells. Another method for measuring the reduced activity of unresponsive T cells includes measuring intracellular and / or extracellular calcium mobilization induced by T cells upon TCR binding. "T cell death" refers to the permanent cessation of substantially all T cell functions.

[0414] On the other hand, this article provides a method for inducing an immune response against HTLV infection (e.g., HTLV-1, HTLV-2, HTLV-3 and / or HTLV-4 infection) in subjects in need, the method comprising administering to the subject a therapeutically effective amount of the composition disclosed herein (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, vectors, cells or binding moieties).

[0415] In some embodiments, generating an immune response includes an increase of approximately 1.5 to 20 times or more in target antigen-specific cytotoxic T lymphocyte (CTL) activity in subjects treated with the compositions of this disclosure compared to a control. In other embodiments, generating an immune response includes an increase of approximately 1.5 to 20 times or more in target-specific CTL activity in subjects treated with the compositions of this disclosure compared to a control. In yet another embodiment, generating an immune response includes an increase of approximately 1.5 to 20 times or more in target antigen-specific cell-mediated immune activity as measured by ELISpot assay of cytokine secretion (such as interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), or other cytokines).

[0416] In another embodiment, generating an immune response includes an increase in target-specific antibody production of 1.5 to 5 times compared to a suitable control. In yet another embodiment, generating an immune response includes an increase in target-specific antibody production of about 1.5 to 20 times or more compared to a control.

[0417] T cell activation can be determined, for example, by measuring changes in the expression levels of cytokines and / or T cell activation markers, and / or by the induction of antigen-specific proliferating cells. Techniques known to those skilled in the art, including but not limited to immunoprecipitation followed by Western blotting, ELISA, flow cytometry, RNA blotting, and RT-PCR, can be used to measure the expression of cytokines and T cell activation markers. Cytokine release can be determined by measuring the secretion of cytokines, including but not limited to interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-12 (IL-12), interleukin-16 (IL-16), PDGF, TGF-α, TGF-β, TNF-α, TNF-β, GCSF, GM-CSF, MCSF, IFN-α, IFN-β, IFN-γ, TFN-γ, IGF-I, and IGF-II.

[0418] T cell regulation can also be assessed by, for example, measuring proliferation (e.g., by ³H-thymidine incorporation, trypan blue cell counting, and fluorescence activated cell sorting (FACS)).

[0419] Antitumor responses of T cells can be determined in xenograft tumor models. Any human cancer cell line expressing the relevant tumor-associated antigen can be used to establish the tumor. To establish a xenograft tumor model, approximately 5 × 10⁻⁶ cells can be injected using, for example, Matrigel (Becton Dickinson). 6 Live cells are injected, for example, subcutaneously into athymic nude mice. The endpoint of the xenograft tumor model can be determined based on tumor size, animal weight, survival time, and histochemical and histopathological examination of the cancer using methods known to those skilled in the art.

[0420] In a related respect, this article discloses a method for treating HTLV-1 infection in subjects in need, the method comprising administering to the subject an effective amount of the composition disclosed herein (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, binding moieties).

[0421] In a related respect, this article discloses a method for preventing or reducing the likelihood of HTLV-1-induced disease or condition in subjects in need, the method comprising administering to the subject an effective amount of the composition disclosed herein (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, carriers, cells, binding moieties).

[0422] HTLV-1 induced diseases or conditions can be, for example, adult T-cell leukemia / lymphoma (ATL) or HTLV-1 myelopathy / tropical spastic paralysis (HAM / TSP). In some implementations, HTLV-1 induced diseases or conditions can be, for example, hypersensitivity reactions, such as, but not limited to, arthritis, uveitis, or HTLV-1-associated infectious dermatitis (IDH).

[0423] Non-limiting examples of ATL include acute ATL, lymphomatous ATL, chronic ATL, and stagnant ATL. The most benign but detectable form of ATL is the asymptomatic preleukemic stage, which can be diagnosed incidentally by, for example, peripheral blood smear examination, which may reveal abnormal lymphocytes with segmented nuclei. Stagnant ATL is the most benign symptomatic form of ATL, characterized by a normal peripheral blood leukocyte count, a small number of circulating leukemic cells, and skin lesions without visceral involvement. The development of chronic ATL is associated with visceral involvement, evidenced by hepatosplenomegaly, lymphadenopathy, and peripheral blood leukocytosis. Patients with acute ATL may present with elevated lactate dehydrogenase and bilirubin levels, peripheral blood leukocytosis, hypercalcemia, and skin and visceral involvement. Acute thrombocytopenic purpura (ATL) can be accompanied by severe immunosuppression, and many ATL patients are highly susceptible to a variety of infections, such as, but not limited to, cryptococcal meningitis, Pneumocystis carinii pneumonia, disseminated cytomegalovirus infection, and candidal esophagitis. Acute ATL is usually unresponsive to conventional chemotherapy, and the median life expectancy for patients with acute ATL is approximately 11 months.

[0424] HTLV-1 is believed to cause approximately 80% of HAM / TSP cases by, for example, weakening the immune system. Most HAM / TSP patients experience few or no symptoms throughout their lives. In addition to neurological symptoms such as weakness, muscle spasms, and / or rigidity, in rare cases, HAM / TSP patients may also experience, for example: arthritis (inflammation of one or more joints); infectious dermatitis (skin inflammation); keratoconjunctivitis sicca (persistent dryness of the cornea and / or conjunctiva); polymyositis (an inflammatory muscle disease); pulmonary lymphocytic alveolitis (inflammation of the lungs); and / or uveitis (inflammation of the uveal canal of the eye).

[0425] When the disease being treated is cancer, the cancer may specifically belong to the following histological types (although it is not limited to these types): malignant tumor; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatric carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; bile duct carcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and bile duct carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma of familial adenomatous colon; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; [The text abruptly ends here, so the translation stops as well.] Chromoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; skin appendage carcinoma; apocrine gland carcinoma; sebaceous gland adenocarcinoma; ceruminous gland adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia (adenocarcinoma w / squamous) Metaplasia); Malignant thymoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant testicular blastoma; Sertoli cell carcinoma; Malignant testicular stromal cell tumor; Malignant lipocytoma; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Angiosarcoma; Malignant melanoma; Amelanotic melanoma; Superficial diffuse melanoma; Malignant melanoma in giant nevus: Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brugia Lennar tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroma; neuroblastoma;Retinoblastoma; olfactory neurogenic tumors; malignant meningiomas; neurofibrosarcomas; malignant schwannomas; malignant granular cell tumors; malignant lymphomas; Hodgkin's disease; Hodgkin's lymphoma; paragranulomas; small lymphocytic malignant lymphomas; diffuse large cell malignant lymphomas; follicular malignant lymphomas; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytic proliferative disorders; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel diseases; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0426] It is anticipated that, when used to treat various diseases, the compositions and methods can be combined with other therapeutic agents suitable for the same or similar diseases. Furthermore, two or more of the embodiments described herein can be co-administered to produce an additive or synergistic effect. When co-administered with a second therapeutic agent, the embodiments described herein and the second therapeutic agent can be administered simultaneously or sequentially (in any order). Due to additive or synergistic effects, the appropriate therapeutically effective dose of each drug may be reduced.

[0427] In some embodiments, the compositions and methods disclosed herein can be used to enhance the efficacy of vaccines against HTLV-1 infection or HTLV-1-induced diseases. Therefore, the compositions and methods described herein can be administered to subjects simultaneously with or before (e.g., 1–30 days prior) administration of reagents intended to elicit an immune response (e.g., to treat HTLV-1, HTLV-2, HTLV-3, and / or HTLV-4 infection or cancer).

[0428] The compositions and methods described herein can also be used in combination with antitumor antibodies or antibodies against pathogenic antigens (e.g., HTLV-1) or allergens.

[0429] The compositions and methods described herein can be used in combination with other immunomodulatory therapies, such as therapeutic vaccines (including but not limited to GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to agents that enhance 41BB, OX40, etc.). The inhibitory therapies described herein can also be used in combination with other therapies capable of modulating NKT function or stability (including but not limited to CD1d, CD1d-fusion proteins, CD1d dimers, or larger polymers of CD1d (unloaded or loaded with antigen), CD1d-chimeric antigen receptors (CD1d-CAR), or any of the other five known CD1 isoforms present in humans (CD1a, CD1b, CD1c, CD1e)) in any of the above forms or formulations, either alone, in combination with each other, or in combination with other agents.

[0430] The treatments described herein can be used in combination with additional immunotherapies and other treatments. For example, when used to treat cancer, the NKT cells described herein can be used in combination with cancer therapies such as surgery, radiation therapy, chemotherapy, or combinations thereof, depending on the type of tumor, the patient's condition, other health problems, and various factors. In some respects, other therapeutic agents used in combination with the inhibitors described herein for cancer treatment include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, platelet-reactive protein-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, soluble bFGF receptor, transforming growth factor β, interferon-α, soluble KDR and FLT-1 receptors, placental proliferator-associated protein, and those listed by Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein may be used in combination with VEGF antagonists or VEGF receptor antagonists, such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, VEGFR tyrosine kinase inhibitors, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0431] Non-limiting examples of chemotherapy compounds that can be used in combination therapy include, for example, aminoglutethimide, acridine, anastrozole, asparaginase, BCG, bicalutamide, bleomycin, busherin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clophosphamide, colchicine, cyclophosphamide, cyproterone acetate, cytarabine, dacarbazine, actinomycin D, daunorubicin, diethylstilbestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estradiol, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, flumethasone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin. Star, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprorelin, levamisole, lomustine, nitrogen mustard, medroxyprogesterone acetate, megestrol acetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilumethoxazole, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, procarbazine, procarbazine, raltitrexed, rituximab, streptozotocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, dichlorodicyclohexane, topotecan, trastuzumab, retinoic acid, vincristine, vinblastine, vinorelbine, and vinorelbine.

[0432] These chemotherapy compounds can be classified according to their mechanisms of action into groups such as: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, fluorouridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone, and vinorelbine. Podophyllotoxin (etoposide, teniposide), DNA damaging agents (actinomycin, acridine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, actinomycin D, daunorubicin, doxorubicin, epirubicin, hexamethylaniline, oxaliplatin, ifosfamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosourea, procainamide, procarbazine, paclitaxel, tessotericin, teniposide, triethylenethiophosphoramide, and etoposide (VP16)); antibiotics such as actinomycin D (actinomycin D (actinomycin D) D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, procainamide (scintillan), and mitomycin; enzymes (L-asparaginase, which systemically metabolizes L-asparagine and deprives cells unable to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (nitrogen mustard, cyclophosphamide and its analogues, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and its analogues, streptozotocin), triazene - dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogues (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, Mitotan, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, niglutethimide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigration agents; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors;Antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (doxorubicin), acridine, camptothecin, daunorubicin, actinomycin D, teniposide, epirubicin, etoposide, idarubicin, mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0433] For the treatment of viral infections, the combination therapy described herein may include co-administration of the compositions and methods described herein with antiviral drugs. Non-limiting examples of useful antiviral drugs include adefovir and entecavir, telbivudine, immune system modulators such as interferon-α, interferon-β, or interferon-γ, doxorinosine, lamivudine, zanamivir, lopinavir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantadine, ribavirin, ganciclovir, foscarnet sodium, and acyclovir, or any salts or variants thereof. See also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., eds., Remington's The Science and Practice of Pharmacy, 20th edition, (2000); Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., eds., Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., eds., The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0434] Reagent test kit

[0435] This disclosure also includes kits that may include any of the various compositions of this disclosure, including peptides, peptide-based molecules (such as complexes containing one or more peptides (e.g., peptide-MHC (pMHC) complexes), fusion proteins or conjugates), nucleic acid molecules, vectors, cells, or binding portions of this disclosure.

[0436] On one hand, this disclosure may include a kit, which may include, for example: (A) a container containing a pharmaceutical composition disclosed herein (e.g., a pharmaceutical composition in solution or lyophilized form); (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; and / or (c) optionally, (i) instructions for use of the solution or (ii) instructions for reconstitution and / or use of the lyophilized formulation.

[0437] In some embodiments, the kit may also include, for example, but not limited to, one or more of (i) buffer solutions, (ii) diluents, (iii) filters, (iv) needles, and / or (v) syringes. As a non-limiting example, the container may be a bottle, vial, syringe, or test tube. In some embodiments, the container may be a multi-purpose container. In some cases, the pharmaceutical composition may be lyophilized.

[0438] The kits disclosed herein may contain the lyophilized formulation of this disclosure, its reconstitution, and / or instructions for use in a suitable container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. Containers may be formed from a variety of materials such as glass or plastic. The kit and / or container may have instructions on or accompanying the container specifying instructions for reconstitution of the lyophilized formulation and / or instructions for use of the kit. For example, a label may indicate that the lyophilized formulation will be reconstituted to a suitable peptide concentration. A label may indicate that the formulation may be used or intended for use in any of the routes of administration disclosed herein, such as the parenteral administration routes disclosed herein.

[0439] The container for the formulation may be a reusable vial that allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The kit may also include a second container containing a suitable diluent (e.g., sodium bicarbonate solution).

[0440] When the diluent and lyophilized formulation are mixed, the final peptide concentration in the reconstituted formulation is achieved. The kit may also include other materials required from a commercial and / or user perspective, including, but not limited to, other buffers, diluents, filters, needles, syringes, and / or packaging inserts (which may include, for example, instructions for use).

[0441] The kit of this disclosure may have a single container containing a formulation of a pharmaceutical composition according to this disclosure (with or without other components, such as other compounds or pharmaceutical compositions of such other compounds), or may have separate containers for each component.

[0442] In some embodiments, the kits of this disclosure may include formulations of this disclosure packaged for use in combination with a second compound (such as an adjuvant (e.g., GM-CSF, a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer or chelator) or a pharmaceutical composition thereof). The components of the combined kit may be pre-announced before administration to a patient, or each component may be present in a separate, different container. The components of the kit may be provided in one or more liquid solutions. The liquid solutions described herein may be aqueous solutions, such as sterile aqueous solutions. The components of the kit may also be provided in solid form, which may be converted into a liquid, for example, by adding a suitable solvent, which may be provided in another, different container.

[0443] The container for the therapeutic kit can be a vial, test tube, flask, bottle, syringe, or any other device for encapsulating solids or liquids. When more than one component is present, the kit may also include a second vial or other container that allows for individual administration. The kit may also include another container for holding a pharmaceutically acceptable liquid. In some embodiments, the kit may include a device (e.g., one or more needles, syringes, droppers, pipettes, etc.) that allows for the administration of the reagents of this disclosure as components of the kit.

[0444] Methods and systems for identifying immunogenic viral-derived peptides

[0445] This disclosure also includes methods and systems for identifying immunogenic viral-derived peptides. Some or all of the steps of the method can be performed by a computing device. In some aspects, some or all of the steps of the method can be stored as computer-readable instructions in a memory, such that the instructions can be executed by one or more processors to perform the functions associated with the method.

[0446] On one hand, this disclosure may include a method for identifying immunogenic viral-derived peptides, the method comprising the steps of: (a) obtaining a plurality of RNA contig sequences derived from an infected subject infected with a virus, wherein the plurality of RNA contig sequences comprise a plurality of viral-derived RNA contig sequences and a plurality of endogenous RNA contig sequences of the infected subject; (b) identifying a plurality of viral-derived RNA contig sequences from the plurality of RNA contig sequences; (c) assembling a viral RNA sequence based on the plurality of viral-derived RNA contig sequences; (d) identifying a protein sequence based on the viral RNA sequence; and (e) identifying an immunogenic viral-derived peptide based at least in part on the identified protein sequence.

[0447] On one hand, a system configured to perform functions related to the aforementioned methods may include a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device. The non-transitory computer-readable medium may contain instructions thereon that, when executed by the one or more processors, cause the computing device to perform the following operations: (a) receiving, as input, a plurality of RNA contig sequences from an infected subject infected with a virus, such that the plurality of RNA contig sequences comprise a plurality of virus-derived RNA contig sequences and a plurality of endogenous RNA contig sequences of the infected subject, wherein the infected subject is infected with a virus; (b) identifying the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences; (c) assembling a viral RNA sequence based on the plurality of virus-derived RNA contig sequences; (d) identifying a protein sequence based on the viral RNA sequence; (e) identifying an immunogenic virus-derived peptide at least partially based on the protein sequence; and (f) providing, as output, the immunogenic virus-derived peptide.

[0448] In some implementations, the multiple RNA contig sequences may be derived from a single infected subject.

[0449] In some implementations, the infected subject can be a human being.

[0450] In some implementations, the plurality of viral RNA contig sequences are derived from the virus that infected the infected subject.

[0451] In some implementations, the endogenous RNA contig sequences of the plurality of infected subjects are derived from the endogenous RNA of the infected subjects.

[0452] In some embodiments, identifying the plurality of viral-derived RNA contig sequences from the plurality of RNA contig sequences (step b) may further include: comparing at least a portion of the contig sequences of the plurality of RNA contig sequences with a reference viral sequence; and identifying the plurality of viral-derived RNA contig sequences such that each contig sequence of the plurality of viral-derived RNA contig sequences contains at least one portion corresponding to the reference viral sequence.

[0453] In some implementations, each contig sequence of the plurality of viral-derived RNA contigs may differ from the endogenous RNA contig sequences of the plurality of infected subjects.

[0454] In some implementations, each of the plurality of viral-derived RNA contig sequences may not contain the endogenous RNA contig sequence of the infected subject.

[0455] In some implementations, the reference viral sequence may include a reference genome.

[0456] In some implementations, the reference genome may include the HTLV-1 viral genome.

[0457] In some embodiments, assembling a viral RNA sequence based on the plurality of viral-derived RNA contiguous sequences (step c) may include: overlapping common sequence portions at the ends of at least a portion of the plurality of viral-derived RNA contiguous sequences such that at least a portion of the plurality of viral-derived RNA contiguous sequences linearly overlap to assemble a viral RNA sequence.

[0458] In some implementations, identifying a protein sequence based on the viral RNA sequence, such that the protein sequence contains the translation product of the viral RNA sequence (step d), may include identifying the protein sequence without comparison with a viral protein database.

[0459] In some embodiments, identifying protein sequences based on the viral RNA sequence such that the protein sequences contain the translation product of the viral RNA sequence (step d) may include: identifying multiple protein sequences, each based on the viral RNA sequence, such that each of the multiple protein sequences contains the translation product of the viral RNA sequence, and identifying the protein sequences as protein sequences that frequently occur among the multiple protein sequences.

[0460] In some implementations, the protein sequence can be identified based on the viral RNA sequence associated with a single infected subject.

[0461] In some embodiments, identifying an immunogenic viral-derived peptide (step 3) based at least in part on the protein sequence may include: identifying an MHC molecule associated with the single infected subject; identifying one or more peptides based at least in part on the protein sequence, such that each of the one or more peptides forms a corresponding MHC-peptide complex with an MHC molecule; and identifying an immunogenic viral-derived peptide based on the one or more peptides.

[0462] Methods and systems for identifying viral gene integration sites within a subject's genome.

[0463] This disclosure also includes methods and systems for identifying viral gene integration sites within the genes of a subject. Some or all of the steps of the method can be performed by a computing device. In some aspects, some or all of the steps of the method can be stored as computer-readable instructions in a memory, such that the instructions can be executed by one or more processors to perform functions associated with the method.

[0464] On one hand, this disclosure may include a method for identifying viral gene integration sites within a subject's genome, the method comprising the steps of: (a) obtaining a plurality of RNA contig sequences derived from a virally infected subject, such that the plurality of RNA contig sequences comprise a plurality of viral-derived RNA contig sequences, a plurality of endogenous RNA contig sequences of the infected subject, and a plurality of heterozygous RNA contig sequences comprising a viral portion and an endogenous portion of the infected subject; (b) identifying the plurality of heterozygous RNA contig sequences from the plurality of RNA contig sequences; (c) comparing the endogenous portion of the infected subject with a test reference genome for at least a portion of the plurality of heterozygous RNA contig sequences; and (d) identifying integration sites comprising the subject's genome based at least in part on the comparison of the endogenous portion of the infected subject with the test reference genome.

[0465] On one hand, a system configured to perform functions related to the aforementioned methods may include a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device. The non-transitory computer-readable medium may contain instructions thereon that, when executed by the one or more processors, cause the computing device to perform the following operations: (a) receiving, as input, a plurality of RNA contig sequences from a virus-infected subject, such that the plurality of RNA contig sequences comprise a plurality of virus-derived RNA contig sequences, a plurality of endogenous RNA contig sequences of the subject, and a plurality of heterozygous RNA contig sequences comprising a viral portion and an endogenous portion of the infected subject; (b) identifying the plurality of heterozygous RNA contig sequences from the plurality of RNA contig sequences; (c) comparing, for at least a portion of the plurality of heterozygous RNA contig sequences, the endogenous portion of the infected subject with a test reference genome; (d) identifying an integration site comprising a subject gene, at least in part based on the comparison of the endogenous portion of the infected subject with the subject reference genome; and (e) providing, as output, the integration site.

[0466] In some implementations, the multiple RNA contig sequences may be derived from a single infected subject.

[0467] In some implementations, the infected subject can be a human being.

[0468] In some implementations, the plurality of viral RNA contig sequences may be derived from viruses that have infected infected subjects.

[0469] In some implementations, the virus may include the HTLV-1 virus.

[0470] In some implementations, the endogenous RNA contig sequences of the plurality of infected subjects may be derived from the endogenous RNA of the infected subjects.

[0471] In some implementations, the test reference genome may include a human genome.

[0472] Some embodiments and implementations of the present disclosure described above are based on systems, methods, and / or computer program products according to exemplary embodiments or implementations of the present disclosure. It will be understood that method steps may be implemented by computer-executable program instructions. Similarly, according to some embodiments or implementations of the disclosed technology, some method steps do not need to be performed in the presented order, may be repeated, or may not need to be performed at all.

[0473] These computer-executable program instructions may be loaded onto a general-purpose computer, special-purpose computer, processor, or other programmable data processing apparatus to produce a particular machine, such that the instructions, which execute on the computer, processor, or other programmable data processing apparatus, create means for implementing one or more functions related to the method steps. These computer program instructions may also be stored in a computer-readable storage medium that can instruct a computer or other programmable data processing apparatus to operate in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of writing comprising instruction means for implementing one or more functions related to the method steps.

[0474] Non-transitory computer-readable media may include, but are not limited to: random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), flash memory or other storage technologies, optical disc ROM (CD-ROM), digital versatile optical disc (DVD) or other optical storage, magnetic tape cassette, magnetic tape, disk storage or other magnetic storage devices, or any other tangible physical medium that can be used to store computer-readable information.

[0475] Embodiments or implementations of the disclosed technology may provide a computer program product comprising a computer-usable medium containing computer-readable program code or program instructions adapted to be executed to perform one or more functions related to the exemplary methods presented herein. Similarly, computer program instructions may be loaded onto a computer or other programmable data processing apparatus to cause a series of operational elements or steps to be performed on the computer or other programmable apparatus, thereby producing a computer-implemented process, such that the instructions, which execute on the computer or other programmable apparatus, provide elements or steps for implementing the functions related to the exemplary methods presented herein.

[0476] Therefore, the illustrations and descriptions of the method support combinations of means for performing a specified function, combinations of elements or steps for performing a specified function, and program instruction means for performing a specified function. It should also be understood that at least some method steps and combinations of method steps can be implemented by a dedicated hardware-based computer system that performs the specified function, element, or step, or by a combination of dedicated hardware and computer instructions.

[0477] Some implementations of the disclosed technologies can be used with client devices that may include mobile computing devices. Those skilled in the art will recognize that there are several categories of mobile devices, often referred to as portable computing devices, which operate on battery power but are not typically classified as laptops. For example, mobile devices may include, but are not limited to, portable computers, tablet PCs, internet tablets, PDAs, ultra-mobile PCs (UMPCs), wearable devices, and smartphones.

[0478] Some implementations of the disclosed technology can be used with medical devices, medical apparatuses and / or related peripheral devices.

[0479] Non-restrictive examples of methods and systems

[0480] In various embodiments, this disclosure provides a method for identifying immunogenic viral-derived peptides, the method comprising:

[0481] A) Obtain multiple RNA contig sequences derived from infected subjects infected with the virus, wherein the multiple RNA contig sequences comprise multiple virus-derived RNA contig sequences and multiple endogenous RNA contig sequences from infected subjects.

[0482] b) Identify multiple viral-derived RNA contig sequences from the plurality of RNA contig sequences;

[0483] c) Assemble viral RNA sequences based on the RNA contiguous group sequences from the multiple viral sources;

[0484] d) Identify the protein sequence based on the viral RNA sequence; and

[0485] e) Identify immunogenic viral peptides based at least in part on the identified protein sequences.

[0486] In some implementations, the multiple RNA contiguous sequences are derived from an infected subject.

[0487] In some implementations, the infected subjects are humans.

[0488] In some implementations, the plurality of viral RNA contig sequences are derived from the virus that infected the infected subject.

[0489] In some implementations, the endogenous RNA contig sequences of the plurality of infected subjects are derived from the endogenous RNA of the infected subjects.

[0490] In some implementations, identifying multiple viral-derived RNA contiguous sequences from the plurality of RNA contiguous sequences includes:

[0491] Compare at least a portion of the contiguous sequences from the plurality of RNA contiguous sequences with a reference viral sequence; and

[0492] Identify the RNA contiguous sequences from the plurality of viral sources such that each contiguous sequence from the plurality of viral sources contains at least one portion corresponding to the reference viral sequence.

[0493] In some implementations, each contig sequence in the plurality of viral-derived RNA contigs is different from the endogenous RNA contig sequence of the plurality of infected subjects.

[0494] In some implementations, each of the plurality of viral-derived RNA contig sequences does not contain an endogenous RNA contig sequence of the infected subject.

[0495] In some implementations, the reference viral sequence comprises a reference genome.

[0496] In some implementations, the reference genome contains the HTLV-1 viral genome.

[0497] In some embodiments, assembling the viral RNA sequence based on the RNA contiguous sequences from the plurality of viral sources includes:

[0498] The common sequence portions at the ends of at least a portion of the contiguous RNA sequences from the plurality of viral sources are overlapped, such that the at least a portion of the plurality of viral RNA contiguous sequences are linearly overlapped to assemble a viral RNA sequence.

[0499] In some embodiments, the identification of a protein sequence based on the viral RNA sequence, such that the identified protein sequence contains the translation product of the viral RNA sequence, includes:

[0500] Protein sequences can be identified without comparison with viral protein databases.

[0501] In some embodiments, the step of identifying a protein sequence based on the viral RNA sequence, such that the identified protein sequence contains the translation product of the viral RNA sequence, further includes:

[0502] Each of the multiple protein sequences identified based on the viral RNA sequence contains a translation product of the viral RNA sequence.

[0503] The protein sequence was identified as a protein sequence that frequently occurs among the plurality of protein sequences.

[0504] In some implementations, protein sequences identified based on the viral RNA sequence are associated with a single infected subject.

[0505] In some embodiments, the identification of immunogenic viral-derived peptides based at least in part on the protein sequence includes:

[0506] Identify MHC molecules associated with the single infected subject;

[0507] At least partially based on the protein sequence, one or more peptides are identified such that each of the one or more peptides forms a corresponding MHC-peptide complex with the MHC molecule; and

[0508] Identification of immunogenic viral peptides based on one or more peptides.

[0509] In various embodiments, this disclosure provides a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium containing instructions thereon that, when executed by the one or more processors, cause the computing device to:

[0510] a) As input, receive multiple RNA contig sequences from an infected subject infected with a virus, such that the multiple RNA contig sequences comprise multiple virus-derived RNA contig sequences and multiple endogenous RNA contig sequences from an infected subject, wherein the infected subject is infected with a virus.

[0511] b) Identify the multiple viral-derived RNA contig sequences from the multiple RNA contig sequences;

[0512] c) Assemble viral RNA sequences based on the RNA contiguous group sequences from the multiple viral sources;

[0513] d) Identify the protein sequence based on the viral RNA sequence;

[0514] E) Identification of immunogenic viral-derived peptides based at least in part on the protein sequence; and

[0515] f) As an output, it provides immunogenic viral-derived peptides.

[0516] In some implementations, the multiple RNA contig sequences are derived from only one infected subject.

[0517] In some implementations, the infected subject includes a human being.

[0518] In some implementations, the plurality of viral RNA contig sequences are derived from the virus that infected the infected subject.

[0519] In some implementations, the endogenous RNA contig sequences of the plurality of infected subjects are derived from the endogenous RNA of the infected subjects.

[0520] In some embodiments, the instructions for causing the computing device to identify the plurality of viral-derived RNA contiguous sequences from the plurality of RNA contiguous sequences further include instructions that, when executed by one or more processors, cause the computing device to perform the following operations:

[0521] Compare at least a portion of the contiguous sequences from the plurality of RNA contiguous sequences with a reference viral sequence; and

[0522] Identify the RNA contiguous sequences from the plurality of viral sources such that each contiguous sequence from the plurality of viral sources contains at least a portion corresponding to the reference viral sequence.

[0523] In some implementations, each contig sequence in the plurality of viral-derived RNA contigs is different from the endogenous RNA contig sequence of the plurality of infected subjects.

[0524] In some implementations, each of the plurality of viral-derived RNA contig sequences does not contain a portion of the endogenous RNA contig sequence of the infected subject.

[0525] In some implementations, the reference viral sequence comprises a reference genome.

[0526] In some implementations, the reference genome includes the HTLV-1 genome.

[0527] In some implementations, the instructions for causing a computing device to assemble a viral RNA sequence based on the plurality of viral-derived RNA contiguous group sequences further include instructions that, when executed by one or more processors, cause the computing device to perform the following operations:

[0528] At least a portion of the common sequence portions at the ends of the contiguous sequences of the plurality of viral-derived RNA contiguous sequences are overlapped, such that at least a portion of the contiguous sequences of the plurality of viral-derived RNA contiguous sequences are linearly overlapped to assemble a viral RNA sequence.

[0529] In some implementations, the instructions for causing a computing device to identify a protein sequence based on the viral RNA sequence, such that the protein sequence contains the translation product of the viral RNA sequence, further include instructions that, when executed by a processor, cause the computing device to perform the following operations:

[0530] The protein sequences can be identified without comparison with viral protein databases.

[0531] In some implementations, the protein sequence identified based on the viral RNA sequence is a novel protein.

[0532] In some implementations, protein sequences identified based on the viral RNA sequence are associated with a single infected subject.

[0533] In some implementations, the instructions for causing a computing device to identify a protein sequence based on the viral RNA sequence, such that the protein sequence contains the translation product of the viral RNA sequence, further include instructions that, when executed by one or more processors, cause the computing device to perform the following operations:

[0534] Each of the plurality of protein sequences is identified based on the viral RNA sequence, such that each of the plurality of protein sequences contains the translation product of the viral RNA sequence; and

[0535] The protein sequence was identified as a protein sequence that frequently occurs among the plurality of protein sequences.

[0536] In some implementations, the instructions for causing the computing device to identify the immunogenic viral-derived peptide at least in part based on the protein sequence further include instructions that, when executed by one or more processors, cause the computing device to perform the following operations:

[0537] Identify the major histocompatibility complex (MHC) molecules associated with the single infected subject;

[0538] At least partially based on the protein sequence, one or more peptides are identified such that each of the one or more peptides can form a corresponding MHC-peptide complex with the MHC molecule; and

[0539] The immunogenic viral-derived peptides are identified based on one or more of the peptides.

[0540] In some implementations, when executed by one or more processors, the instructions also cause the computing device to perform the following operations:

[0541] The protein sequence is stored in a database such that the protein sequence is associated with the infected subject in the database.

[0542] In various embodiments, this disclosure provides a method for identifying integration sites of viral genes within a subject's gene, the method comprising:

[0543] a) Obtain multiple RNA contig sequences derived from infected subjects infected with the virus, such that the multiple RNA contig sequences comprise multiple virus-derived RNA contig sequences, multiple endogenous RNA contig sequences of the infected subjects, and multiple hybrid RNA contig sequences comprising both viral portions and endogenous portions of the infected subjects.

[0544] b) Identify the plurality of heterozygous RNA contig sequences from the plurality of RNA contig sequences;

[0545] c) For at least a portion of the plurality of heterozygous RNA contig sequences, compare the endogenous portion of the infected subject with the tested reference genome; and

[0546] d) Identify integration sites containing the subject's genes, based at least in part on a comparison of the endogenous portion of the infected subject with the test reference genome.

[0547] In some implementations, the multiple RNA contiguous sequences are derived from an infected subject.

[0548] In some implementations, the infected subjects are humans.

[0549] In some implementations, the plurality of viral RNA contig sequences are derived from viruses that have infected infected subjects.

[0550] In some implementations, the virus is HTLV-1.

[0551] In some implementations, the endogenous RNA contig sequences of multiple infected subjects are derived from RNA that is endogenous to the infected subjects.

[0552] In some implementations, the reference genome for the test includes the human genome.

[0553] In various embodiments, this disclosure provides a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium containing instructions thereon that, when executed by the one or more processors, cause the computing device to:

[0554] a) As input, receive multiple RNA contig sequences from an infected subject infected with a virus, such that the multiple RNA contig sequences comprise multiple virus-derived RNA contig sequences, multiple endogenous RNA contig sequences from the subject, and multiple hybrid RNA contig sequences comprising viral portions and endogenous portions from the infected subject.

[0555] b) Identify multiple heterozygous RNA contig sequences from the multiple RNA contig sequences;

[0556] c) For at least a portion of the plurality of heterozygous RNA contig sequences, the endogenous portion of the infected subject is compared with the test reference genome;

[0557] d) Identify integration sites containing the subject's genes, based at least in part on a comparison of the endogenous portion of the infected subject's genome with the tested reference genome; and

[0558] e) Provide an integrated site as output.

[0559] In some implementations, the multiple RNA contig sequences are derived from only one infected subject.

[0560] In some implementations, the infected subject includes a human being.

[0561] In some implementations, the plurality of viral-derived RNA contig sequences are derived from the virus that infected the infected subject.

[0562] In some implementations, the virus includes HTLV-1.

[0563] In some implementations, the endogenous RNA contig sequences of the plurality of infected subjects are derived from RNA that is endogenous to the infected subjects.

[0564] In some implementations, the test reference genome includes a human genome.

[0565] Example

[0566] The present disclosure is also described and illustrated by the following embodiments. However, any use of these and other examples anywhere in this specification is merely illustrative and is in no way intended to limit the scope and meaning of the present disclosure or any exemplary terminology. Similarly, the present disclosure is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the present disclosure will be apparent to those skilled in the art upon reading this specification, and such changes can be made without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is limited only by the terms of the appended claims and the full scope of their equivalents.

[0567] Human T-lymphotropic virus type 1 (HTLV-1) is a pathogenic factor for adult T-cell leukemia / lymphoma (ATL), HTLV-1-associated myelopathy (HAM), HTLV-1-associated infectious dermatitis (IDH), and other inflammatory conditions such as uveitis and arthritis. Although HTLV-1 is the most well-studied of the four HTLV genotypes (HTLV-1, HTLV-2, HTLV-3, and HTLV-4), the immunopeptidome of HTLV-1-infected T cells and cell lines has not been characterized. Surface presentation of HTLV-1-specific peptides by human leukocyte antigen (HLA) can be used to develop immunotherapies targeting infected lymphocytes in ATL. In the examples described below, HLA-I immunopeptidomics was performed on four model HTLV-transformed cell lines: MT-4, C8166, MT-2, and C5 / MJ; primary cells; and ATL patient samples.

[0568] Example 1. Immunopeptidomics of HTLV-transformed cells.

[0569] HLA-I complexes were purified from four HTLV-transformed cell lines (MT-4, C8166, MT-2, and C5 / MJ) using anti-HLA-I W6 / 32 affinity purification. Figure 1AIn short, 100 million cells from each cell line were lysed under mild conditions in 1% NP-40 lysis buffer, and the lysates were incubated with agarose beads conjugated with W6 / 32 in column form. The beads were washed and then HLA eluted with acidic glycine solution (pH 2.7). HLA-related peptides were then separated from HLA molecules by selective elution from C18-based sep-pak using 30% acetonitrile / H2O. Peptides were separated on a 25 cm column and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using a Thermo Orbitrap Fusion Lumos connected online to a nano-LC 1200 (Thermo). Raw mass spectrometry files were searched against the FASTA database containing both human (Uniprot) and HTLV-1 (Uniprot) protein sequences. More than 13,000 peptides were detected from the MT-4 cell line. Figure 1D ), 8,000 to 10,000 peptides were detected in C8166, MT-2, and C5 / MJ cell lines. Figures 1B-1C and Figure 1E Of these, the nonamer is the most abundant peptide length.

[0570] In addition, many unique HTLV peptides were identified. For example, 19 HTLV-1 peptides were detected in MT-4 cells, of which 16 peptides overlapped across three biological replicates. Figure 4 (See Table 5). Similarly, 11 and 32 HTLV-1 specific peptides were detected in C8166 and MT-2 cells, respectively, with high reproducibility among replicates (and Table 5). Figure 2 and Table 3; Figure 3 (and Table 4). For C5 / MJ cells, comparisons were made with and without interferon- The HTLV-1 peptide is presented by HLA upon stimulation. In interferon (IFN) - No significant increase in the number of HTLV peptides was observed after stimulation of C5 / MJ cells. Figure 5 (See Table 6). To increase confidence in the identification of the disclosed peptides, these samples were HLA genotyped, and the panNetMHC 4.0 algorithm was used to predict the binding affinity of the identified HTLV-1 peptides to the HLA alleles of the corresponding cell lines. Most of the HTLV-1 peptides identified from the four cell lines were predicted to be strong binders (panNetMHC 4.0 grade less than 0.5) or weak binders (panNetMHC 4.0 grade between 0.5 and 2.0) to the HLA of the stated cell lines (Tables 3-6).

[0571] Table 3. HTLV-1 peptide detected in C8166 cells

[0572]

[0573] Strongly bound compounds (grade < 0.5) are shown in bold; weakly bound compounds (grade 0.5-2) are shown in bold. italics Display; Len = length

[0574] Table 4. HTLV-1 peptides detected in MT-2 cells

[0575]

[0576]

[0577] Strongly bound compounds (grade < 0.5) are shown in bold; weakly bound compounds (grade 0.5-2) are shown in bold. italics Display; Len = length

[0578] Table 5. HTLV-1 peptides detected in MT-4 cells

[0579]

[0580]

[0581] Strongly bound compounds (grade < 0.5) are shown in bold; weakly bound compounds (grade 0.5-2) are shown in bold. italics Display; Len = length

[0582] Table 6. HTLV-1 peptide detected in C5 / MJ cells treated or untreated with IFN-γ.

[0583]

[0584] Strongly bound compounds (grade < 0.5) are shown in bold; weakly bound compounds (grade 0.5-2) are shown in bold. italics Display; Len = length

[0585] Example 2. Immunopeptidomics of primary ATL cells.

[0586] Next, the HLA-I immunopeptidome of patient-derived primary ATL sample ED41214(-) was analyzed using the same methods discussed in Example 1. Interferon- The total peptide count increased slightly after treatment. Figure 6A and Figure 6C More importantly, from untreated and treated interferon-... Sixteen and 21 unique HTLV-1 peptides were identified in the treated ED41214(-) cells. Figure 6B , Figure 6D and Figure 6E Only a few HTLV peptides were predicted to be conjugates of common HLA alleles in the patient population (see, for example, Table 7).

[0587] Table 7. HTLV-1 peptide detected in C5 / MJ cells with or without IFN-γ treatment

[0588]

[0589]

[0590]

[0591]

[0592] Strong bonds (grade < 0.5) are shown in bold; weak bonds (grade 0.5-2) are shown in italics; Len = length.

[0593] Peptide distributions were obtained for all tested primary HTLV samples; however, HTLV peptides were detected only for ED-41214(-) (combined search for human UniProt + HTLV sequences). Figure 6A and Figures 7A-7D ).

[0594] Example 3. Validation of the heavy analogue and HTLV peptide in cell lines and calculation of copy number.

[0595] Several HTLV peptides were also selected and validated using synthetic heavy analogues. In short, peptides containing leucine (…) 13 C(6) 15 Synthetic peptides from N(1) (Thermo) were mixed with HLA-eluted peptides from HTLV-transformed cell lines and analyzed by LC-MS / MS. Co-elution of endogenous peptides and heavy peptides, as well as the similarity of fragmentation profiles, were used to identify the endogenous peptides.

[0596] Example 4. Identification of HTLV-specific peptides from HLA-I immunopeptidomics of ATL patient samples.

[0597] Samples were obtained from patients with ATL (adult T-cell leukemia). Peripheral blood mononuclear cells (PBMCs) were suspended in lysis buffer containing 1% NP-40 and incubated at 4°C for 1 hour. HLA class I complexes were then purified using a pan-I anti-HLAW6 / 32 antibody covalently conjugated to agarose beads. The beads were then washed, and the HLA complexes were eluted in acidic solution (pH 2.7). HLA proteins were separated from related peptides using C18-based solid-phase extraction, followed by further analysis of HLA proteins by liquid chromatography-mass spectrometry (LC-MS). Of the 23 patient samples, five samples with cell counts in the 50-100 million cell range were analyzed using a Thermo Orbitrap mass spectrometer, while the remaining 18 samples (10-20 million cells) were analyzed using a Bruker Tims TOF SCP mass spectrometer. Peptide sequences were identified from mass spectrometry raw files using PEAKS online (Bioinformatics Solutions Inc.) by searching raw spectra against human Uniprot protein sequences. To identify the HLA-I-restricted HTLV-1 peptide, FASTA-containing sequences from HTLV RNA reads derived from patient samples were combined with human Uniprot. Patient DNA-seq and RNA-seq data were also used to determine the HLA genotyping of individual patient samples.

[0598] Analysis of all ATL patient samples revealed the presence of a large number of nonamelis representing peptides presented by HLA class I. Figures 8A-8D 800-2,300 nonamers were isolated from ATL1-5 using a Thermo Orbitrap mass spectrometer. Figures 8A-8B A much higher count of nonameric peptides was detected in HLA-I peptidomome analysis of ATL6-23 using a Bruker time-to-follow-SCP mass spectrometer. Figures 8C-8D HLA genotyping of ATL patient samples highlighted the diversity of HLA-I in the patient population, with a higher proportion of patients having the HLA-A02 (12 patient samples), HLA-A11 (6 patient samples), and HLA-A24 (9 patient samples) alleles (Table 8).

[0599] Table 8. HLA genotyping of ATL patient samples

[0600]

[0601] Using analysis from a Thermo Orbitrap mass spectrometer, the peptide LPAPHLTLP (SEQ ID NO:53), derived from the HTLV envelope protein, and the predicted HLA-B51 and HLA-B54 conjugates were identified from ATL1C samples, and an octamer derived from the HTLV polymerase protein, ALPELQAL (SEQ ID NO:72), was identified from both ATL1C and ATL2C samples (Table 9). However, the two unique HTLV peptides detected at the highest frequency in ATL patient samples were QSSSFIFHK (SEQ ID NO:143) and EYTNIPISLL (SEQ ID NO:144), as determined by analysis using a Bruker Tims TOF SCP mass spectrometer (Table 10). Both peptides are derived from the HTLV Tax protein and were detected in 2 / 6 of HLA-A11 (33%) and 3 / 6 of HLA-A24 (50%) patient samples, respectively. Using panNetMHC 4.0 combined with a prediction algorithm, HTLV peptides identified from individual patient samples are matched with their respective HLA alleles to further improve the reliability of peptide identification.

[0602] Table 9. HTLV peptides detected from ATL1-5 samples using a Thermo Orbitrap mass spectrometer

[0603]

[0604] Table 10. HTLV peptides detected from ATL6-23 samples using a Bruker timsTOF SCP mass spectrometer

[0605]

[0606] Len = length

[0607] Example 5. Generating a viral sequence database from HTLV-infected patient samples.

[0608] A database of patient-specific HTLV genome sequences was generated using an RNA-seq-based approach, directly reconstructed from transcribed viral RNA sequences amplified from patient samples (Figure 9). First, de novo sequencing was used to convert total human RNA reads into large contigs, and contigs with loose homology to the HTLV reference genome (NCBI accession number NC_001436.1) were labeled as HTLV-specific sequences. Contigs that were fully aligned to the HTLV reference genome were rearranged in a reference-free manner to assemble the patient-specific HTLV genome sequences. Using this method, seven patient-specific HTLV genome sequences were reconstructed. The genome reconstruction was partial, covering 13% to 42% of the HTLV reference genome. All HTLV coding sequences were then extracted and translated using patient-specific viral sequences. Non-canonical protein sequences with start codons embedded in canonical protein sequences were also identified in each sequence. The RNA sequence read coverage of the patient-specific reconstructed genomes is shown in Figure 9. Figure 10 As shown.

[0609] Table 11 provides a summary of the RNAseq data from this embodiment, including the length of the patient-specific reconstructed genome and its percentage (%) of the HTLV reference genome.

[0610] Table 11. Overview of RNAseq data

[0611]

[0612] RNA sequencing

[0613] Total RNA was extracted from human tissues using the MagMAX kit (ThermoFisher). A strand-specific RNA-seq library was prepared from 1 µg of RNA using the KAPA stranded mRNA-Seq kit (KAPA Biosystems). Twelve rounds of PCR were performed to amplify the library. The amplified library was size-selected at 400–600 bp (base pairs) using PippinHT (Sage Science). Sequencing was performed on an Illumina HiSeq® 2500 (Illumina) using 2 x 100 cycles with multiplexed reads.

[0614] Patient RNA sequence mapping into HTLV reference genome

[0615] Batch RNA-seq reads were aligned to an HTLV reference genome (NCBI accession number NC_001436.1) using minimap2 (v2.17) (see, e.g., Li 2018). Alignments were then sorted by coordinates and quality-controlled using samtoolsflagstats (v1.9) (see, e.g., Li et al., 2009) and bedtools genomeCoverageBed (v2.17.0) (see, e.g., Quinlan et al., 2010). Repetitive sequences were then flagged and removed using the Picard toolkit (v2.18.2) (github.com / broadinstitute / picard).

[0616] Workflow for reconstructing the HTLV genome from patient RNAseq data

[0617] The paired-end Illumina RNA reads from each sample were de novo assembled into large contigs using megahit (see, e.g., Li et al., 2015) (parameter settings: --min-count 3 --k-min 27 --k-max 127 --prune-level 2), and these contigs were plotted onto the HTLV reference genome (NCBI accession number NC_001436.1) using BLAST to select HTLV-specific sequences. BLAST parameters used for sequence comparison included output format '7 std sgi stitle'; minimum E-value = 0.001; gap opening penalty = 5; gap extension penalty = 2; best perfect match length = 11; nucleotide match bonus = 2; nucleotide mismatch bonus = -3. Contigs without BLAST matches were discarded, as were BLAST results with an E-value greater than 0.001, a percentage identity less than 79%, or an alignment length less than 50 nucleotides. A custom script is used to merge overlapping contigs and select the final sequence that covers the full or partial length of the reference genome sequence with the highest identity. The final sequence is then aligned to the reference genome to extract and translate all coding sequences. Non-redundant protein sequences are then added to a custom database for peptide identification via mass spectrometry.

[0618] References

[0619] 1. Li, H., Minimap2: pairwise alignment for nucleotidesequences. Bioinformatics, 2018.34(18): p. 3094-3100.

[0620] 2. Heng Li, BH, Alec Wysoker, Tim Fennell, Jue Ruan, Nils Homer, Gabor Marth, Goncalo Abecasis, Richard Durbin, 1000 Genome Project DataProcessing Subgroup, The Sequence Alignment / Map format andSAMtools. Bioinformatics, 2009.25(16): p. 2078-2079.

[0621] 3. Aaron R Quinlan, IMH, BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics, 2010.26(6): p. 841-842.

[0622] 4. Dinghua Li, C.-ML, Ruibang Luo, Kunihiko Sadakane, Tak-Wah Lam,MEGAHIT: an ultra-fast single-node solution for large and complexmetagenomics assembly via succinct de Bruijn graph. Bioinformatics, 2015.31(10): p. 1674-1676.

[0623] Example 6. Identification of HTLV-1 antigen or epitope.

[0624] This embodiment relates to a nucleic acid assay method and composition for identifying HTLV-1 antigens or epitopes.

[0625] RNA or DNA from one or more tumor cells and / or one or more healthy tissues / cells from cancer patients is sequenced to identify sequences that may contain HTLV-1-related insertions in genes expressed in tumor cells.

[0626] HTLV-1-related insertions in tumor material are assessed using sequencing, such as RNA sequencing. Sequencing data is used to study HTLV-1-related insertions expressed in genes. Peptides containing any identified HTLV-1-related insertions are created on a computer and filtered using predictive algorithms, or used to identify MHC-related epitopes via mass spectrometry data. Where appropriate, physiologically relevant HTLV-1 epitope-specific T cell responses are identified within both or either the CD8+ and / or CD4+ T cell populations using a suitable set of MHC-binding HTLV-1 epitopes, through functional assays and / or MHC multimer-based screening.

[0627] HTLV-1 epitopes are identified by sequencing the genome and / or exome of tumor tissue and / or healthy tissue from cancer patients using next-generation sequencing (NGS) methods. NGS technology is used to sequence genes selected based on the presence of HTLV-1-related insertions and their ability to act as antigens. NGS is applied to (but not limited to) genome sequencing, genome resequencing, epigenomic characterization, DNA-protein interaction (ChIP) sequencing, and transcriptome profiling (RNA-Seq). Similar to DNA-based assays using methods such as NGS or massively parallel sequencing (MPS), RNA from tumors is analyzed by converting RNA into cDNA and generating libraries suitable for sequencing.

[0628] Multiple assays are employed to identify HTLV-1 epitopes in biological samples. Suitable assays for identifying HTLV-1 epitopes in biological samples include, but are not limited to, proteomics, NGS, solution hybridization, array hybridization nucleic acid amplification, polymerase chain reaction (PCR), RT-PCR, quantitative PCR, branched DNA (bDNA) assay, rolling circle amplification (RCA), in situ hybridization, Northern hybridization, hybridization protection assay (HPA), single-molecule hybridization detection, Invader assay and / or oligonucleotide ligation assay (OLA), hybridization, and array analysis.

[0629] Sequencing data obtained by identifying the presence of HTLV-1 epitopes in cancer patients were analyzed to predict personal HTLV-1 peptides that can bind to the individual's HLA molecules. The data were analyzed using a computer. Specifically, the presence of HTLV-1 antigens was analyzed from the sequence data.

[0630] The HTLV-1 antigen was evaluated by assessing its affinity for MHC molecules.

[0631] In the prediction algorithm targeting major HLA-A and HLA-B alleles, a neural network-based learning approach using validated binding and non-binding peptides was employed. The algorithm predicts missense mutations that produce peptides that strongly bind to homologous MHC molecules in cancer patients. A set of peptides representing optimal HTLV-1 epitopes was identified for each patient and prioritized. The HTLV-1 epitope prediction algorithm was used to predict the binding of candidate peptides to MHC class I or MHC class II molecules.

[0632] In some cases, peptide binding tools can be one of the following: antibody epitope prediction, ANTIGENIC, BepiPred, CTLPred, DiscoTope, EPIPREDICT, epitope clustering analysis, epitope conservation analysis, EUiPro, HLA peptide binding prediction, HLA Binding, MAPPP, MHCBench, MHC-I processing prediction, Mosaic Vaccine Tool Suite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, antigenic determinant prediction, ProPred, ProPred-1, RankPep, SMM, SVMHC, APPred, VaxiJen, or combinations thereof. Additional exemplary programs such as BIMAS or SYFPEITHI, Rankpep can also be used.

[0633] In some cases, immune epitope databases and analytical resources (IEDB) (Vita R et al., NucleicAcids Res. 2015;43(Database Issues):D405-D412) are used to identify suitable tumor HTLV-1 antigens. Such algorithms are based on artificial neural networks (ANNs) to predict peptide binding to different MHC class I variants, providing predicted IC50 values. 50 As output, NetMHC (Lundegaard C et al., Nucleic Acids Res. 2008;36 (Network Server Special Issue):W509-W512.) was used. Procedures such as SMMPMBEC (Kim Y et al., BMC Bio informatics. 2009; 10:394) and / or SMM (Peters B et al., BMC Bio informatics. 2005; 6:132) were used. These procedures use position-weight matrices to describe the statistical bias from peptide-MHC I binding data. This approach suppresses noise, for example, caused by the finite number of data points present in the training set and / or experimental errors.

[0634] In some cases, single nucleotide polymorphisms (SNPs) can be removed from candidate HTLV-1 antigens or HTLV-1 epitopes. SNPs comprise a range of molecular variations: (1) SNPs, (2) polynucleotide polymorphisms (MNPs), (3) short deletion and insertion polymorphisms (indels / DIPs), (4) microsatellite markers or short tandem repeats (STRs), (5) heterozygous sequences, and (6) named variants.

[0635] Proteomics-based methods, such as direct protein sequencing, are employed to identify tumor-specific HTLV-1 antigens. Protein sequencing of enzymatic digests using multidimensional MS technologies, including tandem mass spectrometry (MS / MS), is used to identify HTLV-1 antigens. High-throughput methods for de novo sequencing of unknown proteins are used, for example, to analyze the proteome of tumors from cancer patients to identify expressed HTLV-1 antigens. In some cases, meta-shotgun protein sequencing is used to identify expressed HTLV-1 antigens.

[0636] MHC multimers are used to identify tumor-specific HTLV-1 antigens to identify HTLV-1 antigen-specific T-cell responses. For example, MHC tetramer-based screening methods can be used for high-throughput analysis of HTLV-1 antigen-specific T-cell responses in cancer patient samples. Such tetramer-based screening methods are used to identify tumor-specific HTLV-1 antigens or as a secondary screening protocol to assess HTLV-1 antigens that patients may have been exposed to, which can aid in the selection of candidate HTLV-1 antigens. Where appropriate, filters are applied to exclude (1) epitopes with binding affinity lower than the corresponding wild-type sequence and / or (2) epitopes predicted to be inefficiently processed by the immunoproteasome. Candidate mutant peptides are synthesized and screened to identify T-cell HTLV-1 antigens.

[0637] Pulsed antigen-presenting cells (APCs) containing relatively long synthetic peptides with minimal T-cell epitopes were used to identify HTLV-1 epitopes. Non-synonymous mutated epitopes were identified in tumors by assessing the response of CD4+ tumor-infiltrating lymphocytes (TILs) to autologous B cells treated with a pulse containing a single mutated peptide. This approach led to the identification of mutated cell epitopes. Peptide screening assays were performed based on a combination of two peptide libraries: (1) overlapping long peptides, and (2) peptides predicted based on MHC binding. Identification of HTLV-1-responsive T cells that led to the screening.

[0638] HTLV-1 epitopes were identified using a tandem microgene screening method. The tandem microgene construct consisted of, for example (but not limited to), 6 to 24 microgenes encoding polypeptides containing mutated amino acid residues, each flanked by, for example, 12 amino acids at the N-terminus and / or C-terminus. The tandem microgene constructs were synthesized and used to transfect cell lines expressing autologous APCs and / or co-expressing autologous HLA molecules. Using this method, HTLV-1 epitopes were identified in cancer patients (e.g., patients with adult T-cell leukemia / lymphoma (ATL) or HTLV-1-associated myelopathy / tropical spastic paraplegia (HAM / TSP)).

[0639] HTLV-1 epitopes were identified using a combination of whole-exome / transcriptome sequencing analysis, MHC binding prediction, and mass spectrometry to detect peptides eluted from HLA molecules. Predicted highly binding peptides were confirmed by mass spectrometry.

[0640] Example 7. Determination of MHC binding capacity.

[0641] The MHC binding affinity (affinity) of candidate peptides according to this disclosure is tested. A single peptide-MHC (pMHC) complex is generated via UV-ligand exchange. The UV-sensitive peptide is cleaved under UV irradiation and exchanges with the target peptide. Peptide candidates that effectively bind and stabilize the MHC molecule in the peptide-accepting state prevent dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA is performed based on the detection of the light chain (β2m) of the stabilized MHC complex. Briefly, a 96-well plate is coated with streptoacidin, washed, and blocked. Refolded HLA-A monomers are used as standards covering a predetermined concentration range. The peptide-MHC monomers from the UV exchange reaction are diluted in blocking buffer. The sample is incubated, washed, incubated with HRP-conjugated anti-β2m, washed again, and detected using a chromogenic substrate solution, stopping the detection according to the manufacturer's protocol. Absorbance is measured, for example, at 450 nm. Candidate peptides exhibiting high exchange yields are preferred for the generation and preparation of antibodies or fragments thereof and / or T-cell receptors or fragments thereof. The candidate peptides demonstrate affinity for MHC molecules and prevent dissociation of the MHC complex.

[0642] Example 8. Preparation of peptide-MHC (pMHC) complex.

[0643] This embodiment relates to a method for preparing soluble recombinant HLA loaded with HTLV-1-derived peptides.

[0644] Using suitable expression vectors, class I HLA molecules (HLA heavy chain and HLA light chain (β2m)) were expressed in *E. coli* as inclusion bodies. The HLA heavy chain additionally contained a C-terminal biotinylated tag, which replaced, for example, transmembrane and / or cytoplasmic domains. *E. coli* cells were lysed, and the inclusion bodies were processed to approximately 80% purity.

[0645] Inclusion bodies of β2m and the heavy chain were denatured separately in denaturing buffer. A refolding buffer was prepared. The synthesized peptide was dissolved to the final concentration and added to the refolding buffer. Then β2m was added, followed by the heavy chain. Refolding was performed until complete.

[0646] The refolded mixture was then dialyzed. The protein solution was then filtered through a filter and loaded onto a pre-equilibrated exchange column. Proteins were eluted using an additional purifier via a linear salt gradient. The HLA-peptide complex was eluted, and the peak fraction was collected. A mixture of protease inhibitors was added, and the fraction was frozen on ice.

[0647] Using a rapid desalting column equilibrated in the same buffer, the biotinylated pHLA molecules were buffer-displaced. After elution, the protein-containing fraction was cooled on ice, and a mixture of protease inhibitors was added. Then, the biotinylation reagent was added. The mixture was then incubated.

[0648] The biotinylated pHLA molecules are further purified, for example, by gel filtration chromatography using a purifier with a column pre-equilibrated with filtered PBS. The biotinylated pHLA mixture is concentrated to a final volume, loaded onto the column, and developed. The biotinylated pHLA molecules elute, for example, as a single peak. The fractions containing the protein are combined, cooled on ice, and a mixture of protease inhibitors is added. The protein concentration is determined, and the aliquots of the biotinylated pHLA molecules are stored frozen.

[0649] Such peptide-MHC (pMHC) complexes are used in soluble form or immobilized on solid supports via their C-terminal biotin moiety for the detection of T cells and T cell receptors bound to the peptide-MHC complex. For example, these complexes are used for panning phage libraries, performing ELISA assays, and / or fabricating sensor chips for measuring affinity and binding kinetics.

[0650] Example 9. Identification of T-cell receptors (TCRs) that bind to the pMHC complex.

[0651] Antigen-binding T-cell receptors (TCRs) were obtained by panning a TCR phage library using peptides disclosed herein. The library was constructed using α- and β-chain sequences obtained from a natural library. Random combinations of these α- and β-chain sequences occurred during library creation, resulting in a non-natural α / β-chain combination library.

[0652] TCRs obtained from the library were assessed by enzyme-linked immunosorbent assay (ELISA) to confirm specific antigen recognition. ELISA plates were coated with streptoacidin and incubated with biotinylated peptide-HLA complexes. Phage clones carrying the TCRs were added to each well and detected using HRP antibody conjugates. Binding antibodies were detected using a peroxidase substrate system. The absence of binding to other peptide-HLA complexes indicated that the TCRs were not highly cross-reactive.

[0653] The ability of isolated TCRs to bind to the disclosed peptide-HLA complex was further confirmed by surface plasmon resonance (SPR). In this case, the α- and β-chain sequences were expressed in *E. coli* as soluble TCRs. The binding of the soluble TCRs to the complex was analyzed by SPR. Biotinylated peptide-HLA monomers were prepared and immobilized on a sensor chip coupled with streptavidin. To measure affinity, serial dilutions of the soluble TCRs were passed through the immobilized peptide-HLA, and the equilibrium response at each concentration was determined. For example, the data were analyzed by plotting specific equilibrium binding against protein concentration and then, assuming a 1:1 interaction, performing least-squares fitting using the Langmuir binding equation.

[0654] TCRs that specifically recognize the peptide-HLA complex disclosed herein were obtained from the library. Data generated from the above experiments confirmed that antigen-specific TCRs could be isolated.

[0655] Example 10. Characterization of MHC binding and stability of the pMHC complex.

[0656] Peptide binding assay based on T2 cells. T2 cells that do not express antigen-processing-associated transporters (TAPs) and therefore do not assemble stable class I MHC on the cell surface were pulsed, washed, and detected with fluorescently labeled antibodies that recognize class I MHC (e.g., the A2 allele) using a FACS scanning analyzer. The difference in MFI (mean fluorescence intensity) corresponding to a given POI concentration between the positive control and the negative control (non-MHC binder) is a function of the amount of stable pMHC complexes displayed on the cell surface. Therefore, at limited concentrations of the peptide, it is primarily responsible for K+. on The measurement, and at the saturation level of the peptide, it is related to K. on and K offBoth are measured. Binding is quantified using two relevant factors: relative affinity (1 / RA) and half-maximal binding (the peptide concentration reaching 50% of the signal corresponding to saturation). Relative affinity RA is the binding normalized to a reference (e.g., wild-type peptide in the case of testing mutant POIs), for example, the ratio between the control and the half-maximal binding of the POI. A higher 1 / RA index indicates a lower half-maximal binding and a higher K0 of the interaction between the POI and MHC. on The higher.

[0657] Binding and stability were characterized by ELISA. Peptide binding, affinity, and dissociation rates are assessed using microtiter plates coated with avidin containing class I monomers loaded with locating peptides. The monomer-coated plates are provided as part of a kit (e.g., the iTopia Epitope Discovery System kit). The kit also provides assay buffer, anti-MHC-FITC mAb, β2-microglobulin, and a control peptide.

[0658] Binding Assay: The ability of the POI to bind to each MHC molecule was first assessed by a binding assay. This assay measures the ability of a single peptide to bind to an HLA molecule under optimal normalized binding conditions. First, the monomer-coated plate was peeled off to release the occupier peptide, leaving only the MHC heavy chain bound to the plate. Then, the test peptide and anti-MHC-FITC monoclonal antibody were introduced under optimal folding conditions. The plate was incubated. The anti-MHC-FITC monoclonal antibody preferentially binds to the refolded MHC complex. Therefore, the fluorescence intensity produced by each peptide is correlated with the peptide's ability to complex with an MHC molecule. The binding of each peptide was assessed relative to the positive control peptide, and the results were expressed as, for example, percentage of binding (%).

[0659] Affinity assay: For the affinity assay, after the initial stripping of the occupier peptide, incremental concentrations of POI were added to a series of wells and incubated under the aforementioned conditions. The plates were read on a fluorometer. A dose-response curve was generated. The amount of peptide required to reach 50% of the maximum value was recorded as the ED50 value.

[0660] Dissociation rate determination: After incubation under the specified conditions, the plate was washed to remove excess peptides. The plate was then incubated on allele-specific monomer plates. The relative fluorescence intensity of the plate was measured at multiple time points (e.g., 0, 0.5, 1, 1.5, 2, 4, 6, and 8 hours). The time required for 50% dissociation of the peptides from the MHC monomer was defined as the T½ value (in hours).

[0661] iScore Calculation: iScore is a multi-parameter calculation provided in the iTopia software. Its value is calculated based on a combination of stability, affinity, and stability data.

[0662] Example 11. Measurement of response to tumor cells.

[0663] A suitable number of mice were immunized by direct inoculation with a plasmid expressing the HTLV-1 peptide disclosed herein. As a non-limiting example, appropriate concentrations of the plasmid were inoculated into the lymph nodes (e.g., inguinal lymph nodes) of mice on day 0 and on subsequent days of the experimental time course (e.g., day 3, day 14, and day 17). In some cases, one or more additional peptide booster immunizations were subsequently performed on subsequent days (e.g., day 28 and day 31) using a negative control peptide and a POI. Splenocytes were stimulated in vitro with the POI and tested for their activity against chromium-51 (C-51) at different E:T ratios. 51 The role of Cr-labeled tumor cells.

[0664] Example 12. In vivo assessment of immune enhancement against HTLV-1 peptide.

[0665] An appropriate number of mice were immunized by direct inoculation with a plasmid expressing the HTLV-1 peptide disclosed herein. As a non-limiting example, an appropriate concentration of plasmid was inoculated into the lymph nodes (e.g., inguinal lymph nodes) of the mice on day 0 and on subsequent days of the experimental time course (e.g., day 3, day 14, and day 17). In some cases, one or more additional peptide booster immunizations were subsequently performed on subsequent days (e.g., day 28 and day 31) using a negative control peptide and a POI.

[0666] To assess the in vivo response to the HTLV-1 peptide, spleen cells were isolated from littermate control mice and incubated with one or more appropriate concentrations of POI for a predetermined period. These cells were then stained with CFSEhi fluorescence and co-injected intravenously into immunized mice along with an equal volume of control spleen cells stained with CFSElo fluorescence. After the predetermined period, the specific elimination of target cells was measured by removing spleens and PBMCs from the attacked animals and measuring CFSE fluorescence by flow cytometry. The relative exhaustion of the population of spleen cells corresponding to the peptide-loaded population was calculated relative to the control (unloaded) population and expressed as the percentage (%) of specific lysis.

[0667] Example 13. Testing the ability of POIs to enhance immunogenicity and overcome tolerance.

[0668] POIs were used in vitro for blood immunization to generate cytotoxic T lymphocytes (CTLs).

[0669] PBMCs from normal donors were purified from the erythrocyte sedimentation rate (ESR) amber layer by centrifugation in a standard sterile medium designed for lymphocyte isolation. Cultures were prepared using autologous plasma (AP). For the in vitro generation of peptide-specific CTLs, autologous dendritic cells (DCs) were used as antigen-presenting cells (APCs). DCs were generated, and CTLs were induced with DCs and peptides derived from PBMCs. Cell fractions enriched with monocytes were cultured to induce maturation. Specific numbers of CD8+-enriched T lymphocytes were co-cultured with peptide-pulsed DCs. Cultures were restimulated with autologous, peptide-pulsed dendritic cells at different days. Immunogenicity was assessed using in vitro cytotoxicity and cytokine production assays.

[0670] Example 14. CD8+ T cell response to peptides.

[0671] Based on predicted or experimentally validated HLA-binding peptides, it is determined whether T cells capable of recognizing tumor-specific peptides can be generated. Peptides with appropriate binding scores are synthesized. To generate T cells with the desired specificity, T cells are stimulated using a predetermined protocol, e.g., in the presence of IL-2 and IL-7, with autologous APCs (such as dendritic cells and / or autologous B cells expanded with CD40L) via peptide pulses (single peptide or peptide library). After multiple rounds of stimulation, expanded CD8+ cells are tested on an ELISpot, and evidence of responsiveness to the peptide is analyzed based on IFNγ secretion.

[0672] Example 15. Assay of cytokine production.

[0673] For cytokines (e.g., IL-2 and IFN-γ) The production assay involved collecting T cells after contact with a peptide pulsed APC, centrifuging them, and simultaneously collecting the cell pellet and supernatant. Cytokine production was measured from the supernatant using ELISA.

[0674] Example 16. In vivo activation of viral peptide-specific T cells as a method to limit viral production.

[0675] For in vivo studies, the ability of viral peptides to enhance CD8+ T cell responses in the context of MHC I molecules (e.g., HLA-A2, HLA-A24, HLA-A11) as a means of limiting viral production after AAV-HTLV-1 challenge was tested in transgenic mice expressing one or more human HLA molecules.

[0676] The resulting molecules (i) exhibit one or more MHC class I molecules displaying the target HTLV-1 viral peptide (POI) or (ii) exhibit OVA 257-264 Control MHC class I molecules for peptides (OVA).

[0677] First, a group of transgenic mice expressing one or more human HLA molecules were injected with either an MHC I molecule / peptide or an MHC I molecule / OVA complex, and the levels of effector and memory CD8+ T cells were monitored at different time points post-injection (from the spleen and / or blood). Mice simulating the injection served as a negative control group. Then, a second group of mice was injected with the MHC I molecule / POI complex, followed by challenge with AAV-HTLV-1 at post-injection time points determined based on measurements of effector and memory CD8+ T cell levels in the first experiment. The control group consisted of (i) mice not injected with the MHC I molecule / POI complex but challenged with AAV-HTLV-1, (ii) mice injected with the MHC I molecule / OVA complex, and (iii) mice injected with the MHC I molecule / POI complex but not challenged with AAV-HTLV-1. Viral load was analyzed at different post-injection time points to determine the limitation of viral infection by MHC I molecule / POI through CD8+ T cell activation.

[0678] Example 17. Activating antigen-specific T cells in vivo using viral peptides as a method to reduce viral production.

[0679] For in vivo studies, the ability of a targeted HTLV-1 viral peptide (POI) against an MHC I molecule (e.g., HLA-A2, HLA-A24, HLA-A11) to enhance T cell responses against HTLV-1 virus after AAV-HTLV-1 challenge was tested in transgenic mice expressing one or more human HLA molecules. More specifically, in vivo activation of peptide-specific CD8+ T cells was compared after (i) POI immunization, (ii) acute challenge with AAV-HTLV-1, and / or (iii) chronic challenge with AAV-HTLV-1.

[0680] The resulting molecules were (i) MHC class I molecules exhibiting the HTLV-1 viral POI or (ii) molecules exhibiting OVA. 257-262 Control MHC class I molecules for peptides (OVA).

[0681] First, transgenic mice expressing one or more human HLA molecules were challenged with AAV-HTLV-1. Then, at 1 and 7 days post-acute challenge, and at 1, 7, and 14 days post-chro...

Claims

1. An isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof, said isolated peptide comprising an amino acid sequence having at least 90% identity with an amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146, wherein said isolated peptide is 5-20 amino acids in length.

2. The isolated peptide according to claim 1, wherein the isolated peptide comprises the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

3. The isolated peptide according to claim 1 or 2, wherein the isolated peptide is substantially composed of the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

4. The isolated peptide according to any one of claims 1-3, wherein the isolated peptide consists of the amino acid sequence of any one of SEQ ID NO: 1-89 and 143-146.

5. An isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof, said isolated peptide comprising two or more amino acid sequences selected from any one of SEQ ID NO: 1-89 and 143-146.

6. The isolated peptide according to any one of claims 1-5, wherein the isolated peptide comprises one or more reverse peptide bonds, one or more non-peptide bonds, one or more D-isomers of amino acids, one or more chemical modifications, or any combination thereof.

7. The isolated peptide according to any one of claims 1-6, wherein the isolated peptide is produced by expression in a heterologous host cell.

8. The isolated peptide according to any one of claims 1-6, wherein the isolated peptide is synthesized.

9. The isolated peptide according to any one of claims 1-8, wherein the isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof, when presented on the surface of antigen-presenting cells (APCs) in a complex with a major histocompatibility complex (MHC) molecule, induces a human T-lymphotropic virus type 1 (HTLV-1)-specific immune response in a subject.

10. A fusion protein comprising one or more isolated peptides according to any one of claims 1-9 fused to one or more heterologous molecules.

11. The fusion protein of claim 10, wherein the one or more heterologous molecules enhance peptide-specific immune responses in the subject.

12. The fusion protein of claim 10, wherein the one or more heterologous molecules mediate the delivery of the peptide to a specific site in the body of the subject.

13. The fusion protein according to any one of claims 10-12, wherein the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

14. A conjugate comprising one or more isolated peptides according to any one of claims 1-9 conjugated to one or more heterologous molecules.

15. The conjugate of claim 14, wherein the one or more heterologous molecules enhance peptide-specific immune responses in the subject.

16. The conjugate of claim 14, wherein the one or more heterologous molecules mediate the delivery of the peptide to a specific site within the body of the subject.

17. The conjugate according to any one of claims 14-16, wherein the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

18. The conjugate according to any one of claims 14-16, wherein the one or more peptides are conjugated to the particles.

19. An oligomeric complex comprising two or more isolated peptides according to any one of claims 1-9.

20. A non-covalent complex comprising a separated peptide and an MHC molecule, or a fragment or derivative thereof, according to any one of claims 1-9.

21. The non-covalent complex of claim 20, wherein the MHC molecule or a fragment thereof is a class I MHC molecule.

22. The non-covalent complex according to claim 21, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

23. The non-covalent complex of claim 20, wherein the MHC molecule or a fragment thereof is a class II MHC molecule.

24. The non-covalent complex according to claim 23, wherein the type II MHC molecule is a type II HLA molecule.

25. A fusion protein comprising a separated peptide and an MHC molecule, or a fragment or derivative thereof, according to any one of claims 1-9.

26. The fusion protein of claim 25, wherein the MHC molecule or a fragment thereof is a class I MHC molecule.

27. The fusion protein of claim 26, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

28. The fusion protein of claim 25, wherein the MHC molecule or a fragment thereof is a class II MHC molecule.

29. The fusion protein of claim 28, wherein the class II MHC molecule is a class II HLA molecule.

30. A conjugate comprising the isolated peptide and MHC molecule, or fragments or derivatives thereof, according to any one of claims 1-9.

31. The conjugate according to claim 30, wherein the MHC molecule or a fragment thereof is a class I MHC molecule.

32. The conjugate according to claim 31, wherein the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

33. The conjugate according to claim 30, wherein the MHC molecule or a fragment thereof is a class II MHC molecule.

34. The conjugate according to claim 33, wherein the type II MHC molecule is a type II HLA molecule.

35. A pharmaceutical composition comprising (i) one or more isolated peptides according to any one of claims 1-9, one or more fusion proteins according to any one of claims 10-13 and 25-29, one or more conjugates according to any one of claims 14-18 and 30-34, one or more oligomeric complexes according to claim 19, or one or more non-covalent complexes according to any one of claims 20-24, or any combination thereof; and (ii) a pharmaceutically acceptable carrier or excipient.

36. The pharmaceutical composition according to claim 35, further comprising an adjuvant.

37. An isolated molecule that combines an isolated peptide according to any one of claims 1-9, a fusion protein according to any one of claims 10-13 and 25-29, a conjugate according to any one of claims 14-18 and 30-34, an oligomeric complex according to claim 19, or a non-covalent complex according to any one of claims 20-24.

38. The isolated molecule according to claim 37, wherein the molecule is an antibody or an antigen-binding fragment thereof.

39. The isolated molecule according to claim 38, wherein the antibody is a bispecific antibody.

40. The isolated molecule of claim 37, wherein the molecule is a replacement scaffold.

41. The isolated molecule according to claim 37, wherein the molecule is a chimeric antigen receptor (CAR).

42. The isolated molecule according to claim 37, wherein the molecule is a T-cell receptor (TCR).

43. An isolated cell comprising the CAR according to claim 41.

44. The isolated cells according to claim 43, wherein the isolated cells are immune cells.

45. The isolated cells according to claim 44, wherein the immune cells are T cells, NK cells, or macrophages.

46. ​​An isolated cell comprising the TCR according to claim 42.

47. The isolated cells according to claim 46, wherein the isolated cells are immune cells.

48. The isolated cells according to claim 47, wherein the immune cells are T cells, NK cells, or macrophages.

49. A pharmaceutical composition comprising (i) an isolated molecule according to any one of claims 38-43, or an isolated cell according to any one of claims 43-48; and (ii) a pharmaceutically acceptable carrier or excipient.

50. An isolated polynucleotide comprising a nucleotide sequence encoding one or more isolated peptides according to any one of claims 1-9 or a fusion protein according to any one of claims 10-13 and 25-29.

51. The isolated polynucleotide of claim 50, wherein the nucleotide sequence is operatively linked to a promoter.

52. The isolated polynucleotide of claim 50 or claim 51, wherein the isolated polynucleotide comprises DNA.

53. The isolated polynucleotide according to claim 50 or claim 51, wherein the isolated polynucleotide comprises RNA.

54. The isolated polynucleotide according to claim 53, wherein the RNA is mRNA.

55. The isolated polynucleotide according to claim 53, wherein the RNA is a self-replicating RNA.

56. A vector comprising isolated polynucleotides according to any one of claims 50-55.

57. The carrier according to claim 56, wherein the carrier is an expression carrier.

58. The vector according to claim 56 or 57, wherein the vector is a viral vector.

59. A host cell comprising an isolated polynucleotide according to any one of claims 50-55 or a vector according to any one of claims 56-58.

60. The host cell according to claim 59, wherein the host cell is a prokaryotic cell.

61. The host cell according to claim 59, wherein the host cell is a eukaryotic cell.

62. The host cell of claim 61, wherein the host cell is an APC.

63. A pharmaceutical composition comprising (i) an isolated polynucleotide according to any one of claims 50-55, or a carrier according to any one of claims 56-58; and (ii) a pharmaceutically acceptable carrier or excipient.

64. The pharmaceutical composition of claim 63, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle carrier.

65. A method for inducing an immune response against HTLV-1 infection in a subject in need, the method comprising administering to the subject a therapeutically effective amount of: a) one or more isolated peptides according to any one of claims 1-9; b) The fusion protein according to any one of claims 10-13 and 25-29; c) The conjugate according to any one of claims 14-18 and 30-34; d) The oligomeric complex according to claim 19; e) A non-covalent complex according to any one of claims 20-24; f) The pharmaceutical composition according to any one of claims 35, 36, 49, 63 and 64; g) The molecule according to any one of claims 37-42; h) Cells isolated according to any one of claims 43-48 and 59-62; i) isolated polynucleotides according to any one of claims 50-55; or j) The carrier according to any one of claims 56-58.

66. A method for inducing an immune response against HTLV-1 infection in a subject in need, the method comprising administering to the subject a therapeutically effective amount of one or more isolated peptides according to any one of claims 1-9.

67. A method for inducing an immune response against HTLV-1 infection in a subject in need, the method comprising administering activated T cells to the subject, the activated T cells being generated by contacting the T cells with an APC, the APC presenting a separated peptide complexed with an MHC molecule according to any one of claims 1-9.

68. A method of treating an HTLV-1-induced disease or condition in a subject in need, the method comprising administering an effective amount of: a) one or more isolated peptides according to any one of claims 1-9; b) The fusion protein according to any one of claims 10-13 and 25-29; c) The conjugate according to any one of claims 14-18 and 30-34; d) The oligomeric complex according to claim 19; e) A non-covalent complex according to any one of claims 20-24; f) The pharmaceutical composition according to any one of claims 35, 36, 49, 63 and 64; g) The molecule according to any one of claims 37-42; h) Cells isolated according to any one of claims 43-48 and 59-62; i) isolated polynucleotides according to any one of claims 50-55; or j) The carrier according to any one of claims 56-58.

69. A method for preventing or reducing the likelihood of HTLV-1-induced disease or condition in a subject in need, the method comprising administering an effective amount of: a) one or more isolated peptides according to any one of claims 1-9; b) The fusion protein according to any one of claims 10-13 and 25-29; c) The conjugate according to any one of claims 14-18 and 30-34; d) The oligomeric complex according to claim 19; e) A non-covalent complex according to any one of claims 20-24; f) The pharmaceutical composition according to any one of claims 35, 36, 49, 63 and 64; g) The molecule according to any one of claims 37-42; h) Cells isolated according to any one of claims 43-48 and 59-62; i) isolated polynucleotides according to any one of claims 50-55; or j) The carrier according to any one of claims 56-58.

70. A method for treating HTLV-1 induced disease or condition in a subject in need, the method comprising administering to the subject an effective amount of one or more isolated peptides according to any one of claims 1-9.

71. A method for preventing or reducing the likelihood of HTLV-1-induced disease or condition in a subject in need, the method comprising administering to the subject an effective amount of one or more isolated peptides according to any one of claims 1-9.

72. The method according to any one of claims 69-71, wherein the HTLV-1 induced disease or condition is adult T-cell leukemia / lymphoma (ATL) or HTLV-1 myelopathy / tropical spastic paralysis (HAM / TSP).

73. A reagent kit comprising: (i) a) one or more isolated peptides according to any one of claims 1-9; b) The fusion protein according to any one of claims 10-13 and 25-29; c) The conjugate according to any one of claims 14-18 and 30-34; d) The oligomeric complex according to claim 19; e) A non-covalent complex according to any one of claims 20-24; f) The pharmaceutical composition according to any one of claims 35, 36, 49, 63 and 64; g) The molecule according to any one of claims 37-42; h) Cells isolated according to any one of claims 43-48 and 59-62; i) isolated polynucleotides according to any one of claims 50-55; or j) The carrier according to any one of claims 56-58; and (ii) Its packaging and / or instructions for use.

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