Viral Peptide and Its Use

Abstract

The present disclosure provides isolated peptides derived from hepatitis B virus (HBV), peptide-based molecules (e.g., peptide-MHC (pMHC) complexes), polynucleotides and vectors encoding these peptides or peptide-based molecules, pharmaceutical compositions (e.g., vaccine compositions), and uses thereof for the treatment or prevention of HBV infection and / or diseases induced by HBV. The present disclosure also provides binding moiety structures that bind to the peptides or peptide-based molecules disclosed herein, and uses thereof for the treatment or prevention of HBV infection and / or diseases induced by HBV. The present disclosure further provides methods and systems for identifying immunogenic virus-derived peptides.

Description

Cross - Reference to Related Applications

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 398,305, filed on August 16, 2022, and U.S. Provisional Patent Application No. 63 / 349,804, filed on June 7, 2022. The disclosures of these provisional patent applications are hereby incorporated by reference in their entirety for all purposes. Sequence Listing

[0002] This application includes a sequence listing, which is submitted electronically in XML file format and is hereby incorporated by reference in its entirety. This XML copy, created on May 30, 2023, has a file name of 250298_000482_SL.xml and a size of 98,432 bytes.

Technical Field

[0003] The present disclosure relates to methods for isolated peptides derived from hepatitis B virus (HBV), compositions comprising such isolated peptides, and the use of such methods for the treatment or prevention of HBV infection and / or diseases induced by HBV.

Background Art

[0004] Chronic hepatitis B virus (HBV) infection affects approximately 250 million people worldwide and can lead to severe complications including acute and chronic hepatitis and hepatocellular carcinoma (HCC). Vaccination against HBV is effective in preventing infection, but currently, there is no treatment for patients chronically infected with HBV.

[0005] The persistence or control of HBV infection is mainly determined by the host immune response mediated by cytotoxic T cells, which mediate adaptive immunity by recognizing HLA-peptide complexes presented on the surface of infected cells through interactions via the T cell receptor (TCR). Proteogenomics can be applied to the identification of HLA-related variant peptides in cancer, but HBV-HCC tumor cells may contain fragments of HBV DNA inserted into the host genome, which ultimately makes it difficult to identify HBV epitopes. Further complicating this endeavor is the existence of thousands of HBV strains distributed across 10 different HBV genotypes, which differ from each other by at least 8% at the nucleotide level. Therefore, the development of methods for identifying HBV epitopes may be useful for patients infected with HBV.

Summary of the Invention

Problems to be Solved by the Invention

[0006] As described in the "Background Art" section above, there is a high demand in the art for the development of methods for identifying HBV epitopes that may be useful for patients infected with HBV. This application addresses these and other needs.

Means for Solving the Problems

[0007] In one aspect, provided herein is an isolated peptide comprising an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the length of the isolated peptide is 8-12 amino acids.

[0008] In some embodiments, the isolated peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112.

[0009] In some embodiments, the isolated peptide consists essentially of any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112.

[0010] In some embodiments, the isolated peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112.

[0011] In another aspect, provided herein is an isolated peptide comprising two or more amino acid sequences selected from any one of SEQ ID NOs: 1-54 and 110-112, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0012] In some embodiments, the isolated peptide consists of the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), where X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H, or L, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0013] 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.

[0014] In some embodiments, the isolated peptide is produced by expression in a heterologous host cell.

[0015] In some embodiments, the isolated peptide is generated by synthesis.

[0016] In some embodiments, an isolated peptide, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, elicits a hepatitis B virus (HBV)-specific immune response in a subject when presented as a complex with a major histocompatibility complex (MHC) molecule on the surface of an antigen presenting cell (APC).

[0017] In another aspect, provided herein is a fusion protein comprising one or more isolated peptides disclosed herein fused to one or more heterologous molecules.

[0018] In some embodiments, the one or more heterologous molecules enhance a peptide-specific immune response in a subject.

[0019] In some embodiments, the one or more heterologous molecules mediate delivery of the peptide to a specific site in a subject.

[0020] In some embodiments, the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

[0021] In another aspect, provided herein is a conjugate comprising one or more isolated peptides disclosed herein conjugated to one or more heterologous molecules.

[0022] In some embodiments, the one or more heterologous molecules enhance a peptide-specific immune response in a subject.

[0023] In some embodiments, the one or more heterologous molecules mediate delivery of the peptide to a specific site in a subject.

[0024] In some embodiments, the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

[0025] In some embodiments, one or more peptides are conjugated to the particles.

[0026] In another aspect, provided herein is an oligomeric complex comprising two or more isolated peptides disclosed herein.

[0027] In another aspect, provided herein is a non-covalent complex comprising an isolated peptide disclosed herein and an MHC molecule or a fragment or derivative thereof.

[0028] In some embodiments, the MHC molecule or a fragment thereof is a class I MHC molecule.

[0029] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

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

[0031] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0032] In another aspect, provided herein is a fusion protein comprising an isolated peptide disclosed herein and an MHC molecule or a fragment or derivative thereof.

[0033] In some embodiments, the MHC molecule or a fragment thereof is a class I MHC molecule.

[0034] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

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

[0036] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0037] In another aspect, provided herein is a conjugate comprising an isolated peptide disclosed herein and an MHC molecule or a fragment or derivative thereof.

[0038] In some embodiments, the MHC molecule or a fragment thereof is a class I MHC molecule.

[0039] In some embodiments, the class I MHC molecule is a class I human leukocyte antigen (HLA) molecule.

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

[0041] In some embodiments, the class II MHC molecule is a class II HLA molecule.

[0042] In another aspect, provided herein is a pharmaceutical composition comprising (i) one or more isolated peptides disclosed herein, one or more fusion proteins disclosed herein, one or more conjugates disclosed herein, one or more oligomeric complexes disclosed herein, or one or more non-covalent complexes disclosed herein, or any combination thereof; and (ii) a pharmaceutically acceptable carrier or excipient.

[0043] In some embodiments, the pharmaceutical composition disclosed herein further comprises an adjuvant.

[0044] In another aspect, provided herein is an isolated molecule that binds to an isolated peptide, a fusion protein, a conjugate, an oligomeric complex, or a non-covalent complex disclosed herein.

[0045] In some embodiments, the molecule is an antibody or an antigen-binding fragment thereof.

[0046] In some embodiments, the antibody is a bispecific antibody.

[0047] In some embodiments, the molecule is an alternative scaffold.

[0048] In some embodiments, the molecule is a chimeric antigen receptor (CAR).

[0049] In some embodiments, the molecule is a T cell receptor (TCR).

[0050] In another aspect, provided herein is an isolated cell comprising a CAR disclosed herein.

[0051] In some embodiments, the isolated cell is an immune cell.

[0052] In some embodiments, the immune cell is a T cell, an NK cell, or a macrophage.

[0053] In another aspect, provided herein is an isolated cell comprising a TCR disclosed herein.

[0054] In some embodiments, the isolated cell is an immune cell.

[0055] In some embodiments, the immune cell is a T cell, an NK cell, or a macrophage.

[0056] In another aspect, provided herein is a pharmaceutical composition comprising (i) an isolated molecule disclosed herein, or an isolated cell disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0057] In another aspect, provided herein is an isolated polynucleotide comprising a nucleotide sequence encoding one or more of the isolated peptides disclosed herein or a fusion protein disclosed herein.

[0058] In some embodiments, the nucleotide sequence is operably linked to a promoter.

[0059] In some embodiments, the isolated polynucleotide comprises DNA.

[0060] In some embodiments, the isolated polynucleotide comprises RNA.

[0061] In some embodiments, the RNA is mRNA.

[0062] In some embodiments, the RNA is self-replicating RNA.

[0063] In another aspect, provided herein is a vector comprising an isolated polynucleotide disclosed herein.

[0064] In some embodiments, the vector is an expression vector.

[0065] In some embodiments, the vector is a viral vector.

[0066] In another aspect, provided herein is a host cell comprising an isolated polynucleotide disclosed herein or a vector disclosed herein.

[0067] In some embodiments, the host cell is a prokaryotic cell.

[0068] In some embodiments, the host cell is a eukaryotic cell.

[0069] In some embodiments, the host cell is an APC.

[0070] In another aspect, provided herein is a pharmaceutical composition comprising: (i) an isolated polynucleotide disclosed herein, or a vector disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0071] In some embodiments, the pharmaceutically acceptable carrier is a lipid nanoparticle carrier.

[0072] In another aspect, provided herein is a method of inducing an immune response against hepatitis B virus (HBV) infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: a) one or more isolated peptides disclosed herein; b) a fusion protein disclosed herein; c) a conjugate disclosed herein; d) an oligomeric complex disclosed herein; e) a non-covalent complex disclosed herein; f) a pharmaceutical composition disclosed herein; g) a molecule disclosed herein; h) an isolated cell disclosed herein; i) an isolated polynucleotide disclosed herein; or j) a vector disclosed herein comprising the step of administering.

[0073] In another aspect, provided herein is a method of inducing an immune response against hepatitis B virus (HBV) infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more isolated peptides disclosed herein.

[0074] In another aspect, provided herein is a method of inducing an immune response against HBV infection in a subject in need of inducing an immune response against HBV infection, the method comprising administering activated T cells to the subject, wherein the activated T cells are generated by contacting T cells with an APC that presents an isolated peptide disclosed herein as a complex with an MHC molecule.

[0075] In another aspect, provided herein is a method of treating a disease or disorder induced by HBV in a subject in need of treating a disease or disorder induced by HBV, the method comprising administering to the subject an effective amount of: a) one or more isolated peptides disclosed herein; b) a fusion protein disclosed herein; c) a conjugate disclosed herein; d) an oligomer complex disclosed herein; e) a non-covalent complex disclosed herein; f) a pharmaceutical composition disclosed herein; g) a molecule disclosed herein; h) an isolated cell disclosed herein; i) an isolated polynucleotide disclosed herein; or j) a vector disclosed herein comprising the step of administering.

[0076] In another aspect, provided herein is a method of preventing or reducing the likelihood of a disease or disorder induced by HBV in a subject in need of preventing or reducing the likelihood of a disease or disorder induced by HBV, the method comprising administering to the subject an effective amount of: a) one or more isolated peptides disclosed herein; b) a fusion protein disclosed herein; c) a conjugate disclosed herein; d) The oligomeric complexes disclosed herein; e) The non-covalent complexes disclosed herein; f) The pharmaceutical compositions disclosed herein; g) The molecules disclosed herein; h) The isolated cells disclosed herein; i) The isolated polynucleotides disclosed herein; or j) The vectors disclosed herein comprising the step of administering.

[0077] In another aspect, provided herein is a method of treating a disease or disorder induced by HBV in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides disclosed herein.

[0078] In another aspect, provided herein is a method of preventing or reducing the likelihood of a disease or disorder induced by HBV in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides disclosed herein.

[0079] In some embodiments, the disease or disorder induced by HBV is hepatitis, liver fibrosis, cirrhosis, or liver cancer.

[0080] In some embodiments, the liver cancer is hepatocellular carcinoma (HCC).

[0081] In another aspect, provided herein is a kit, the kit comprising: (i) a) one or more isolated peptides disclosed herein; b) the fusion proteins disclosed herein; c) the conjugates disclosed herein d) an oligomeric complex disclosed herein; e) a non-covalent complex disclosed herein; f) a pharmaceutical composition disclosed herein; g) a molecule disclosed herein; h) an isolated cell disclosed herein; i) an isolated polynucleotide disclosed herein; or j) a vector disclosed herein; and (ii) the packaging and / or instructions for use of the above kit comprising.

[0082] In another aspect, provided herein is a method for identifying an immunogenic virus-derived peptide, the method comprising: a) obtaining a plurality of RNA contig sequences derived from an infected subject infected with a virus, the plurality of RNA contig sequences comprising a plurality of virus-derived RNA contig sequences and a plurality of infected subject endogenous RNA contig sequences; b) identifying the plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences; c) performing assembly of the viral RNA sequence based on the plurality of virus-derived RNA contig sequences; d) identifying a protein sequence based on the viral RNA sequence; and e) identifying an immunogenic virus-derived peptide based at least in part on the identified protein sequence comprising.

[0083] In some embodiments, the plurality of RNA contig sequences are derived from a single infected subject.

[0084] In some embodiments, the infected subject is human.

[0085] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from the virus that infected the subject.

[0086] In some embodiments, the plurality of subject endogenous RNA contig sequences are derived from the endogenous RNA of the subject.

[0087] In some embodiments, the step of identifying a plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences comprises: comparing at least a portion of the contig sequences of the plurality of RNA contig sequences to a reference virus sequence; and identifying the plurality of virus-derived RNA contig sequences such that each contig sequence of the plurality of virus-derived RNA contig sequences comprises at least a portion corresponding to the reference virus sequence including.

[0088] In some embodiments, each contig sequence of the plurality of virus-derived RNA contigs is different from the plurality of subject endogenous RNA contig sequences.

[0089] In some embodiments, each contig sequence of the plurality of virus-derived RNA contig sequences does not include a subject endogenous RNA contig sequence.

[0090] In some embodiments, the reference virus sequence includes a reference genome.

[0091] In some embodiments, the reference genome includes a hepatitis B virus genome.

[0092] In some embodiments, the step of performing an assembly of the viral RNA sequence based on the plurality of virus-derived RNA contig sequences comprises: Performing assembly of the viral RNA sequence by overlapping common sequence portions at the ends of at least a portion of a plurality of virus-derived RNA contig sequences such that at least a portion of the plurality of virus-derived RNA contig sequences overlap linearly comprising.

[0093] In some embodiments, the step of identifying a protein sequence based on a viral RNA sequence such that the identified protein sequence includes translation of the viral RNA sequence is: the step of identifying the protein sequence without the need for comparison with a database of viral proteins comprising.

[0094] In some embodiments, the step of identifying a protein sequence based on a viral RNA sequence such that the identified protein sequence includes translation of the viral RNA sequence is: identifying a plurality of protein sequences, each based on the viral RNA sequence, such that each of the plurality of protein sequences includes translation of the viral RNA sequence; and identifying a protein sequence as a protein sequence that frequently occurs within the plurality of protein sequences further comprising.

[0095] In some embodiments, the protein sequence identified based on the viral RNA sequence is related to an infected subject.

[0096] In some embodiments, the step of identifying an immunogenic virus-derived peptide based at least in part on a protein sequence is: identifying MHC molecules related to the infected subject; identifying, based at least in part on the protein sequence, one or more peptides such that each of the one or more peptides forms an MHC-peptide complex with the MHC molecule; and The step of identifying an immunogenic virus-derived peptide based on the one or more peptides described above is included.

[0097] In another aspect, provided herein is a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium including instructions that, when executed by the one or more processors, cause the computing device to: a) receive, as input, a plurality of RNA contig sequences derived from an infected subject infected with a virus, wherein the plurality of RNA contig sequences includes a plurality of virus-derived RNA contig sequences and a plurality of infected subject endogenous RNA contig sequences, and the infected subject is infected with the virus; b) identify the plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences; c) perform an assembly of the viral RNA sequence based on the plurality of virus-derived RNA contig sequences; d) identify a protein sequence based on the viral RNA sequence; e) identify an immunogenic virus-derived peptide based at least in part on the identified protein sequence; f) provide the immunogenic virus-derived peptide as an output.

[0098] In some embodiments, the plurality of RNA contig sequences are derived from only one infected subject.

[0099] In some embodiments, the infected subject includes a human.

[0100] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from the virus with which the infected subject is infected.

[0101] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from the endogenous RNA of the infected subject.

[0102] In some embodiments, instructions that cause a computing device to identify a plurality of virus-derived RNA contig sequences from among a plurality of RNA contig sequences, when executed by one or more processors, cause the computing device to: compare at least a portion of the contig sequences of the plurality of RNA contig sequences with a reference virus sequence; further include instructions to identify a plurality of virus-derived RNA contig sequences such that each contig sequence of the plurality of virus-derived RNA contig sequences includes at least a portion corresponding to the reference virus sequence.

[0103] In some embodiments, each contig sequence of the plurality of virus-derived RNA contigs is different from a plurality of endogenous RNA contig sequences of an infected subject.

[0104] In some embodiments, each contig sequence of the plurality of virus-derived RNA contig sequences does not include a portion of an endogenous RNA contig sequence of an infected subject.

[0105] In some embodiments, the reference virus sequence includes a reference genome.

[0106] In some embodiments, the reference genome includes a hepatitis B virus genome.

[0107] In some embodiments, instructions that cause a computing device to perform an assembly of a viral RNA sequence based on a plurality of virus-derived RNA contig sequences, when executed by one or more processors, cause the computing device to: further include instructions to perform an assembly of the viral RNA sequence by overlapping common sequence portions at the ends of at least a portion of the plurality of virus-derived RNA contig sequences such that at least a portion of the plurality of virus-derived RNA contig sequences linearly overlap.

[0108] In some embodiments, instructions to cause a computing device to identify a protein sequence based on a viral RNA sequence such that the protein sequence includes a translation of the viral RNA sequence, when executed by a processor, cause the computing device to: further include instructions to identify the protein sequence without requiring a comparison to a database of viral proteins.

[0109] In some embodiments, the protein sequence identified based on the viral RNA sequence is a novel protein.

[0110] In some embodiments, the protein sequence identified based on the viral RNA sequence is associated with an infected subject.

[0111] In some embodiments, instructions to cause a computing device to identify a protein sequence based on a viral RNA sequence such that the protein sequence includes a translation of the viral RNA sequence, when executed by one or more processors, cause the computing device to: identify a plurality of protein sequences based on the viral RNA sequence such that each of the plurality of protein sequences includes a translation of the viral RNA sequence; further include instructions to identify a protein sequence as a protein sequence that frequently occurs within the plurality of protein sequences.

[0112] In some embodiments, instructions to cause a computing device to identify an immunogenic virus-derived peptide based at least in part on a protein sequence, when executed by one or more processors, cause the computing device to: identify major histocompatibility complex (MHC) molecules associated with the infected subject; identify one or more peptides based at least in part on the protein sequence such that each of the one or more peptides is capable of forming an MHC-peptide complex with the MHC molecules; Further comprising an instruction to identify an immunogenic virus-derived peptide based on the one or more peptides.

[0113] In some embodiments, the instruction further causes the computing device, when executed by one or more processors, to: Store a protein sequence in a database such that the protein sequence is associated with an infected subject within the database.

[0114] In another aspect, provided herein is a method for identifying an integration site of a viral gene within a subject's gene, the method comprising: a) Obtaining a plurality of RNA contig sequences from an infected subject infected with a virus, such that the plurality of RNA contig sequences includes a plurality of virus-derived RNA contig sequences, a plurality of infected subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences comprising a viral portion and an infected subject endogenous portion; b) Identifying the plurality of hybrid RNA contig sequences from within the plurality of RNA contig sequences; c) Comparing the infected subject endogenous portion with a subject reference genome for at least a portion of the plurality of hybrid RNA contig sequences; and d) Identifying an integration site comprising the subject's gene based at least in part on a comparison of the infected subject endogenous portion with the subject reference genome. Including.

[0115] In some embodiments, the plurality of RNA contig sequences are from a single infected subject.

[0116] In some embodiments, the infected subject is human.

[0117] In some embodiments, the plurality of virus-derived RNA contig sequences are from the virus that infected the infected subject.

[0118] In some embodiments, the virus is hepatitis B virus.

[0119] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from the endogenous RNA of an infected subject.

[0120] In some embodiments, the subject reference genome includes the human genome.

[0121] In another aspect, provided herein is a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium including instructions that, when executed by the one or more processors, cause the computing device to: a) obtain, as input, a plurality of RNA contig sequences from an infected subject infected with a virus, wherein the plurality of RNA contig sequences includes a plurality of virus-derived RNA contig sequences, a plurality of infected subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences including viral and infected subject endogenous portions; b) identify the plurality of hybrid RNA contig sequences from within the plurality of RNA contig sequences; c) compare an infected subject endogenous portion with a subject reference genome for at least a portion of the plurality of hybrid RNA contig sequences; d) identify an integration site including a gene of the subject, at least in part based on a comparison of the infected subject endogenous portion with the subject reference genome; e) provide the integration site as output.

[0122] In some embodiments, the plurality of RNA contig sequences are derived from only one infected subject.

[0123] In some embodiments, the infected subject includes a human.

[0124] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from the virus that infected the subject.

[0125] In some embodiments, the virus includes hepatitis B virus.

[0126] In some embodiments, the plurality of subject endogenous RNA contig sequences are derived from the endogenous RNA of the subject.

[0127] In some embodiments, the subject reference genome includes the human genome.

[0128] These and other aspects of the invention will become apparent to those of ordinary skill in the art in the following description, claims, and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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BRIEF DESCRIPTION OF THE INVENTION

[0130] Chronic HBV infection can significantly reduce liver function by causing hepatitis, fibrosis, and cirrhosis, and may ultimately lead to hepatocellular carcinoma (HCC). Approximately 25-40% of HBV carriers develop HCC. Vaccination and antiviral agents (such as reverse transcriptase inhibitors and interferon therapy) are preferred preventive and treatment options in the management of HBV infection. However, once infection is established, antiviral agents can only control viral transcription and have little effect on the serum clearance of hepatitis B surface antigen (HBsAg). Furthermore, when HBV DNA fragments are integrated into the host genome, gene damage and chromosomal instability may be induced, which may further contribute to the development of HCC. Worldwide, chronic HBV infection is estimated to account for approximately 50% of primary liver cancer.

[0131] The persistence or control of HBV infection is determined by the host immune response mainly mediated by the adaptive immune system after HBV evades the innate immune system. Cytotoxic CD8+ T cells of the adaptive immune system protect the host from intracellular pathogens and tumors. Therefore, cytotoxic CD8+ T cells play an important role in the defense against HBV infection, mainly responsible for the removal of the virus in acute infection and the prevention of the development of HCC. In the case of chronic HBV infection, HBV-specific CD8+ T cells are often undetectable, which is thought to be the result of high antigen load and chronic exposure of the liver to immunotolerogenic hepatocytes.

[0132] Since CD8+ T cells play an important role in the defense against viral infections, the use of CD8+ T cells has become an important aspect of immunotherapy. Cytotoxic T cells mediate adaptive immunity by recognizing HLA-peptide complexes presented on the surface of infected cells through their interaction with T cell receptors (TCRs) expressed on their surface. These HLA-peptide complexes can be used as targets for the development of various immunotherapies, including, for example, bispecific antibody therapy and CAR-T therapy.

[0133] Proteogenomics can be applied to the identification of HLA-related variant peptides in cancer. HBV-HCC tissues may lack HBV covalently closed circular DNA (cccDNA), which is the template for viral protein synthesis. Instead, tumor cells may contain fragments of HBV DNA inserted into the host genome, which can be transcribed and ultimately express viral antigens on the cell surface. This makes it difficult to identify HBV epitopes because the exact region of the integrated HBV genome can vary from patient to patient. Compounding this effort is the existence of thousands of HBV strains distributed across 10 different HBV genotypes (genotypes A-J), which differ from each other by at least 8% at the nucleotide level.

[0134] The present disclosure particularly provides isolated peptides derived from hepatitis B virus (HBV), and fragments or derivatives thereof. Various peptide-based molecules are also provided, including, for example, complexes comprising the peptide (e.g., peptide-MHC (pMHC) complexes), fusion proteins, and conjugates. Further provided herein are polynucleotides and vectors encoding the peptides or peptide-based molecules described herein. 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 the present disclosure can be used to induce an immune response against HBV infection and / or for the treatment or prevention of diseases or disorders induced by HBV. In one aspect, the present disclosure also provides a method for identifying immunogenic virus-derived peptides.

[0135] Lymphocytes such as T cells play important roles in adaptive anti-infection, anti-tumor, autoimmune, and transplant rejection responses. Generally, T cell-mediated immune responses require close contact between T cells and antigen-presenting cells (APCs), such as an immunological synapse. The formation of an immunological synapse involves the pairing of multiple molecules, including, but not limited to: (a) the T cell receptor (TCR) on the T cell that specifically binds to a peptide presented in the peptide-binding groove of the major histocompatibility complex (MHC) molecule on the APC; and (b) CD28 (on the T cell) that pairs with the B7 molecule on the APC. The TCR forms a TCR complex together with the CD3 molecule, and when the TCR pairs with a peptide-MHC (peptide-MHC: pMHC) complex, a signal is transmitted through CD3. T cells are activated by signal transduction through both the TCR complex and CD28 on the T cell.

[0136] The T cell receptor is a heterodimeric structure composed of two types of chains (α (alpha) and β (beta) chains, or γ (gamma) and δ (delta) chains). The α chain is encoded by a nucleic acid sequence located within the α locus (on human or mouse chromosome 14), which also encompasses the entire δ locus encoding the δ chain. The β chain is encoded by a nucleic acid sequence located within the β locus (on mouse chromosome 6 or human chromosome 7). The majority of T cells have αβ TCR, while a minority of T cells have γδ TCR. The α polypeptide and β polypeptide (similarly, the γ polypeptide and δ polypeptide) of the T cell receptor are linked to each other via disulfide bonds. Each of the two polypeptides constituting the TCR contains an extracellular domain, including a constant region, a variable region, a transmembrane domain, and a cytoplasmic tail (the transmembrane domain and cytoplasmic tail are also part of the constant region).

[0137] The variable region of each TCR contains a unique and characteristic structure that determines the specificity of the TCR, namely an idiotope or idiotype. Generally, a TCR binds to a pMHC complex only when it contains an idiotype that recognizes the presentation of a peptide in the context of MHC, for example, the unique three-dimensional structure of a specific pMHC complex.

[0138] Immunotherapy approaches for the treatment of diseases act to regulate the activity of T cells in vivo, for example, to enhance anti-infection and anti-tumor responses, or to downregulate, for example, autoimmune and transplant rejection responses. However, such methods may lack specificity. This is because immunotherapy can target signal transduction by the TCR complex through binding to CD3 and / or engagement of costimulatory molecules. Such an approach may result in unwanted side effects, such as an overactive immune response or systemic immunosuppression. Therefore, therapies that utilize the unique specific interaction between the TCR and the pMHC complex can provide the ability to specifically regulate the activity of specific T cells in vivo and can provide treatments based on the regulation of T cells.

[0139] Presentation of virus-derived peptides by MHC molecules on the surface of infected cells, recognition of these pMHC complexes by CD8+ cytotoxic T cells, and subsequent activation of CD8+ cytotoxic T cells provide important mechanisms for immune-based protection against viruses. Infected cells, including those that have progressed to cancer cells, can express various HBV-related antigens. Peptides derived from these antigens can be displayed on the cell surface as complexes with MHC molecules. Detection of HBV-derived peptides presented by MHC can lead to targeted killing of infected cells by T cells having the corresponding TCR. However, due to the selection processes that occur during T cell maturation in the thymus, T cells that recognize HBV-derived peptides with a sufficiently high level of affinity are often lacking in the circulating repertoire. As a result, infected cells often escape elimination by the immune system.

[0140] By identifying HBV-derived peptides presented on infected cells (e.g., HBV-induced cancer cells), immunotherapeutic reagents can be developed that are designed to specifically target and destroy HBV-infected cells (e.g., HBV-induced cancer cells). Such reagents can be reagents having a partial structure that binds to an HBV-derived peptide and / or a pMHC complex, and a partial structure that can function, for example, by inducing a T cell response. For example, such reagents can be based on antibodies, TCRs, and / or CARs.

[0141] The present disclosure is based, in part, on a proteogenomic approach for detecting MHC-associated HBV peptides from HBV-infected cells. The repertoire of HLA-I-HBV peptides expressed in the livers of infected patients characterized herein can provide an accurate representation of HBV epitopes within the population.

[0142] HLA-restricted viral peptides as potential targets can be utilized to deliver immunotherapeutic agents (such as antibodies (e.g., bispecific antibodies), engineered TCRs, or CAR-based cell therapies, etc.) to infected tissues. However, the genomic variability among viral strains such as HBV, along with differences in the HLA alleles of patients, can pose challenges in the development of therapeutic agents against these peptide targets. To address this challenge, in one aspect, the present disclosure provides a proteogenomic approach for generating a patient-specific database, which enables comprehensive identification of viral peptides, such as HBV-derived peptides, based on the viral transcriptase sequenced from the samples of individual patients. The HBV-HLA-related peptides disclosed herein can be used in the development of immunotherapeutic agents (such as antibodies (e.g., bispecific antibodies), engineered TCRs, or CAR-based cell therapies, etc.) for the treatment of HBV-related diseases or disorders including hepatocellular carcinoma (HCC), and can also be used to improve clinical monitoring for monitoring patient-specific HBV diversity and provide information for vaccine development. The proteogenomic discovery platform described herein provides a method for identifying viral-derived peptides as targets for anti-viral related immunotherapy.

[0143] Definitions The 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, unless otherwise defined.

[0144] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to one of ordinary skill in the art upon reading this disclosure.

[0145] The term "about" or "approximately" includes being within a statistically significant range of a value. Such a range can be within one digit of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and still even more preferably within 5%. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system under study, which can be readily understood by those skilled in the art.

[0146] The term "antigen" includes any factor (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, a portion thereof, or a combination thereof) that, when introduced (either directly or, for example, as expressed in a DNA or RNA vaccine) into an immunocompetent host, is recognized by the host's immune system and can induce an immune response in the host. T cell receptors (TCRs) recognize peptides presented in the context of the major histocompatibility complex (MHC) as part of an immune synapse. Peptide-MHC (pMHC) complexes are recognized by TCRs, and the peptide (epitope) and TCR idiotype provide the specificity of this interaction. Thus, the term "antigen" includes peptides presented in the context of MHC, such as pMHC complexes. Peptides displayed in MHC may also be referred to as "epitopes" or "antigenic determinants". The terms "peptide", "antigenic determinant", "epitope", etc. include not only those naturally presented by antigen-presenting cells (APCs), but also any desired peptide as long as it is recognized by immune cells, such as when appropriately presented to cells of the immune system. For example, a peptide having an artificially prepared amino acid sequence can also be used as an epitope.

[0147] An antigen (such as an antigenic polypeptide) may have two or more epitopes. An epitope can be defined structurally or functionally. A functional epitope is generally a subset of a structural epitope and is defined as the residues that directly contribute to the affinity of the interaction between an MHC molecule and an antigen. An epitope may have a three-dimensional structure, i.e., it may consist of non-linear amino acids. In certain embodiments, an epitope may include determinants that are chemically active surface groups of a molecule such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group, and in certain embodiments, may have specific three-dimensional structural features and / or specific charge features. An epitope formed from contiguous amino acids is typically retained even when exposed to a denaturing solvent, whereas an epitope formed by tertiary folding is typically lost upon treatment with a denaturing solvent.

[0148] The terms "major histocompatibility complex" and "MHC" include the terms "human leukocyte antigen" or "HLA" (which are generally reserved for human MHC molecules), naturally occurring MHC molecules (e.g., MHC class I molecules including MHC class I α (heavy) chain and β2-microglobulin; MHC class II molecules including MHC class II α chain and MHC class II β chain), individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, MHC class II α chain, and MHC class II β chain), individual subunits of these chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1-α2 subunits of the MHC class II α chain, β1-β2 subunits of the MHC class II β chain), and these portions (e.g., peptide-binding portions, e.g., peptide-binding grooves), variants, and various derivatives (including fusion proteins), where the above-mentioned portions, variants, and derivatives retain the ability to display antigen peptides for recognition by a TCR, such as an antigen-specific TCR. MHC class I molecules contain a peptide-binding groove formed by the α1 and α2 domains of the heavy chain that can accommodate peptides of about 8-10 amino acids. Despite the fact that both classes of MHC bind to the core of about 9 amino acids (e.g., 5-17 amino acids) within the peptide, the unrestricted nature of the MHC class II peptide-binding groove (the α1 domain of the class II MHCα polypeptide bound to the β1 domain of the class II MHCβ polypeptide) allows for a wider range of peptide lengths. Although the length of peptides that bind to MHC class II is usually 13-17 amino acids, shorter or longer lengths are not uncommon. As a result, the peptide can shift within the MHC class II peptide-binding groove, changing which 9-mer is directly seated 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. Conventional designations for specific MHC variants are used herein.For example, HLA-A11 refers to the human leukocyte antigen from position number 11 of the gene locus (known as the gene locus) of the A gene group (and thus MHC class I); the gene HLA-DR11 refers to the human leukocyte antigen encoded by the gene from position number 11 of the DR region (and thus MHC class II) gene locus.

[0149] "MHC-peptide complex", "peptide-MHC complex", "pMHC complex", "peptide-in-groove", etc. include (i) an MHC molecule, such as a human and / or non-human animal MHC molecule, or a portion thereof (e.g., its peptide-binding groove and, e.g., its extracellular portion), and (ii) an antigenic peptide (e.g., a peptide derived from HBV), where the MHC molecule and the antigenic peptide are complexed in a manner such that the pMHC complex can specifically bind to a T cell receptor. The pMHC complex includes a pMHC complex expressed on the cell surface and a soluble pMHC complex.

[0150] "HLA-peptide complex", "peptide-HLA complex", "pHLA complex", etc. refer to an MHC-peptide complex in which the MHC molecule is a human leukocyte antigen (HLA) molecule.

[0151] The term "T cell" or "T lymphocyte" is used herein in its broadest sense to refer to any type of immune cell expressing 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 (Treg), 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 (γδT cells)", which refers to a specialized population of T cells with different TCRs on their surface, and unlike most T cells whose TCRs are composed of two glycoprotein chains called the α-TCR chain and the β-TCR chain, the TCR of γδT cells is composed of the γ chain and the δ chain.

[0152] The term "antigen presenting cell" or "APC" refers to any cell that presents antigens associated with major histocompatibility complex molecules, i.e., MHC class I molecules, or MHC class II molecules, or both, on the surface of the cell.

[0153] The terms "antibody (antibody, antibodies)", "immunoglobulin", etc. refer to molecules that contain an antigen-binding site that specifically binds to an antigen, whether they are immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., whether they are natural or are produced in part or in whole by synthesis. 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-bonded Fv (sdFv), intrabodies, minibodies, diabodies, and anti-idiotype (anti-Id) antibodies (including, e.g., anti-Id antibodies against antigen-specific TCRs), as well as epitope-binding fragments of any of the foregoing. The term "antibody (antibody, antibodies)" also refers to covalent diabodies such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, which is incorporated herein by reference in its entirety, and Ig-DARTS such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910, which is incorporated herein by reference in its entirety. Antibodies that can be used in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. The 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.

[0154] The terms "specifically binds", "binds in a specific manner", "antigen-specific", etc. refer to the fact that multiple molecules involved in this specific binding can form a relatively stable complex under physiological conditions, and cannot non-specifically form a stable complex with other molecules outside of this specific binding pair. Therefore, for a HBV-derived peptide or a peptide-based molecule (a complex containing the described peptide (e.g., pMHC complex), fusion protein, or conjugate, etc.), a peptide-binding partial structure that binds in a specific manner (e.g., an antibody, alternative scaffold, CAR, or TCR) means that the above peptide-binding partial structure forms a stable intermolecular non-covalent bond with the above HBV-derived peptide or peptide-based molecule (a complex containing the described peptide (e.g., pMHC complex), fusion protein, or conjugate, etc.). Specific binding can be characterized by an equilibrium dissociation constant (K D ) in the range of low micromolar to picomolar (i.e., the smaller the K D , the stronger the binding). High specificity can be seen in the low nanomolar range, and extremely high specificity can be seen in the picomolar range. For example, the peptide-binding partial structure can exhibit binding to a HBV-derived peptide or a peptide-based molecule (a complex containing the described peptide (e.g., pMHC complex), fusion protein, or conjugate, etc.) with a K D of about 3000 nM or less, about 2000 nM or less, about 1000 nM or less, about 500 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 1 nM or less, or about 0.5 nM or less. Methods for determining whether two molecules specifically bind to each other are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0155] The terms "protein" and "polypeptide", which are used interchangeably herein, encompass all types of natural and synthetic proteins, including protein fragments of any length, fusion proteins, and modified proteins. Modified proteins include, but are not limited to, glycoproteins and any other type of modified protein (e.g., proteins obtained from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as "peptides".

[0156] The terms "polynucleotide" and "nucleic acid", which are used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. 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, unnatural, or derivatized nucleotide bases.

[0157] The terms "operably linked", etc., refer to juxtaposition in such a relationship that a plurality of described components can function in their intended manner. For example, a control sequence "operably linked" to a coding sequence is joined in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. "Operably linked" sequences include both an expression control sequence contiguous with the gene of interest and an expression control sequence that acts in trans or at a distance for the control of the gene of interest (or sequence of interest). The term "expression control sequence" includes polynucleotide sequences necessary to affect the expression and processing of the coding sequences to which they are ligated. "Expression control sequences" include: appropriate transcription start, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and, when desired, sequences that enhance the secretion of the polypeptide. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences generally include a promoter, ribosome binding site, and transcription termination sequence, and in eukaryotes, such control sequences typically include a promoter and transcription termination sequence. The term "control sequence" is intended to include components essential for expression and processing, and may also include additional components, such as leader sequences and fusion partner sequences, whose presence is advantageous.

[0158] The term "isolated" refers to a homogeneous population of molecules (such as polynucleotides or polypeptides) that are substantially separated from and / or purified from other components of the system in which the molecules are produced, as well as proteins that have been subjected to at least one purification or isolation step. "Isolated" refers to a molecule that is substantially free of other cellular materials and / or chemical substances, and includes molecules that are isolated to a relatively high degree, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0159] 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 described below and include, by way of example, insertions, substitutions, deletions, transversions, and / or inversions at one or more positions in the amino acid or nucleotide sequence.

[0160] "To "treat" a condition, disorder, disease, or medical condition, or "treatment" thereof, means: (1) preventing, delaying, or reducing the incidence or likelihood of the appearance of at least one clinical or potential symptom of a condition, disorder, disease, or medical condition that has occurred in a subject who has or may have a predisposition to the condition, disorder, disease, or medical condition but who has not yet experienced or manifested the clinical or potential symptoms of the condition, disorder, disease, or medical condition; or (2) inhibiting the condition, disorder, disease, or medical condition, i.e., preventing, reducing, or delaying the occurrence of the disease or its recurrence, or of at least one of its clinical or potential symptoms; or (3) alleviating the condition, disorder, disease, or medical condition, which may include causing regression of the condition, disorder, disease, or medical condition, or of at least one of the clinical or potential symptoms of the condition, disorder, disease, or medical condition. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0161] The terms "individual", "subject", or "animal" refer to humans, domestic animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.

[0162] The term "effective" as applied to a dosage or amount refers to an amount of a compound or pharmaceutical composition that is sufficient to provide the desired activity when administered to a subject in need thereof. It should be noted that when administering a combination of multiple active ingredients, the effective amount of the combination may or may not include the amounts of each ingredient that were effective when administered individually. The exact amount required will vary from subject to subject depending on the species, age, and general condition of the subject, the severity of the condition being treated, the one or more specific drugs employed, the mode of administration, etc.

[0163] When used in connection with the compositions described herein, the phrase "pharmaceutically acceptable" refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and typically produce no harmful reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, more particularly in humans.

[0164] The terms "administration" and the like refer to and include the administration of a composition to a subject or system (e.g., to a cell, organ, tissue, organism, or related set of these components or multiple components). One of ordinary skill in the art will understand that the route of administration can vary depending on, for example, the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, and the like. For example, in certain embodiments, administration to a subject that is an animal (e.g., to a human or a rodent) can be by bronchial (including by bronchial instillation), buccal, enteral, intercutaneous, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by tracheal instillation), transdermal, vaginal, and / or intravitreal administration. In some embodiments, administration can include intermittent dosing. In some embodiments, administration can include continuous dosing (e.g., perfusion) over at least a selected period of time.

[0165] According to the disclosure in this specification, conventional molecular biology, microbiology, and recombinant DNA techniques within the scope of the relevant technical field can be employed. Such techniques are well-described in the literature. For example, see Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, F.M. et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include 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 No. 5,789,166 and U.S. Patent No. 5,932,419, Hogrefe, Strategies l4. 3: 74-75 (2001), U.S. Patent No. 5,702,931, U.S. Patent No. 5,780,270, and U.S. Patent No. 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, Protein Engineer. 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 et al., Meth. Molec. Biol. 67: 209-218, including site-directed mutagenesis as described therein.

[0166] The peptides disclosed herein In one aspect, the present disclosure provides an isolated peptide comprising an amino acid sequence derived from hepatitis B virus (HBV).

[0167] The HBV genome exhibits genetic variability at an estimated rate of 1.4 - 3.2×10 -5 nucleotide substitutions per site per year. A significant number of viral variants arise due to incorrect incorporation of nucleotides in the absence of the proofreading ability of the viral polymerase. This variability gives rise to viral subtypes. HBV is classified into multiple genotypes based on differences between groups of at least 8% of the full genome sequence, each of which has a defined geographical distribution. For example, genotype A is common in sub-Saharan Africa, northern Europe, and West Africa; genotypes B and C are widely distributed in Asia; genotype C is mainly present in Southeast Asia; genotype D is found in Africa, Europe, Mediterranean countries, and India; genotype G has been observed in France, Germany, and the United States; genotype H is mainly found in Central and South America. Genotype I has recently been observed in Laos and Vietnam. Genotype J has been reported in the Ryukyu Islands of Japan.

[0168] HBV is an enveloped DNA virus and a member of the Hepadnaviridae family. HBV has a small, partially double-stranded (DS), relaxed-circular DNA (rcDNA) genome that is replicated by reverse transcription of the pregenomic RNA (pgRNA), which is an RNA intermediate. The circular DNA genome of HBV is considered atypical because the DNA is not fully double-stranded. One end of the full-length strand is ligated to the viral DNA polymerase. The length of the full-length genomic strand is approximately 3020 - 3320 nucleotides, and the length of the short genomic strand is 1700 - 2800 nucleotides. The negative-sense (i.e., non-coding) strand is complementary to the viral mRNA.

[0169] There are four genes encoded by the HBV genome: gene C, X, P, and S. The core protein is encoded by gene C (HBcAg), and there is an upstream in-frame AUG start codon before the start codon of gene C, from which the precore protein is generated. HBeAg is generated by proteolytic processing of the precore protein. DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg (hepatitis B core antigen)). The HBsAg gene is a single long open reading frame containing three in-frame start codons (ATG) that subdivide the gene into three components: preS1, preS2, and S. The presence of multiple start codons results in the production of three different sized proteins called Large (preS1 / preS2 / S), Middle (preS2 / S), and Small (S). Gene X may be involved in the development of liver cancer and may stimulate genes that promote cell growth and inactivate growth regulatory molecules.

[0170] Viral DNA is localized in the nucleus after cell infection. The partially double-stranded DNA becomes completely double-stranded by completion of the plus strand, removal of proteins from the minus strand, and removal of short sequences of RNA from the plus strand. Non-coding bases are removed from the ends of the minus strand and the ends are religated.

[0171] The life cycle of HBV begins when the virus attaches to host cells and then enters the cells. Sodium-taurocholate co-transporting polypeptide (NTCP) can serve as a functional receptor in HBV infection. The relaxed circular DNA (rcDNA) of the virion is transported to the nucleus, where the virion rcDNA is repaired and covalently closed circular DNA (cccDNA) is formed. The episomal cccDNA acts as a template for the transcription of the pregenomic RNA (pgRNA) and other viral mRNAs by host RNA polymerase II. Subsequently, the transcripts are exported to the cytoplasm for the translation of subsequent viral proteins. Reverse transcriptase (RT) binds to the pgRNA and activates the assembly of core proteins into immature RNA-containing nucleocapsids. The immature nucleocapsids mature upon reverse transcription of the pgRNA by RT to become mature rcDNA. A unique feature of hepadnavirus reverse transcription is the initiation of minus-strand DNA synthesis by RT priming, whereby RT covalently attaches to the 5' end of the minus-strand DNA.

[0172] Subsequently, the mature rcDNA-containing nucleocapsids are either enveloped by viral surface proteins and secreted as virions via the secretory pathway or recycled to the nucleus for further amplification of the cccDNA pool via the recycling pathway.

[0173] The HBV-derived amino acid sequences disclosed herein may include naturally occurring proteogenic amino acids, non-proteogenic amino acids, and non-naturally occurring amino acids such as amino acid analogs. In some embodiments, amino acids that can be used in the practice of the present disclosure include, for example but not limited to, naturally occurring proteogenic (L)-amino acids, their optical (D)-isomers; chemically modified amino acids including amino acid analogs such as selenocysteine (Sec), penicillamine (3-mercapto-D-valine), pyroglutamic acid (5-oxoproline), etc.; naturally occurring non-proteogenic amino acids such as norleucine; and chemically synthesized amino acids having properties known in the art as characteristics of amino acids; and amino acid equivalents.

[0174] In some embodiments, the isolated peptides of the present disclosure comprise an amino acid sequence that is 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% identical to any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, the isolated peptides of the present disclosure comprise any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptides of the present disclosure consist essentially of any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptides of the present disclosure consist of any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptide comprises two or more sequences selected from any one of SEQ ID NOs: 1-54 and 110-112, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0175] A non-limiting list of examples of HBV-derived peptides is provided in Table 1 below.

Table 1-1

Table 1-2

[0176] In some embodiments, the isolated peptide of the present disclosure comprises the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), where X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H, or L, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof. In some embodiments, the isolated peptide of the present disclosure consists of the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), where X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H, or L, or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof.

[0177] The peptides of the present disclosure may be produced by synthesis or by hydrolysis. Peptides produced by synthesis may include: randomly generated peptides; specially designed peptides; and peptides in which at least some of the amino acid positions are conserved among multiple peptides and the remaining positions are random. Alternatively, the peptides of the present disclosure may be produced by expression in a heterologous host cell.

[0178] In nature, peptides produced by hydrolysis are hydrolyzed before the antigen binds to the MHC molecule. Class I MHC typically presents peptides derived from proteins actively synthesized in the cytoplasm of cells. In contrast, class II MHC typically presents peptides derived from exogenous proteins that enter the endocytic pathway of cells or from proteins synthesized in the endoplasmic reticulum (ER). By intracellular delivery, peptides become capable of binding to MHC molecules.

[0179] By binding to the MHC peptide binding groove, the peptide can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR. Such spatial control is due in part to the hydrogen bonds formed between the peptide and the MHC molecule. Based on the knowledge of how peptides bind to various MHC molecules, the major MHC anchor amino acids and the amino acids exposed on the surface, which differ among various peptides, can be determined.

[0180] Preferably, the length of the peptide that binds to MHC is about 5 to about 40 amino acid residues, more preferably about 6 to about 30 amino acid residues, still more preferably about 8 to about 20 amino acid residues, and still more preferably about 9 to 11 amino acid residues, and any size of amino acids with a length of 5 to 40 amino acids is included, and the increments of the above lengths are in integer units (i.e., 5, 6, 7, 8, 9…40). Peptides that bind to MHC class II in the natural state vary from about 9 to 40 amino acids, but in almost all cases, the peptides can be truncated to about 9 to 11 amino acids without losing MHC binding activity or T cell recognition.

[0181] In some embodiments, the length of the isolated peptide of the present disclosure may be about 8 to 12 amino acids. For example, the length of the peptide of the present disclosure is 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids.

[0182] The peptides of the present disclosure may include 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.

[0183] In some embodiments, the peptide may be modified to include one or more reverse peptide bonds or non-peptide bonds. Such modifications can induce a stronger immune response by improving the stability of the peptide and / or the binding of the peptide to MHC molecules. In a reverse peptide bond, amino acid residues are not linked by a peptide (-CO-NH-) bond, and the peptide bond is reversed. Such retro-inverso peptide mimetics can be prepared using methods known in the art, such as the method described in Meziere et al., 1997 (Meziere C., et al. J Immunol 1997). This approach involves the preparation of peptidomimetics that include changes involving the backbone rather than the orientation of the side chains. Such peptidomimetics can be useful, for example, for desired MHC binding and / or T helper cell responses. Retro-inverso peptides that include an NH-CO bond instead of a CO-NH peptide bond are highly resistant to proteolysis. Further non-peptide bonds that can be used are, for example, -CH2-NH, -CH2S-, -CH2CH2-, -CH=CH-, -COCH2-, -CH(OH)CH2-, and -CH2SO-.

[0184] The amino acid residues comprising the peptides of the present disclosure may be chemically modified. Non-limiting examples of chemical modifications include, for example, phosphorylation, acetylation, deamidation, acylation, amidation, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification of carboxyl groups, and sulfhydryl modification by oxidation of cysteine to cysteic acid with performic acid, formation of mercury derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanate under alkaline pH. The chemical modifications do not have to correspond to those that may exist in vivo.

[0185] For example, the modification of arginyl residues in a protein may be based on the reaction of adjacent dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form adducts. Another example is the reaction of methylglyoxal with arginine residues. Cysteine can be modified without simultaneously modifying other nucleophilic sites such as lysine and histidine. Selective reduction of disulfide bonds in a protein can also be carried out. Disulfide bonds can be formed and oxidized during the heat treatment of biopharmaceuticals. Specific glutamic acid residues may be modified using Woodward reagent K. Intramolecular cross-links between lysine residues and glutamic acid residues can be formed using N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide. For example, histidyl residues in a protein can be modified using diethylpyrocarbonate and 4-hydroxy-2-nonenal. The reaction of lysine residues with other α-amino groups is useful, for example, for the attachment of peptides to a surface or for protein / peptide cross-linking. Lysine is an attachment site for poly(ethylene) glycol and is also a major modification site in the glycosylation of proteins. Methionine residues in a protein can be modified, for example, with iodoacetamide, bromoethylamine, and chloramine T. Tetranitromethane and N-acetylimidazole can be used for the modification of tyrosine residues. Cross-linking by the formation of dityrosine can be achieved with hydrogen peroxide / copper ions. In recent studies, N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skator) have been used for the modification of tryptophan. The good modification of therapeutic proteins and peptides with PEG can extend the circulation half-life, while the cross-linking of proteins / peptides with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde can be used for the preparation of hydrogels. Chemical modification of allergens for immunotherapy can be achieved by carbamylation with potassium cyanate.

[0186] The peptides of the present disclosure may be synthesized such that additional chemical groups are present at their N-terminus and / or C-terminus in order to enhance the stability, bioavailability, and / or affinity of the peptides.

[0187] N-terminal modifications include methylation (e.g., -NHCH3 or -N(CH3)2), acetylation (e.g., acetic acid or its halogenated derivatives such as α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid), addition of a benzyloxycarbonyl (Cbz) group, or blocking of the amino terminus with any blocking group containing a carboxylate 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. Also, by incorporating a desamino acid at the N-terminus (so that the N-terminal group is absent), the sensitivity to proteases can be reduced, or the conformation of the peptide can be restricted. Further, a hydrophobic group such as a carbobenzoxyl group, dansyl group, or t-butyloxycarbonyl group may be added to the N-terminus. Similarly, an acetyl group or a 9-fluorenylmethoxycarbonyl group may be placed at the N-terminus.

[0188] Examples of C-terminal modifications include substitution of the free acid with a carboxamide group, or introduction of a structural constraint by formation of a cyclic lactam at the carboxy terminus. Also, by cyclizing the peptides of the present disclosure or incorporating a desamino residue or a descarboxy residue at the terminus of the peptide so that the terminal amino group or carboxyl group is absent, the sensitivity to proteases can be reduced, or the conformation of the peptide can be restricted. Examples of C-terminal functional groups of the compounds of the present disclosure include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, and their lower ester derivatives, and their pharmaceutically acceptable salts. Further, a hydrophobic group, a t-butyloxycarbonyl group, or an amide group may be added to the C-terminus.

[0189] Additional examples of non-natural modifications include: incorporation of uncoded α-amino acids, photoreactive crosslinking amino acids, N-methylated amino acids, and β-amino acids; backbone reduction; retro-inverso conversion using D-amino acids; and C-terminal amidation and PEGylation.

[0190] 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 with a substituted amino acid residue at the same position. The inserted amino acid residue may be inserted at any position, and part or all of the inserted amino acid residues may be inserted so that they are directly adjacent to each other, or the inserted amino acid residues may be inserted so that they are not directly adjacent to another inserted amino acid residue. One or more (e.g., 1, 2, 3, or 4) amino acids from any one of the sequences of SEQ ID NOs: 1-54 and 110-112 may have substitutions and / or insertions and / or deletions. Substitutions and / or insertions and / or deletions may each occur at any position of any one of the sequences of SEQ ID NOs: 1-54 and 110-112.

[0191] In some embodiments, the peptides of the present 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 the sequences of SEQ ID NOs: 1-54 and 110-112. The peptides of the present disclosure may contain an amino acid sequence of any one of the sequences of SEQ ID NOs: 1-54 and 110-112, except for one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, insertions, or deletions.

[0192] The inserted amino acids and the amino acids for substitution may be either naturally occurring amino acids or non-naturally occurring amino acids. For example, they may contain non-natural side chains and / or may be joined together by non-native peptide bonds. Such modified peptide ligands are further described 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 the references therein. When two or more amino acid residues are substituted and / or inserted, the amino acid residues for substitution / insertion may be the same as or different from each other. Each amino acid for substitution may have a side chain different from the amino acid to be substituted.

[0193] D-amino acids may be used to substitute the L-amino acids in the antigen peptides of the present disclosure. Further, non-standard amino acids (i.e., amino acids other than common naturally occurring protein-forming amino acids such as β-γ-δ-amino acids and many derivatives of L-α-amino acids) can be used for substitution or addition to generate the peptides of the present disclosure.

[0194] Amino acid substitutions may be conservative, where "conservative" means that the amino acid for substitution has chemical properties similar to the original amino acid. For example, the following groups of amino acids share 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.

[0195] Significant changes in function (e.g., affinity for MHC molecules and / or TCRs) can be obtained by selecting substitutions that are less conserved compared to those described above, i.e., by selecting residues that differ more significantly in terms of the structure of the peptide backbone in the region of the substitution (e.g., sheet or helical conformation), the bulk of the side chain, or the effect on maintaining the charge or hydrophobicity of the peptide at positions involved in MHC or TCR binding. Substitutions that are generally expected to result in the greatest change in peptide properties are (a) substitutions that replace a hydrophilic residue, e.g., Ser, with a hydrophobic residue, e.g., Leu, Ile, Phe, Val, or Ala (or vice versa); (b) substitutions that replace a residue with a positively charged side chain, e.g., Lys, Arg, or His, with a residue with a negatively charged side chain, e.g., Glu or Asp (or vice versa); or (c) substitutions that replace a residue with a bulky side chain, e.g., Phe, with a residue without a side chain, e.g., Gly (or vice versa).

[0196] The naturally occurring side chains of 20 genetically encoded amino acids (or stereoisomeric D-amino acids) can also be substituted with other side chains such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, or 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and its lower ester derivatives, as well as 4-, 5-, 6-, or 7-membered heterocycles. 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 may be saturated or unsaturated, and in the case of unsaturation, it may 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, furyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl. These heterocyclic groups may or may not be substituted. When the group is substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or substituted or unsubstituted phenyl.

[0197] Other examples of amino acid substitutions include stereoisomers (e.g., D-amino acids), as well as non-natural amino acids such as L-ornithine, L-homocysteine, L-homoserine, L-citrulline, 3-sulphino-L-alanine, N-(L-arginino)succinate, 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-methylpropanoate, 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, norleucine, and other similar amino acids and imino acids, etc.

[0198] Amino acid residues that do not contribute much to the interaction with the T cell receptor can be modified by substitution with other amino acids that, when incorporated, do not substantially affect T cell reactivity and whose binding to the relevant MHC is not eliminated.

[0199] Peptides may also include isosteres of two or more residues. As used herein, "isostere" refers to a sequence of two or more residues whose three-dimensional structure can replace a second sequence by fitting into a binding site specific for the second sequence. The term specifically includes modifications of the peptide backbone known to those skilled in the art. Such modifications include: modification of the amide nitrogen, α-carbon, amide carbonyl; complete substitution of the amide bond; extension; deletion; or cross-linking of the backbone.

[0200] Combinations of multiple substitutions / additions / deletions at two or more positions can be developed and tested to determine whether the combination results in an additive or synergistic effect on the immunogenicity of the peptide. In some embodiments, no more than four positions within the peptide are modified simultaneously.

[0201] Preferably, the peptides of the present disclosure bind to MHC molecules within the peptide-binding groove of the MHC molecule. Generally, the amino acid modifications described above do not impair the ability of the peptide to bind to the MHC molecule. In some embodiments, the amino acid modifications improve the ability of the peptide to bind to the MHC molecule. For example, the peptide may be mutated at the positions where it binds to the MHC molecule. Such anchoring positions and the preferred residues at these positions for peptides that bind particularly to HLA-A*02 may include, for example, the amino acid residues at positions 2 and / or the C-terminus of the peptide, which can be considered as the major anchoring positions. The preferred anchoring residues may vary for each HLA type. As a non-limiting example, for HLA-A*02, the preferred amino acid at position 2 is Leu, Ile, Val, or Met, and the preferred amino acid at the C-terminus is Val or Leu. For stable peptide binding to HLA-A*02, multiple positions including positions 2, 3, 5-7, and 9 may be important. The anchoring residues at positions 2 and 9 may be the most important for peptide binding to HLA-A2. However, other peptide side chains, such as the peptide side chain at position 3, may contribute to the stability of the interaction. In some cases, the optimal length of the peptide bond may be longer than 9 residues.

[0202] The immunological properties of the peptide can be described as a function of its binding to MHC molecules (K on and K off ) and its binding to TCRs (the affinity of the interaction between the TCR and the MHC-peptide complex). Modification of the primary MHC anchor residues exhibits considerable predictability with respect to the overall effect on binding to MHC molecules. Modification of the secondary MHC anchoring residues can affect the affinity of the interaction of the MHC-peptide complex with TCRs, as well as K on and K off relating to the peptide-MHC interaction.

[0203] When the HBV peptide is a variant peptide, T cell lines against the native (non-mutated) epitope are generated and an immunization strategy is used that has sufficient potency to generate useful responses in transgenic mice bearing the immunization strategy human MHC (such as the A2 allele). The HBV peptide is examined ex vivo in the presence of competing APCs, and the functional effect of T cells specific for the native (non-mutated) epitope is measured. (Activated at a limited concentration and inhibited at a higher concentration by antigen-induced cell death (AICD)) In the case of cross-reactive peptides, since the expected effect is biphasic, evaluation is performed at various HBV peptide concentrations. Using the measurement of the following three parameters, the basic and useful characteristics of the HBV peptide are defined:

[0204] 1. The minimum concentration of the HBV metric required to induce an effect (such as cytokine (such as IFN-γ) production) that serves as an indicator of T cell activation; 2. The maximum (peak value) effect (such as cytokine (such as IFN-γ) production) at any HBV peptide concentration; and 3. The HBV peptide concentration (such as cytokine (such as IFN-γ) concentration) at the peak value of the activation effect.

[0205] As a non-limiting example, an HBV peptide with low values related to the first and third parameters and a high value related to the second parameter may be useful. Using the native epitope and / or an irrelevant non-cross-reactive peptide as a reference is useful for identifying classes of peptides that may be valuable. Peptides with characteristics quantitatively equivalent to those of the native epitope, or slightly weakened characteristics from those of the native epitope, may still be useful because while retaining cross-reactivity, they may exhibit immunological characteristics different from those of the native peptide, such as a reduced ability to break tolerance or re-establish responsiveness in vivo, or a reduced tendency to induce AICD.

[0206] In addition to practicality and speed, further advantages of this screening approach include, but are not limited to, the use of more relevant polyclonal T cell lines as readouts instead of potentially biased T cell clones, and the use of composite values integrating parameters such as K on , K off , and TCR affinity. Since these parameters can be predictors of immunological properties in vivo, they can define a useful panel of peptides for further evaluation, optimization, and practical applications. Peptides that bind to MHC and retain cross-reactivity to TCRs specific for nominal wild-type peptides are predicted to induce measurable effects in this assay.

[0207] The peptides of the present disclosure, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof, can be used to induce an immune response. In this case, it is important that the immune response be specific for the intended target (e.g., HBV) in order to avoid the risk of unwanted side effects that may be associated with "off target" immune responses. Accordingly, the amino acid sequences of the peptides of the present disclosure preferably do not match the amino acid sequences of any other one or more endogenous proteins, particularly the amino acid sequences of other human proteins. Also, the amino acid modifications described herein should not impair the ability of the peptides to induce an antigen-specific immune response when presented as a complex with MHC molecules on the surface of antigen-presenting cells (APCs).

[0208] The peptides can also be modified, for example, by PEGylation, glycosylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, or acylation to improve half-life and / or bioavailability.

[0209] The peptides of the present disclosure can also function as structural models of non-peptide compounds having equivalent biological activities. Compounds having the desired biological activity identical or equivalent to the lead peptide compound, but having more favorable activities than the lead peptide compound with respect to solubility, stability, and susceptibility to hydrolysis and proteolysis, can be constructed using a variety of techniques. These techniques include substituting the peptide backbone with a backbone composed of amidates, phosphonates, carbamates, sulfonamides, secondary amines, and N-methyl amino acids.

[0210] The plurality of peptides described herein may be operably linked to each other. Thus, in one aspect, the present disclosure provides an isolated peptide or polypeptide, or a derivative thereof, or a pharmaceutically acceptable salt thereof, comprising two or more amino acid sequences selected from SEQ ID NOs: 1-54 and 110-112. For example, such a multi-epitope peptide or polypeptide may comprise 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 of the single-epitope peptides described herein (e.g., SEQ ID NOs: 1-54 and 110-112). These single-epitope peptides (e.g., SEQ ID NOs: 1-54 and 110-112) may be arranged in any order and may be the same or different.

[0211] In some embodiments, the present disclosure provides an isolated peptide or polypeptide, or a derivative thereof, or a pharmaceutically acceptable salt thereof, comprising 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, 52, 53, or 54 amino acid sequences selected from SEQ ID NOs: 1-54 and 110-112.

[0212] These single epitope peptides may be linked via a linker. The linker may be composed of relatively small neutral molecules such as amino acids or amino acid mimetics that have substantially no charge under physiological conditions. The linker can be selected, for example, from Table 2, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the linker, if present optionally, need not be composed of the same residues and may be a hetero- or homo-oligomer. If a linker is present, this will usually be at least one or two residues, more generally three to six residues.

[0213] The peptides of the present disclosure can be synthesized, for example, by solid-phase synthesis. Therefore, the peptides can be immobilized on a solid support such as, for example, beads. The peptides of the present disclosure can be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis. Temporary protection of the N-amino group is provided by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repeated cleavage of this base-labile protecting group is carried out 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), butyloxycarbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cysteine), and 4-methoxy-2,3,6-trimethylbenzenesulfonyl derivatives (in the case of arginine). When glutamine or asparagine is the C-terminal residue, the 4,4'-dimethoxybenzhydryl group is used for protection of the side-chain amide functional group. The solid support is based on a polydimethyl-acrylamide polymer composed of three monomers, namely dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (cross-linking agent), and acryloylsarcosine methyl ester (functionalizing agent). The agent used here to cleavably link the peptide and the resin is a 4-hydroxymethyl-phenoxyacetic acid derivative that is extremely acid-labile. All amino acid derivatives are added as their preformed symmetric anhydride derivatives, except for asparagine and glutamine, which are added using an inverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isothiocyanate test procedures. When the synthesis is complete, the peptide is cleaved from the resin support, and simultaneously, the side-chain protecting groups are removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture.Commonly used scavengers include ethanedithiol, phenol, anisole, and water, and the exact choice depends on the constituent amino acids of the peptide to be synthesized. A combination of solid-phase and liquid-phase methods for peptide synthesis is also possible.

[0214] Trifluoroacetic acid is removed by vacuum evaporation, and then the crude peptide is provided by dispersion in diethyl ether. Any scavenger present is removed by a simple extraction procedure of lyophilizing the aqueous phase to provide a scavenger-free crude peptide.

[0215] Purification can be carried out by techniques such as recrystallization, ion-exchange chromatography, size-exclusion chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography using, for example, an acetonitrile / water gradient separation, or a combination thereof.

[0216] The peptide can be analyzed using thin-layer chromatography, electrophoresis, especially capillary electrophoresis, solid-phase extraction (CSPE), reverse-phase high-performance liquid chromatography, amino acid analysis after acid hydrolysis, and also by fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

[0217] Alternatively, the peptide may be produced by recombinant expression in a heterologous host cell. Such methods typically involve using a vector containing a nucleic acid sequence encoding the peptide to be expressed to express the polypeptide in vivo, for example, in bacteria, yeast, insects, or mammalian cells.

[0218] In a further embodiment, an in vitro cell-free system may be used. The peptide can be isolated and / or provided in a substantially pure form. For example, the peptide can be provided in a form substantially free of other peptides or proteins.

[0219] The peptide-MHC (pMHC) complex disclosed in this specification In another aspect, the present disclosure provides a complex of a peptide of the present disclosure and an MHC molecule. Preferably, the peptide binds to the peptide-binding groove of the MHC molecule. In some embodiments, the peptide and the MHC molecule form a non-covalent complex. In other embodiments, the peptide and the MHC molecule may be covalently linked, for example, via a linker.

[0220] MHC molecules are generally classified into two categories: class I MHC molecules and class II MHC molecules. MHC class I molecules are integral membrane proteins that contain a glycoprotein heavy chain, also called an α chain, having three extracellular domains (i.e., α1, α2, and α3) and two intracellular domains (i.e., a transmembrane domain (TM) and a cytoplasmic domain (CYT)). This heavy chain non-covalently associates with a soluble subunit called β2-microglobulin (β2m or Β2M). MHC class II molecules or MHC class II proteins are heterodimeric integral membrane proteins that contain one non-covalently associated α chain and one β chain. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (a TM domain and a CYT domain). The β chain contains two extracellular domains (β1 and β2) and two intracellular domains (a TM domain and a CYT domain).

[0221] The domain composition of class I and class II MHC molecules forms the antigenic determinant binding site of the MHC molecule, such as the peptide-binding portion or peptide-binding groove. The peptide-binding groove refers to a portion of the MHC molecule that forms a cavity to which a peptide, such as an antigenic determinant, can bind. The three-dimensional structure of the peptide-binding groove can be modified upon binding of the peptide, thereby allowing proper alignment of amino acid residues important for binding of the TCR to the peptide-MHC (pMHC) complex.

[0222] In some embodiments, the MHC molecule comprises a fragment of the MHC chain sufficient to form the peptide-binding groove. For example, the peptide-binding groove of a class I protein can comprise portions of the α1 and α2 domains of the heavy chain capable of forming two β-pleated sheets and two α-helices. By including a portion of the β2-microglobulin chain, the MHC class I molecule is stabilized. For most versions of the MHC class II molecule, the interaction between the α and β chains can occur in the absence of peptide, but the two-chain molecule of MHC class II is unstable until the binding groove is filled with peptide. The peptide-binding groove of a class II protein can comprise portions of the α1 and β1 domains capable of forming two β-pleated sheets and two α-helices. The first portion of the α1 domain forms the first β-pleated sheet, and the second portion of the α1 domain forms the first α-helix. The first portion of the β1 domain forms the second β-pleated sheet, and the second portion of the β1 domain forms the second α-helix. X-ray crystallographic structures of class II proteins with a peptide engaged in the binding groove of the protein show that one or both ends of the engaged peptide can project beyond the MHC protein. Thus, the ends of the α1 and β1 α-helices of class II form an open cavity, such that the ends of the peptide bound in the binding groove do not bury into the cavity. Further, X-ray crystallographic structures of class II proteins show that the N-terminus of the MHC β chain clearly projects in an unstructured form from the side of the MHC protein, because the first four amino acid residues of the β chain could not be assigned by X-ray crystallography.

[0223] The peptides of the present disclosure can bind to MHC molecules in such a manner that the pMHC complex can preferably bind to the TCR in a specific manner. In certain embodiments, binding of the pMHC complex to the TCR can induce a T cell response.

[0224] Whether a peptide forms a complex with an MHC molecule can be determined by evaluating whether the MHC can be refolded in the presence of the peptide using, for example, the process described in WO 2018 / 083505 (which is incorporated herein by reference in its entirety for all purposes). If the peptide does not form a complex with the MHC, the MHC will not be folded. Refolding can be confirmed using an antibody that recognizes only the folded state of the MHC. Alternatively, the ability of a peptide to stabilize the MHC on the surface of an antigen processing associated transporter (TAP) - deficient cell line such as T2 cells (which lack the ability to translocate cytosolic peptides into the endoplasmic reticulum (ER) via TAP for loading onto MHC class I molecules), or other biophysical methods for determining interaction parameters, can be determined.

[0225] The peptides according to the present disclosure can be provided as MHC groove - binding peptides. In some embodiments, the MHC groove - binding peptides can be designed such that the peptide can vary at some or all of the positions involved in MHC binding. For example, MHCBN is a comprehensive database of MHC - binding and non - binding peptides collected from published literature and existing databases. The latest version of this database has 25,860 entries, which include 20,717 MHC binders and 4,022 MHC non - binders related to more than 450 MHCs. This database includes (a) the source protein of the peptide and (b) the sequence and structural data of the MHC. MHCBN has a number of web tools including (i) mapping of peptides on a query sequence; (ii) searching in any field; (iii) creation of datasets; and (iv) online data submission.

[0226] In some cases, peptide binding tools for predicting 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, HLABinding, MAPPP, MHCBench, MHC-I Processing Predictions, Mosaic Vaccine Tool Suite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, Prediction of Antigenic Determinants, ProPred, ProPred-1, RankPep, SMM, SVMHC, TAPPred, VaxiJen, or combinations thereof. Additional exemplary programs such as BIMAS, SYFPEITHI, or Rankpep are used.

[0227] In a specific embodiment, a library of modified peptides is generated by genetically engineering the library using polymerase chain reaction (PCR), or any other suitable technique for constructing a DNA fragment encoding the peptide. In the PCR technique, by using oligonucleotides randomly mutated within a specific triplet codon, the resulting pool of fragments encodes all possible combinations of codons at these positions. Preferably, certain of the amino acid positions are kept constant, and these are conserved amino acids that do not contact the T cell receptor (TCR) necessary for binding to the MHC peptide binding groove.

[0228] In some embodiments, when generating a library of modified peptides by genetically engineering a library using polymerase chain reaction (PCR) or any other suitable technique for constructing DNA fragments encoding the peptides, the target TCR is a TCR for which it is desired to identify the epitope of the peptide recognized by the receptor. In some embodiments, the target TCR is from a patient with HBV infection and / or a disease or disorder induced by HBV, such as hepatocellular carcinoma (HCC). In some embodiments, the TCR comprises an α-chain and a β-chain.

[0229] Examples of MHC molecules used in the pMHC complexes described herein include naturally occurring full-length MHC molecules, as well as individual chains of MHC molecules (e.g., MHC class I α (heavy) chain, β2-microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC (e.g., α1, α2, and / or α3 subunits of the MHC class I α chain, α1 and / or α2 subunits of the MHC class II α chain, β1 and / or β2 subunits of the MHC class II β chain), and fragments, variants, and various derivatives thereof (including fusion proteins, such as fusions with viral envelope proteins or fusogens). The fragments, variants, and derivatives as described above retain the ability to display antigenic determinants for recognition by antigen-specific TCRs. In a specific embodiment, the MHC comprises a transmembrane domain embedded in the lipid envelope of liposomes, recombinant virus particles, or virus-like particles (VLPs).

[0230] Naturally occurring MHC molecules are encoded by gene clusters on human chromosome 6 or mouse chromosome 17. MHC is also called H-2 in mice and human leukocyte antigen (HLA) in humans. MHC class I molecules specifically bind to CD8 molecules expressed on cytotoxic T lymphocytes (CD8+ T cells), and 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 specificities 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 specificities include 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.

[0231] In some embodiments, the MHC molecule of the pMHC complex of the present disclosure is a human leukocyte antigen (HLA) molecule. The MHC molecule may be a human HLA molecule selected from the group consisting of 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-limiting examples of HLA-B alleles 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-limiting 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-limiting 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.

[0232] In some embodiments, the MHC class I molecule can 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, and HLA-C*07. Allelic variants of the HLA types described above also exist, and all of them are included in the present disclosure. In some embodiments, the MHC molecule can be HLA-A*02 or HLA-A*11.

[0233] As used herein, the MHC molecule may be derived from any other mammalian or avian species, such as non-human primates, rodents (e.g., mice), rabbits, horses, cows, dogs, cats, pigs, etc.

[0234] Naturally occurring MHC class I molecules bind to peptides derived from proteins degraded by proteolysis in the cell, particularly endogenously synthesized proteins. The small peptides thus obtained are transported to the endoplasmic reticulum, where, after binding to nascent MHC class I molecules, they pass through the Golgi apparatus and are displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0235] Naturally occurring MHC class I molecules consist of an α (heavy) chain bound to β2-microglobulin. This heavy chain consists of subunits α1-α3. The β2-microglobulin protein binds to the α3 subunit of the heavy chain. In certain embodiments, the β2-microglobulin and the α3 subunit are covalently bound. In certain embodiments, the β2-microglobulin and the α3 subunit are non-covalently bound. The α1 and α2 subunits of the heavy chain fold to form a groove for peptides, such as epitopes, that are displayed and recognized by the TCR.

[0236] Class I molecules can bind peptides that are approximately 8-10 amino acids in length. All humans have 3-6 different class I molecules, each of which can bind a number of different types of peptides.

[0237] In some embodiments, the MHC contained in the pMHC complex of the present disclosure comprises (i) a class I MHC polypeptide, or a fragment, variant, or derivative thereof, and optionally (ii) a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof. In certain specific embodiments, the class I MHC polypeptide is linked to the β2 microglobulin polypeptide by a peptide linker.

[0238] In certain specific embodiments, the class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G. In another specific embodiment, the class I MHC polypeptide is a mouse class I MHC polypeptide selected from the group consisting of H-2K, H-2D, H-2L, H2-IA, H2-IB, H2-IJ, H2-IE, and H2-IC.

[0239] In some embodiments, the peptides disclosed herein form a complex with one or more MHC class I α chains. In some embodiments, the MHC class I α chain is fully human. In some embodiments, the MHC class I α chain is humanized. Humanized MHC class I α chains are described, for example, in US Patent Application Publication Nos. 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467. In some embodiments, the MHC class I α 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 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 can be the HLA-A*0201 sequence. In various aspects, the peptide-MHC can include all domains of the MHC class I heavy chain.

[0240] In some embodiments, the MHC molecule comprises β2-microglobulin. In some embodiments, the β2-microglobulin is fully human. In some embodiments, the β2-microglobulin is humanized.

[0241] In some embodiments, the MHC class I molecule comprises mutations in the β2-microglobulin (β2m or Β2M) polypeptide and the heavy chain sequence, thereby creating a disulfide bond between Β2M and the heavy chain. Optionally, the heavy chain is HLA, and the disulfide bond links to one of the following pairs of residues: Β2M residue 12, HLA residue 236; Β2M residue 12, HLA residue 237; Β2M residue 8, HLA residue 234; Β2M residue 10, HLA residue 235; Β2M residue 24, HLA residue 236; Β2M residue 28, HLA residue 232; Β2M residue 98, HLA residue 192; Β2M residue 99, HLA residue 234; Β2M residue 3, HLA residue 120; Β2M residue 31, HLA residue 96; Β2M residue 53, HLA residue 35; Β2M residue 60, HLA residue 96; Β2M residue 60, HLA residue 122; Β2M residue 63, HLA residue 27; Β2M residue Arg3, HLA residue Gly120; Β2M residue His31, HLA residue Gln96; Β2M residue Asp53, HLA residue Arg35; Β2M residue Trp60, HLA residue Gln96; Β2M residue Trp60, HLA residue Asp122; Β2M residue Tyr63, HLA residue Tyr27; Β2M residue Lys6, HLA residue Glu232; Β2M residue Gln8, HLA residue Arg234; Β2M residue Tyr10, HLA residue Pro235; Β2M residue Ser11, HLA residue Gln242; Β2M residue Asn24, HLA residue Ala236; Β2M residue Ser28, HLA residue Glu232; Β2M residue Asp98, HLA residue His192; and Β2M residue Met99, HLA residue Arg234, first linker position Gly2, heavy chain (HLA) position Tyr84; light chain (Β2M) position Arg12, HLA Ala236; and / or Β2M residue Arg12, HLA residue Gly237.

[0242] In some embodiments, the amino acid sequence of the epitope can be that of the peptides described herein, which can be presented by MHC class I molecules. In certain embodiments, the sequence can comprise about 8 to about 15 contiguous amino acids. In certain embodiments, the sequence can comprise about 8 to about 12 contiguous amino acids.

[0243] In some embodiments, at least one chain of the MHC and the peptide are included within a fusion protein. In certain embodiments, the MHC and the peptide are separated by a linker sequence. For example, a single-chain molecule can include, from the amino terminus towards the carboxy terminus, an epitope, a β2-microglobulin sequence, and a class I α (heavy) chain sequence. Alternatively, a single-chain molecule can include, from the amino terminus towards the carboxy terminus, an epitope, a class I α (heavy) chain sequence, and a β2-microglobulin sequence. The single-chain molecule can further include a signal peptide sequence at the amino terminus. In certain embodiments, a linker sequence may be present between the peptide sequence and the β2-microglobulin sequence. In certain embodiments, a linker sequence may be present between the β2-microglobulin sequence and the class I α (heavy) chain sequence. The single-chain molecule can further include 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 certain embodiments, the β2-microglobulin and class I α (heavy) chain sequences can be of human, mouse, or porcine origin.

[0244] In some embodiments, the single-chain molecule can include a first flexible linker between the peptide ligand segment and the β2-microglobulin segment. For example, the linker can extend from the carboxy terminus of the peptide ligand segment to connect the carboxy terminus to the amino terminus of the β2-microglobulin segment. Preferably, the linker is configured such that the linked peptide ligand can be folded into the binding groove to obtain a functional MHC-antigen peptide. In some embodiments, this linker can include at least about 10 amino acids and up to about 15 amino acids. In some embodiments, the single-chain molecule can include a second flexible linker between the β2-microglobulin segment and the heavy chain segment. For example, the linker can extend from the carboxy terminus of the β2-microglobulin segment to connect the carboxy terminus to the amino terminus of the heavy chain segment. In certain embodiments, by folding the β2-microglobulin and the heavy chain into the binding groove, a molecule that can function in promoting T cell proliferation can be obtained.

[0245] Suitable linkers for use with MHC can be any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids (including from 4 amino acids to 10 amino acids, from 5 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 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 (e.g., including (GS)n, (GSGGS)n (SEQ ID NO: 105), and (GGGS)n (SEQ ID NO: 106), where n is an integer of 1 or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine, and glycine-serine polymers can be used, and since both Gly and Ser are relatively unstructured, they can function as neutral tethers between components. Glycine polymers can be used, and glycine has significantly greater access to a wider phi-psi space than alanine and far fewer restrictions compared to residues with longer side chains. Exemplary linkers can include amino acid sequences including, but not limited to, those listed in Table 2. In some embodiments, the linker peptide includes a cysteine residue capable of forming a disulfide bond with a cysteine residue present in a second polypeptide.

[0246]

Table 2

[0247] In certain embodiments, the single-chain molecule can comprise a peptide covalently bound to an MHC class I α (heavy) chain via a disulfide bridge (i.e., a disulfide bond between two cysteines). In certain embodiments, this disulfide bond comprises a first cysteine comprising a linker extending from the carboxy terminus of the antigen peptide and a second cysteine comprising an MHC class I heavy chain (e.g., an MHC class I α (heavy) chain having a non-covalent binding site for the antigen peptide). In certain embodiments, the second cysteine can be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In certain embodiments, the single-chain molecule can comprise one continuous polypeptide chain and a disulfide bridge. In certain embodiments, the single-chain molecule can comprise two continuous polypeptide chains joined via a disulfide bridge as the only covalent bond. In some embodiments, the linker sequence can comprise at least one amino acid comprising one or more Gly residues, one or more Ala residues, and / or one or more Ser residues in addition to the Cys residue.

[0248] In certain embodiments, when the pMHC complex comprises a first cysteine in a Gly-Ser linker extending between the C-terminal peptide of the peptide and β2-microglobulin and a second cysteine at the proximal heavy chain position, the disulfide bridge can link the antigen peptide described herein within the class I groove of the pMHC complex.

[0249] Linking a peptide to an MHC class I or MHC class II molecule via a flexible linker can help ensure that the peptide occupies and remains bound to the MHC molecule during biosynthesis, transport, and display. However, there can be situations where this linker might interfere with peptide binding to the MHC molecule or recognition of the complex by the TCR. As an alternative approach, in some embodiments, the MHC molecule and the peptide are expressed separately.

[0250] In some embodiments, the β2-microglobulin sequence can include the full-length β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks a leader peptide sequence. Thus, in some configurations, the β2-microglobulin sequence can include about 99 amino acids and can be a mouse β2-microglobulin sequence (e.g., GenBank accession number X01838). In some other configurations, the β2-microglobulin sequence can include about 99 amino acids and can be a human β2-microglobulin sequence (e.g., GenBank accession number AF072097.1).

[0251] In some embodiments, the pMHC complex can comprise an MHC sequence as disclosed in U.S. Patent No. 4,478,823; U.S. Patent No. 6,011,146; U.S. Patent No. 8,518,697; U.S. Patent No. 8,895,020; U.S. Patent No. 8,992,937; International Publication No. 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. Nat’l 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 (each of these references is hereby incorporated by reference in its entirety).

[0252] In some embodiments, the MHC comprises a class II MHC polypeptide, or a fragment, variant, or derivative thereof. In certain specific embodiments, the MHC comprises the α and β polypeptides of a class II MHC complex, or a fragment, variant, or derivative thereof. In certain specific embodiments, the α polypeptide and the β polypeptide are linked by a peptide linker. In certain specific embodiments, the MHC comprises the α and β polypeptides of a human class II MHC complex selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO. In another specific embodiment, the MHC comprises the α and β polypeptides of a mouse H-2A or H-2E class II MHC complex.

[0253] Naturally occurring MHC class II molecules can comprise two polypeptide chains, namely α and β. These chains can be derived from the DP, DQ, or DR gene groups. There are approximately 40 various known human MHC class II molecules. Although they all have the same nucleotide structure, their molecular structures may vary slightly. MHC class II molecules can bind to peptides that are 13 - 18 amino acids in length.

[0254] In some embodiments, this MHC class II α chain is fully human. In some embodiments, this MHC class II α chain is humanized. Humanized MHC class II α chains are described, for example, in U.S. Patent No. 8,847,005, U.S. Patent No. 9,043,996, and U.S. Patent No. 10,154,658 (these documents 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.

[0255] In some embodiments, the peptides of the present disclosure form a complex with one or more MHC class II β-chains. In some embodiments, this MHC class II β-chain is fully human. In some embodiments, this MHC class II β-chain polypeptide is humanized. Humanized MHC class II β-chain polypeptides are described, for example, in U.S. Patent No. 8,847,005, U.S. Patent No. 9,043,996, and U.S. Patent No. 10,154,658 (these documents 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-DOA, 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.

[0256] The pMHC complexes of the present disclosure may be isolated and / or in a substantially pure form. For example, the complex may be provided substantially free of other peptides or proteins. The MHC molecules disclosed herein can include recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC (including derivatives or variants thereof), as long as the peptide binding is retained. For example, the MHC molecule may be fused to a therapeutic moiety, attached to a solid support, in a soluble form, attached to a tag, biotinylated, and / or in a multimeric form. The peptides disclosed herein may covalently bind to the MHC.

[0257] Methods for generating soluble recombinant MHC molecules capable of forming complexes with the peptides disclosed herein include, but are not limited to, growth and purification from E. coli cells or insect cells. Alternatively, MHC molecules can also be generated synthetically or using a cell-free system.

[0258] The peptides disclosed herein may be presented on the surface of cells as complexes with MHC. Accordingly, the present disclosure also provides cells presenting the pMHC complexes disclosed herein. Such cells may be mammalian cells, preferably cells of the immune system, and specialized antigen-presenting cells (APCs) such as dendritic cells or B cells. Other preferred cells include T2. Cells presenting the peptides or pMHC complexes of the present disclosure may preferably be isolated in the form of a homogeneous population or provided in a substantially pure form. Such cells may not naturally present the complexes of the present disclosure, or the cells may present the complexes at a higher level than natural. Such cells can be obtained by pulsing the cells with one or more peptides of the present disclosure (e.g., 2 - 50, 2 - 40, 2 - 30, 5 - 25, 5 - 20, or 10 - 15 peptides) or by genetically modifying the cells to express one or more peptides of the present disclosure (by DNA or RNA transfection). Pulsing typically involves incubating the cells with the peptide for several hours using a peptide concentration in the range of 10 -5 ~10 -12 M. Such cells can be further transduced with HLA molecules such as HLA-A*02 to further induce the presentation of one or more peptides. The cells may be generated recombinantly. Cells presenting the peptides of the present disclosure can be used to isolate T cells and TCRs that are activated by or bind to the cells.

[0259] The fusion proteins, conjugates, and oligomeric complexes disclosed herein The peptides or pMHC complexes disclosed herein may be fused or conjugated to one or more heterologous molecules. The peptides or pMHC complexes of the present disclosure may be in multimeric form. Accordingly, the present disclosure also provides fusion proteins, conjugates, and oligomeric complexes comprising the peptides or pMHC complexes of the present disclosure.

[0260] In some embodiments, the peptide is fused or conjugated to one or more heterologous molecules comprising an MHC molecule (or a fragment thereof).

[0261] Suitable heterologous molecules for genetic fusion and / or chemical conjugation with the peptides or pMHC complexes of the present disclosure include, but are not limited to, peptides, polypeptides, small molecules, polymers, nucleic acids, lipids, sugars, and the like. One or more heterologous molecules may be fused to the N-terminus and / or C-terminus of the peptide within the pMHC complex and / or another polypeptide chain.

[0262] Heterologous peptides and polypeptides include epitopes (e.g., FLAG) or tag sequences (e.g., His6 (SEQ ID NO: 109), etc.) that enable detection and / or isolation of fusion proteins; transmembrane receptor proteins or fragments thereof, such as extracellular domains or transmembrane and intracellular domains; ligands that bind to transmembrane receptor proteins or portions thereof; enzymes having catalytic activity or portions thereof; polypeptides or peptides that promote oligomerization, such as leucine zipper domains; polypeptides or peptides that improve stability, such as immunoglobulin constant regions (e.g., Fc domain); 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 mainly hydrophilic or mainly hydrophobic fusion partners for fusion proteins, including half-life extension sequences; functional or non-functional antibodies (e.g., antibodies specific for dendritic cells), or heavy or light chains thereof; and polypeptides having activities such as therapeutic activities different from the fusion proteins of the present disclosure. In some embodiments, one or more heterologous molecules enhance a peptide-specific immune response in a subject's body. In some embodiments, one or more heterologous molecules mediate peptide delivery to a specific site in a subject's body.

[0263] In some embodiments, the fusion proteins of the present disclosure may include one or more affinity tags, for example, to enable affinity purification or coupling to another molecule. Examples of affinity tags include, but are not limited to, His6 tag (SEQ ID NO: 109), Avi tag, biotin, hemagglutinin (HA) tag, FLAG tag, Myc tag, GST tag, MBP tag, chitin-binding protein tag, calmodulin tag, V5 tag, streptavidin-binding tag, green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, SUMO tag, and ubiquitin tag.

[0264] In some embodiments, the fusion proteins of the present disclosure may include one or more epitopes that are not present in the antigen. One such example is the use of a fusion peptide in which a promiscuous T helper epitope is covalently linked to a peptide sequence (e.g., via a polypeptide linker or spacer). Non-limiting examples of promiscuous T helper epitopes include the PADRE peptide, the tetanus toxoid peptide (830-843), or influenza hemagglutinin, HA (307-319).

[0265] The peptides or pMHC complexes of the present disclosure may be conjugated to additional substructures such as carrier molecules or adjuvants for use as vaccines. Examples of adjuvants used in vaccines include bacteria such as Bacillus Calmette-Guerin (BCG), and / or substances produced by bacteria such as Detox B (an oil-in-water emulsion of monophosphoryl lipid A and mycobacterial cell wall skeleton). KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), and the E2 core protein of the pyruvate dehydrogenase complex are examples of suitable carrier proteins used in vaccine compositions. Further examples of carrier proteins suitable for use in the compositions of the present disclosure include, but are not limited to, ovalbumin (OVA), Blue Carrier Protein (BCP), thyroglobulin (THY), soybean trypsin inhibitor (STI), and multiple antigen peptide (MAP), albumin, serum albumin, C-reactive protein, conalbumin, lactalbumin, ion carrier protein, acyl carrier protein, signal transduction adapter protein, androgen-binding protein, calcium-binding protein, calmodulin-binding protein, ceruloplasmin, cholesterol ester transfer protein, F-box protein, fatty acid-binding protein, follistatin, follistatin-related protein, GTP-binding protein, insulin-like growth factor-binding protein, iron-binding protein, latent TGFβ-binding protein, light-harvesting protein complex, lymphocyte antigen, membrane transport protein, neurophysin, periplasm-binding protein, phosphate-binding protein, phosphatidylethanolamine-binding protein, phospholipid transport protein, retinol-binding protein, RNA-binding protein, S-phase kinase-associated protein, sex hormone-binding globulin, thyroxine-binding protein, transcobalamin, transcortin, transferrin-binding protein, and / or vitamin D-binding protein.

[0266] As a further example, the peptides or pMHC complexes of the present disclosure can be fused, for example, to the 80 N-terminal amino acids of the 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.

[0267] The peptides or pMHC complexes of the present disclosure may be covalently or non-covalently linked (e.g., via a linker) to a substructure capable of inducing a therapeutic effect, such as an antibody, or cytokines such as interleukin 2, interferon α, and granulocyte macrophage colony-stimulating factor. Alternatively, or further, the peptide or pMHC complex may be encapsulated in liposomes.

[0268] Other suitable heterologous molecules include, but are not limited to, fluorescent or luminescent labels, radioactive labels, nucleic acid probes, and contrast reagents, antibodies, or enzymes that produce detectable products. Methods for detecting heterologous molecules can include flow cytometry, microscopy, electrophoresis, or scintillation counting.

[0269] In some embodiments, the peptides or pMHC complexes of the present disclosure may be conjugated to fluorocarbons to enhance the immunogenicity of cells. When linking the peptide of the pMHC complex or another polypeptide chain to a fluorocarbon, the ends of the peptide or polypeptide chain, for example, the end not conjugated to the fluorocarbon, or other attachment sites, can be modified, for example, to promote the solubility of the fluorocarbon-peptide / polypeptide construct by micelle formation. To facilitate the large-scale synthesis of this construct, the N- or C-terminal amino acid residues of the peptide of the pMHC complex or another polypeptide chain can be modified. If the desired peptide of the pMHC complex or another polypeptide chain is particularly sensitive to cleavage by peptidases, normal peptide bonds can be replaced with non-cleavable peptide mimics. Such coupling and synthetic methods are known in the art.

[0270] The peptides or pMHC complexes of the present disclosure may be provided in a soluble form or may be immobilized by attachment to a suitable solid support. Examples of solid supports include, but are not limited to, beads, membranes, sepharose, magnetic beads, plates, tubes, columns. The pMHC complex may be attached to an ELISA plate, magnetic beads, or a surface plasmon resonance biosensor chip. Methods for attaching peptides or pMHC complexes to solid supports are known to those skilled in the art and include, for example, using affinity binding pairs such as biotin and streptavidin, or an antibody and an antigen. In some embodiments, the peptide or pMHC complex is labeled with biotin and attached to a surface coated with streptavidin.

[0271] The peptides or pMHC complexes of the present disclosure may be in a multimeric form, such as a dimer, tetramer, pentamer, or octamer, or even a multimer with a larger number of monomers. Thus, in some aspects, the present disclosure provides an oligomeric complex comprising the peptides or pMHC complexes of the present disclosure. As used herein, the terms “oligomer,” “oligomeric,” “oligomerize,” and “oligomerization,” etc. include dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, or even various polymeric monomers that make up the peptide or pMHC complex. Having multiple copies of the peptide or pMHC complex in a large complex can enhance their biological activities, such as immunogenic activity.

[0272] For example, the peptides of the present disclosure can be oligomerized using the biotin / streptavidin system. Biotinylated analogs of peptide monomers can be synthesized by standard techniques. For example, the C-terminus of the peptide can be biotinylated. Next, these biotinylated peptide monomers are oligomerized by incubating them with streptavidin (e.g., at a molar ratio of 4:1, at room temperature, in phosphate-buffered saline (PBS) or HEPES-buffered RPMI medium for 1 hour). In a variant of this embodiment, the biotinylated peptide monomers can be oligomerized by incubating them with an anti-biotin antibody (e.g., goat anti-biotin IgG).

[0273] Generally, oligomeric pMHC complexes can be generated using pMHC that is tagged with biotin residues and complexed with fluorescently labeled streptavidin. For example, a biotinylation site may be introduced into a pMHC complex to which biotin can be added using the BirA enzyme. Alternatively, oligomeric pMHC complexes can be formed by using an immunoglobulin as a molecular scaffold. In this system, the extracellular domain of the MHC molecule is fused to the constant region of an immunoglobulin heavy chain separated by a short amino acid linker. Oligomeric pMHC complexes have also been generated using carrier molecules such as dextran. Oligomeric pMHC complexes can be useful for improving the detection of binding moieties such as T cell receptors that bind to the complexes described above due to their binding activity effects.

[0274] In other embodiments, the peptides or pMHC complexes of the present disclosure can be oligomerized by covalent attachment to at least one linker. The linker substructure can be a peptide linker such as those described herein (e.g., in Table 2). In some embodiments, polyethylene glycol (PEG) can function as a linker for oligomerizing peptide monomers. For example, a single PEG substructure can be simultaneously attached to the N-termini of the two peptide chains of a peptide dimer.

[0275] Alternatively, the oligomeric peptide or pMHC complex may contain one or more intramolecular disulfide bonds between the cysteine residues of the peptide or pMHC monomer. Preferably, the two monomers contain at least one intramolecular disulfide bond. Most preferably, the two monomers contain one intramolecular disulfide bond and thus each monomer contains a cyclic group. Such disulfide bonds can be formed by oxidation of the cysteine residues of the peptide core sequence. In one embodiment, control of the formation of the disulfide bond is effected by selecting an oxidizing agent of the type and concentration effective to optimize the formation of the desired isomer. For example, when the oxidizing agent is DMSO, oxidation of the peptide dimer to form two intramolecular disulfide bonds (one in each peptide chain) is achieved preferentially (over the formation of intramolecular disulfide bonds). Formation of the cysteine bond can be controlled by the selective use of thiol protecting groups during peptide synthesis.

[0276] In some embodiments, the peptides or pMHC complexes described herein may be fused or conjugated to a dimerization substructure. The dimerization substructure may include, for example, an immunoglobulin domain such as that derived from an IgG antibody (e.g., human IgG), which connects two monomers to generate a homodimer or heterodimer molecule. As a non-limiting example, the dimerization motif of the protein according to the present disclosure may be constructed to include a hinge region and an immunoglobulin domain (e.g., Cy3 domain), such as a carboxy-terminal C domain (CH3 domain), or a sequence substantially identical to the C domain. The hinge region may be of Ig origin and contributes to dimerization by forming one or more inter-chain covalent bonds, such as one or more disulfide bridges. Further, such a homodimer or heterodimer molecule may further include one or more targeting substructures that bind to target molecules present on antigen-presenting cells (APCs) such as dendritic cells or B cells. In such cases, the hinge region can function as a flexible spacer between domains and can simultaneously bind two targeting units to two target molecules on the APCs expressed at variable intervals. The immunoglobulin domain contributes to dimerization by non-covalent interactions, such as hydrophobic interactions. In a preferred embodiment, the CH3 domain is derived from IgG. These dimerization substructures can also be exchanged, for example, with other multimerization substructures derived from other Ig isotypes / subclasses. Preferably, the dimerization motif is derived from a native human protein such as human IgG. Examples of such homodimer protein constructs are described in U.S. Patent No. 10,590,195, which is incorporated herein by reference in its entirety.

[0277] Nucleic Acids and Vectors In another aspect, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence encoding one or more peptides and / or peptide-based molecules of the present disclosure (such as complexes comprising the peptides described herein (e.g., pMHC complexes), fusion proteins, or conjugates, etc.). The polynucleotide may be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, RNA, or a combination thereof, or a polynucleotide in native form or a stabilized form (e.g., a polynucleotide having a phosphorothioate backbone, etc.), and may or may not contain introns as long as it encodes a peptide.

[0278] In some embodiments, the polynucleotides described herein encode a peptide, or a fragment or derivative thereof, having an amino acid sequence that is 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% identical to any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the polynucleotides described herein encode a peptide, or a fragment or derivative thereof, comprising any one of the amino acid sequences of SEQ ID NOs: 1-54 and 110-112.

[0279] In some embodiments, the polynucleotides described herein encode two or more peptides selected from any one of SEQ ID NOs: 1-54 and 110-112, or fragments thereof. For example, the polynucleotides described herein may encode 2-50, 2-40, 2-30, 5-25, 5-20, or 10-15 peptides, or fragments thereof, described herein (e.g., of SEQ ID NOs: 1-54 and 110-112). The peptides may be arranged in any order and may be the same or different.

[0280] In some embodiments, the polynucleotides described herein encode two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-one, fifty-two, fifty-three, or fifty-four amino acid sequences selected from SEQ ID NOs: 1-54 and 110-112, or fragments thereof.

[0281] In some embodiments, the polynucleotides described herein are DNA molecules.

[0282] Examples of methods for delivering DNA to a subject include direct delivery as naked DNA. Delivery can also be achieved by nanoparticles; gene guns, microneedle arrays, and in situ electroporation. Nucleic acids can also be administered using ballistic delivery. Particles composed of only DNA can be administered. Alternatively, DNA can be attached to particles such as gold particles.

[0283] In some embodiments, the polynucleotides described herein are RNA molecules. For example, the RNA molecule may be mRNA or self-replicating RNA.

[0284] The polynucleotides encoding RNA disclosed in this specification can be used for the production of vaccines. Since RNA cannot be integrated into the genome and thus has little carcinogenicity, RNA can be useful for the production of vaccines. Also, in contrast to DNA which needs to enter the nucleus, RNA only needs to enter the cytoplasm. The RNA molecules disclosed in this specification may be chemically modified and / or can incorporate modified nucleosides in order to overcome sensitivity to degradation. RNA vaccines may include 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 into liposomes or nanoparticles.

[0285] Nucleic acids (DNA or RNA) can also be delivered as a complex with a cationic compound such as a cationic lipid. Lipid-mediated gene delivery methods are described, for example, in WO 91 / 06309, WO 93 / 24640, WO 96 / 18372, US Patent No. 5,279,833 (these documents are hereby incorporated by reference in their entirety into this application).

[0286] The nucleic acid molecules described herein may be generated by synthesis. One method is the phosphoramidite method. Without wishing to be bound by theory, in this chemical reaction, a phosphoramidite (a nucleoside having side chain protecting groups that preserve the integrity of the sugar, phosphodiester bond, and base during the chain elongation step) is coupled via its reactive 3’ phosphorus group to the 5’ hydroxyl group of a nucleoside immobilized on a solid support column. The steps of oligonucleotide synthesis can include: (1) detritylation, in which the dimethoxytrityl (DMT or trityl) group of the 5’ hydroxyl of the support-side nucleoside is removed by treatment with trichloroacetic acid (TCA). (2) In the coupling step, the phosphoramidite, made reactive by tetrazole (a weak acid), is chemically coupled to the base last added to the column support material. (3) In the capping step, any free 5’ hydroxyl groups of unreacted nucleotides on the column are acetylated by treatment with acetic anhydride and N-methylimidazole. (4) In the oxidation step, the labile internucleotide phosphate bond between the already-coupled base and the last-added base is oxidized by treatment with iodine and water to form a more stable phosphotriester bond. After coupling all of the bases in the sequence of the oligonucleotide, the completed nucleic acid strand 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.

[0287] As a non-limiting example, the synthesis cycle may include the growth of a nucleotide chain on a solid support from a first protected nucleoside derivatized via its terminal 3'-hydroxyl. Reagents and solvents can be pumped through the support to separate the reactivity of specific chemical substructures on the monomer and stepwise add them to the growing oligonucleotide chain by inducing sequential removal and addition of sugar protecting groups. The assembly of the protected oligonucleotide chain can be carried out in chemical steps including, but not limited to, deblocking, activation / coupling, oxidation, and capping. Thereafter, a single-stranded nucleic acid appears by cleavage and deprotection.

[0288] The nucleic acid synthesis methods disclosed herein can include, for example, oligonucleotide synthesis, column-based oligonucleotide synthesis, microarray-based oligonucleotide synthesis, gene synthesis from oligonucleotides, gene synthesis from oligonucleotide pools derived from arrays, and any one of various error correction and sequence verification steps, or any combination thereof.

[0289] The chemical synthesis of RNA can be similar to that used for DNA. In some embodiments, the chemical synthesis method of RNA may include additional protecting groups on the 2'-hydroxyl of ribose. The position of the 2'-hydroxyl of ribose may be protected with a tert-butyldimethylsilyl group, which can be made stable throughout the synthesis and removed by adding a basic fluoride ion such as tetrabutylammonium fluoride (TBAF) in the final deprotection step. The remaining positions of both the sugar and the base can be protected in the same manner as in the case of DNA. By adjusting multiple parameters of the DNA synthesis protocol, including, but not limited to, coupling time, monomer delivery rate, frequency of washing steps, and type of capping reagent, a stepwise coupling efficiency of up to 99% can be obtained.

[0290] Viral nucleic acid synthesis can be catalyzed by both viral and host enzymes, and their relative contributions can be determined by the virus type and specific molecules. Viruses with RNA genomes, except retroviruses, synthesize mRNA and replicate their genomes using virus-encoded RNA-dependent RNA polymerases. In contrast, retroviruses use a virion-encoded RNA-dependent DNA polymerase (reverse transcriptase) to synthesize a double-stranded complementary DNA (cDNA) copy of their single-stranded RNA genome. In subsequent steps, the retroviral cDNA is integrated into the host chromosome and transcribed by host-encoded DNA-dependent RNA polymerase II (pol II) to obtain viral messages and genomic RNA. DNA viruses, except poxviruses, also use host-encoded pol II for the transcription of their messages. Poxviruses replicate in the cytoplasm and do not have access to pol II, so they assemble a novel transcriptase composed of multiple poxvirus-specific (and derived from one or more hosts) subunits. Most DNA virus families (e.g., Poxviridae, Iridoviridae, Herpesviridae, Adenoviridae) synthesize virus-encoded DNA-dependent DNA polymerases. However, two families (i.e., Parvoviridae and Papovaviridae) utilize host DNA polymerases, and the Hepadnaviridae replicates viral DNA via an RNA intermediate using a virus-encoded transcriptase.

[0291] Due to the degeneracy of the genetic code, nucleic acid molecules with different nucleotide sequences can encode the same amino acid sequence. For expression in various hosts, the polynucleotide may be codon-optimized.

[0292] In a further aspect, the present disclosure provides a vector comprising a nucleic acid sequence according to the third aspect of the present disclosure. This vector may comprise one or more additional nucleic acid sequences encoding one or more additional peptides in addition to the nucleic acid sequence encoding only the peptide of the present disclosure. Such additional peptides, when expressed, may be fused to the N-terminus or C-terminus of the peptide of the present disclosure. Examples of such additional peptides have been detailed in multiple previous sections. In one embodiment, the vector comprises a nucleic acid sequence encoding a peptide or protein tag such as a biotinylation site, FLAG tag, MYC tag, HA tag, GST tag, Strep tag, or poly-histidine tag, etc.

[0293] In the context of the present disclosure, it is desirable for the vector to comprise sequences suitable for introduction into cells. For example, the vector may be an expression vector, a vector in which the coding sequence of the polypeptide is under the control of its own cis-acting regulatory elements, a vector designed to facilitate gene integration or gene replacement in a host cell, etc.

[0294] In the context of the present disclosure, the term "vector" includes DNA molecules such as plasmids, bacteriophages, phagemids, viruses, or other mediators that contain one or more heterologous or recombinant nucleotide sequences (e.g., the above-described nucleic acid molecule of the present disclosure under the control of a functional promoter and optionally an enhancer) and can function as a vector in a sense understood by those skilled in the art.

[0295] The following vectors can be cited as examples: bacteriophages such as lambda(X) bacteriophage, EMBL bacteriophage; 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 transposon and PiggyBac transposon.

[0296] In some embodiments, the vector is a viral vector. The viral vector may be derived from a naturally occurring viral genome that has typically been modified to be replication-incompetent, for example non-replicating. Non-replicating viruses require the supply of proteins in trans for replication. Typically, these proteins are expressed stably or transiently in a viral production cell line, thereby enabling viral replication. Thus, viral vectors are typically infectious and non-replicating.Viral vectors may be adenoviral vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 5 and type 2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE-SIN chimeras), herpesvirus vectors (e.g., vectors derived from cytomegalovirus such as rhesus cytomegalovirus (RhCMV)), arenavirus vectors (e.g., lymphocytic choriomeningitis virus (LCMV) vectors), measles virus vectors, poxvirus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipoxvirus vectors (canarypox virus (ALVAC) and fowlpox virus (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retroviral vectors, lentiviral vectors, simian virus 40 (SV40), bovine papillomavirus, Epstein-Barr virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, Rous sarcoma virus, poxvirus-like particles, baculovirus vectors, and may be bacterial spores.

[0297] As a further example, an adenovirus vector may be derived not only from human adenovirus (Ad), but also from adenoviruses that infect 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 adenoviruses derived from great apes such as the genus Pan, Gorilla, Pongo, Pan paniscus, and Pan troglodytes. A poxvirus (Poxviridae) vector may be derived from variola virus (smallpox), vaccinia virus, cowpox virus, or monkeypox virus. Examples of vaccinia virus are Copenhagen vaccinia virus (W), New York Attenuated Vaccinia Virus (NYVAC), ALVAC, TROVAC, and Modified Vaccinia Ankara (MVA).

[0298] In the art, a number of expression systems are known, including bacteria (e.g., Escherichia coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., the genus Aspergillus), plant cells, animal cells (e.g., mammalian cells), and insect cells.

[0299] In yet another aspect, the present disclosure provides a host cell comprising a vector of the present disclosure. This host cell may be a prokaryotic cell or a eukaryotic cell. Bacterial cells can be preferred prokaryotic host cells depending on the situation, and typically are strains of Escherichia coli such as Escherichia coli strains DH5 and RR1. Non-limiting examples of eukaryotic host cells include yeast, insect, and mammalian cells (e.g., 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 embryo cells NIH / 3T3, monkey kidney-derived COS-1 cells, and 293 cells which are human fetal kidney cells. An example of an insect cell is Sf9 cells which can be transfected with a baculovirus expression vector.

[0300] Transformation of a suitable cell host with the DNA construct of the present disclosure is typically achieved by known methods depending on the type of vector used. A well-transformed cell, i.e., a cell containing the DNA construct of the present disclosure, can be identified, for example, by PCR. Alternatively, the presence of a protein in the supernatant can be detected using an antibody.

[0301] It will be understood that certain host cells of the present disclosure are useful for the preparation of the peptides or peptide-based molecules of the present disclosure, such as bacterial, yeast, and insect cells. However, other host cells may also be useful in certain therapeutic methods. For example, antigen-presenting cells (APCs) such as dendritic cells or B cells can be used to express a peptide of the present disclosure such that the peptide can be loaded onto appropriate MHC molecules.

[0302] A further aspect of the present disclosure provides a method for generating a peptide or peptide-based molecule of the present disclosure, the method comprising culturing a host cell and isolating the peptide or peptide-based molecule from the host cell or its culture medium.

[0303] Peptide and pMHC binding substructure Using the peptides, pMHC complexes, or other peptide-based molecules of the present disclosure (such as complexes, fusion proteins, or conjugates containing the peptides of the present disclosure), binding substructures that specifically bind to the peptides, pMHC complexes, or other peptide-based molecules of the present disclosure can be identified and / or isolated. Such binding substructures can be used as immunotherapy reagents and can include, for example, antibodies (or antigen-binding fragments thereof), alternative scaffolds, TCRs, and CARs.

[0304] In one aspect, the present disclosure provides a peptide binding substructure that binds to the peptide of the present disclosure. Preferably, this peptide binding substructure binds to the peptide when the peptide is complexed with MHC. In the latter case, the peptide binding substructure may bind partially to MHC as long as the peptide binding substructure also binds to the peptide. The peptide binding substructure may bind only to the peptide, and this binding may be specific. The peptide binding substructure may bind only to the pMHC complex, and this binding may be specific.

[0305] The present disclosure also provides a method for identifying a peptide binding substructure that binds to the pMHC complex of the present disclosure. The method includes contacting a peptide binding substructure candidate with the pMHC complex and determining whether the peptide binding substructure candidate binds 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, and microscopy. Alternatively, or additionally, binding can be determined by a functional assay that detects a biological response, such as cytokine release or apoptosis of cells, upon binding.

[0306] The peptide binding substructure candidate may be a peptide binding substructure of the type already described, such as an antibody or TCR.

[0307] For example, antibodies and TCRs can be obtained from display libraries in which libraries are panned using the pMHC complexes of the present disclosure. TCRs can be displayed, for example, on the surfaces of phage particles and yeast particles, and such libraries have been used for the isolation of high-affinity variants of TCRs derived from T cell clones. TCR phage libraries can be used for the isolation of TCRs having new antigen specificities. Such libraries can be constructed with α-chain sequences and β-chain sequences corresponding to those found in the natural repertoire. However, random combinations of these α-chain sequences and β-chain sequences that occur during library creation may generate TCR repertoires that do not occur naturally.

[0308] In some embodiments, the pMHC complexes of the present disclosure can be used to screen a library of diverse TCRs displayed on the surface of phage particles. The TCRs displayed by the library may not correspond to those included in the natural repertoire. For example, they may include pairings of α-chains and β-chains that do not exist in vivo, and / or the TCRs may include non-natural variants, and / or the TCRs may be in a soluble form. Screening may include the step of panning the phage library with the pMHC complex of the present disclosure and the step of isolating 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 the pMHC complex bound with phage is isolated by a magnet or by chromatography, respectively. The panning step may be repeated multiple times. The isolated phage may be further propagated in E. coli cells. The isolated phage particles may be tested for specific binding to the pMHC complex of the present disclosure. Binding can be detected using techniques including, but not limited to, ELISA or SPR using, for example, equipment manufactured by Biacore. The DNA sequence of the T cell receptor displayed by the pMHC-binding phage can be further identified by PCR.

[0309] Alternatively, antigen-binding T cells and TCRs can be isolated from fresh blood obtained from a patient or a healthy donor. Such methods include the steps of stimulating T cells with autologous dendritic cells (DCs), followed by autologous B cells, and then pulsing with the peptides disclosed herein. Multiple rounds of stimulation, such as 3 or 4 rounds, may be performed. Thereafter, the activated T cells may be tested for specificity by measuring cytokine release in the presence of T2 cells pulsed with the peptides of the present disclosure (e.g., using an IFNγ ELISpot assay). Next, the activated cells may be sorted by fluorescence-activated cell sorting (FACS) using labeled antibodies for detecting the expression of intracellular cytokine production (e.g., IFNγ) or cell surface markers (such as CD137). The sorted cells may be expanded and further verified, for example, by ELISpot assay, and / or cytotoxicity against target cells, and / or staining with peptide-MHC tetramers. And the TCR chains from the verified clones may be amplified by rapid amplification of cDNA ends (RACE) and sequenced.

[0310] Peptide binding moieties disclosed herein include, but are not limited to, for example, antibodies, TCRs, or CARs.

[0311] In some embodiments, the peptide binding moiety of the present disclosure may be an antibody or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof include antibody derivatives, functional equivalents, and homologs, humanized antibodies, which are any polypeptides containing immunoglobulin binding domains whether natural or fully or partially synthetic, and any polypeptides or proteins having a binding domain that is an antibody binding domain or is homologous to an antibody binding domain. Thus, also included are chimeric molecules containing an immunoglobulin binding domain, or equivalents thereof, fused to another polypeptide. A humanized antibody may be a modified antibody having a variable region of a non-human antibody, such as a mouse antibody, and a constant region of a human antibody. Examples of antibodies include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotype subclasses; or fragments containing antigen-binding domains, such as Fab, scFv, Fv, dAb, Fd; and diabodies. The antibody may be a polyclonal antibody or a monoclonal antibody. In the present specification, a monoclonal antibody may sometimes be referred to as "mAb".

[0312] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. The bispecific antibody may include a second targeting moiety that targets a desired cell or tissue, such as the liver, or another desired antigen associated with the same or a similar disease or disorder, such as a liver cancer antigen.

[0313] One can collect an antibody, such as a monoclonal antibody, and use recombinant DNA technology to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may include the step of introducing DNA encoding the immunoglobulin variable region or complementary determining region (CDR) of the antibody into the constant region of a different immunoglobulin, or into the constant region and framework region. Hybridomas (or other cells producing antibodies) can be subject to genetic mutations or other changes, which may or may not alter the binding specificity of the antibodies produced.

[0314] It has been shown that fragments of whole antibodies can function to bind antigens. Examples of binding fragments include: (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) an isolated CDR region; (vi) an F(ab’)2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH domain and the VL domain are linked by a peptide linker capable of associating these two domains to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer; (ix) a “diabody,” a multivalent or multispecific fragment constructed by gene fusion; and (x) a VHH or VNAR antibody, also known as a single-domain antibody or nanobody (Nb), which may be derived from heavy-chain antibodies from, for example, human camels, llamas, alpacas, or sharks.

[0315] A diabody is a multimer of polypeptides, each polypeptide comprising a first domain containing a binding region of an immunoglobulin light chain and a second domain containing a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen-binding site, the antigen-binding site being formed by the association of a first domain of a polypeptide within the multimer with a second domain of another polypeptide within the multimer (WO 94 / 13804). When bispecific antibodies are used, these can be produced in a variety of ways, for example, they can be conventional bispecific antibodies that can be prepared chemically or from hybrid hybridomas, or they can be any of the bispecific antibody fragments described above. In some cases, it may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed using only variable domains without using the Fc region, which may reduce the influence of anti-idiotypic reactions. Another form of bispecific antibody is the single-chain "Janusin". In contrast to bispecific whole antibodies, bispecific diabodies can be useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) with appropriate binding specificities can be readily selected from libraries using phage display. If one arm of the diabody is maintained in a constant state, for example, having specificity directed against an antigen of interest, a library with a variable other arm can be created and an antibody with appropriate specificity can be selected. An "antigen binding domain" is a part of an antibody that specifically binds to and is complementary to part or all of a particular antigen, and includes a region that specifically binds to part or all of an antigen. If the antigen is large, the antibody may bind only to a specific part of the antigen, which is called an epitope. The antigen binding domain can be provided by one or more antibody variable domains. The antigen binding domain may include the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH).

[0316] In some embodiments, the peptide-binding substructure may be an antibody-like molecule designed to specifically bind to a peptide or peptide-MHC complex of the present disclosure. In some embodiments, the peptide-binding substructure may include a TCR-mimicking antibody. In some embodiments, such a TCR-mimicking antibody can include a high-affinity soluble antibody molecule that has been imparted with TCR-like specificity for a tumor or viral epitope that can target tumor cells and / or virus-infected cells and mediate their specific killing.

[0317] The present disclosure also encompasses binding substructures based on engineered protein scaffolds or "alternative scaffolds". Alternative scaffolds are derived from stable soluble native protein structures that have been modified to provide a binding site for a target molecule of interest. Examples of alternative scaffolds include, but are not limited to, Affibodies based on the Z domain of staphylococcal protein A that provides a binding interface on two of its α helices; Anticalins derived from lipocalins in which a binding site for a small ligand is incorporated at the open end of the β barrel fold; Monobodies designed to incorporate the fibronectin type III domain (FN3) of fibronectin or tenascin as a protein scaffold, or a synthetic FN3 domain (e.g., Tencon); Nanobodies; and DARPins. Further alternative scaffolds include Adnectin™, iMab, EETI-II / AGRP, Kunitz domains, thioredoxin peptide aptamers, Affilins, Tetranectins, Fynomere, and Avimers. Alternative scaffolds are typically potential therapeutic agents intended to bind to the same antigenic proteins as antibodies. These can function as inhibitors or antagonists, or as an in vivo delivery vehicle for a target molecule such as a toxin to a specific tissue. Short peptides can also be used to bind to target proteins. A phylomer is a natural structured peptide derived from a bacterial genome. This peptide represents diverse protein structural folds and can be used to inhibit / interfere with protein-protein interactions in vivo.

[0318] Alternative scaffolds are typically single-chain polypeptide frameworks that include a highly structured core associated with variable domains having a high conformational tolerance that allows for insertions, deletions, or other substitutions within the variable domains. Libraries that introduce diversity into one or more variable domains and, optionally, the structured core can be generated using known protocols, and the resulting libraries can be screened for binding to the peptides and / or pMHC complexes of the present disclosure, and the identified binders can be further characterized for their specificity using known methods. Alternative scaffolds can be derived from Protein A, particularly its Z domain (affibody), ImmE7 (immune protein), BPTI / APPI (knotted domain), CTLA-4, calibrachoa toxin (scorpion venom), Min-23 (notchkin), lipocalin (anticalin), Ras-binding protein AF-6 (PDZ-domain), neocarzinostatin, fibronectin domain, ankyrin consensus repeat domain, or thioredoxin.

[0319] In some embodiments, the antibodies or alternative scaffolds described herein can be immobilized on a viral vector. Such modified recombinant viral vectors can be useful for the targeted introduction of the genetic material encoded by the viral vector into cells and / or tissues (e.g., hepatocytes and / or liver tissue). Various means can be used to immobilize the antibody or alternative scaffold on the viral vector, for example, means using affinity binding pairs such as c-Myc / anti-Myc antibody, streptavidin / biotin, or means using the 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)).

[0320] In some embodiments, the peptide bond moiety may be a TCR. TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and the IMGT public database of TCR sequences.

[0321] The TCRs of the present disclosure may be of any format. For example, the TCR may be an αβ heterodimer, or an αα or ββ homodimer.

[0322] The α / β heterodimer TCR has an α chain and a β chain. Generally, these chains each contain a variable region, a joining region, and a constant region, and the β chain usually also contains a short diversity region between the variable region and the joining region, which is often considered part of the joining region. Each variable region contains three hypervariable CDRs (complementary determining regions) embedded in a framework sequence, and CDR3 is considered the main mediator of antigen recognition. There are multiple types of α-chain variable (Vα) regions and β-chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences, and partially defined CDR3 sequences.

[0323] The TCRs of the present disclosure may not correspond to naturally occurring TCRs. For example, they may include combinations of α and β chains that do not exist in the natural repertoire. Alternatively, or further, the TCRs described herein may be soluble, and / or the α-chain and / or β-chain constant domains may be truncated such that, for example, there is no C-terminal transmembrane domain and intracellular region compared to the native / naturally occurring TRAC / TRBC sequences. Such truncation may remove cysteine residues from TRAC / TRBC that form native interchain disulfide bonds.

[0324] Furthermore, the TRAC / TRBC domain may include modifications. For example, the extracellular sequence of the α-chain may include modifications to native / naturally occurring TRAC, such that when referring to the IMGT numbering, amino acid T48 of TRAC is replaced by C48. Similarly, the extracellular sequence of the β-chain may include modifications to native / naturally occurring TRBC1 or TRBC2, such that when referring to the IMGT numbering, S57 of TRBC1 or TRBC2 is replaced by C57. Such cysteine substitutions to the native α-chain and β-chain extracellular sequences allow for the formation of non-native inter-chain disulfide bonds, thereby stabilizing the refolded soluble TCR, i.e., the TCR formed by the refolding of the extracellular α-chain and β-chain. This non-native disulfide bond promotes the display of the correctly folded TCR in phage. Furthermore, by using the disulfide-bonded stable TCR, the binding affinity and binding half-life can be more conveniently evaluated. Alternative positions for the formation of non-native disulfide bonds include, for example, Thr45 in exon 1 of TRAC*01, and Ser77 in exon 1 of TRBC1*01 or TRBC2*01; Tyr10 in exon 1 of TRAC*01, and Ser17 in exon 1 of TRBC1*01 or TRBC2*01; Thr45 in exon 1 of TRAC*01, and Asp59 in exon 1 of TRBC1*01 or TRBC2*01; and Ser15 in exon 1 of TRAC*01, and Glu15 in exon 1 of TRBC1*01 or TRBC2*01. The TCR having non-native disulfide bonds may be full-length or truncated.

[0325] The TCR of the present disclosure may be in single-chain form. The single-chain TCR comprises any of the following types of αβ TCR polypeptides: Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ, or Vα-Cα-L-Vβ-Cβ, optionally in the reverse orientation, where 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. The single-chain TCR may contain non-native disulfide bonds. The TCR may be in soluble form (i.e., having no transmembrane or cytoplasmic domain) or may contain full-length α and β chains. The TCR may be provided on the surface of cells such as T cells.

[0326] The TCR of the present disclosure may be engineered to contain mutations. Methods for generating mutated high-affinity TCR variants, such as phage display methods and site-directed mutagenesis. Preferably, mutations for improving affinity are made within the variable regions of the α and / or β chains. More preferably, mutations for improving affinity are made within the CDRs. There may be 1 to 15 mutations in the α and / or β chain variable regions.

[0327] The TCR of the present disclosure may be labeled with an imaging compound, for example a label suitable for diagnostic purposes. Such labeled high-affinity TCRs can be used in a method for detecting TCR ligands selected from CD1-antigen complexes, bacterial superantigens, and MHC-peptide / superantigen complexes, the method comprising contacting the TCR ligand with a high-affinity TCR (or multimeric high-affinity TCR complex) specific for the TCR ligand; and detecting binding to the TCR ligand. In a multimeric high-affinity TCR complex (formed, for example, using biotinylated heterodimers), a detectable label can be provided using fluorescent streptavidin. The fluorescently labeled multimer is suitable for use in FACS analysis, for example, for detecting antigen-presenting cells that hold a peptide to which the high-affinity TCR is specific.

[0328] Alternatively, or additionally, the TCRs (or multivalent complexes thereof) of the present disclosure may be conjugated (e.g., by covalent bonding or other means) to a therapeutic agent, which may be a toxic moiety for use, for example, in cell killing, or an immunostimulatory agent such as interleukin or cytokine. The multivalent high-affinity TCR complexes of the present disclosure can improve the binding ability to TCR ligands as compared to non-multimeric wild-type or high-affinity T cell receptor heterodimers. Thus, the multivalent high-affinity TCR complexes according to the present disclosure are particularly useful for tracking or targeting cells presenting a specific antigen in vitro or in vivo, and are also useful as intermediates for the production of further multivalent high-affinity TCR complexes having such uses. Accordingly, the high-affinity TCRs or multivalent high-affinity TCR complexes can be provided as pharmaceutically acceptable formulations for in vivo use.

[0329] The high-affinity TCRs of the present disclosure can be used for the production of soluble bispecific reagents. A preferred embodiment is a reagent comprising a soluble TCR fused via a linker to an anti-CD3 specific antibody fragment.

[0330] In a further aspect, the present disclosure provides a nucleic acid encoding a TCR of the present disclosure, a TCR expression vector comprising the nucleic acid encoding a TCR of the present disclosure, and a cell harboring such a vector. The TCR can be encoded by either a single open reading frame or two different open reading frames. Also within the scope of the present disclosure are cells harboring a first expression vector comprising a nucleic acid encoding the α-chain of a TCR of the present disclosure and a second expression vector comprising a nucleic acid encoding the β-chain of a TCR of the present disclosure. Alternatively, one vector may encode both the α-chain and the β-chain of a TCR of the present disclosure.

[0331] A further aspect of the present disclosure provides a cell displaying the TCR of the present disclosure on its surface. The cell may be a T cell or other immune cell. The T cell may be modified so as not to correspond to a naturally occurring T cell. For example, the cell may be transfected with a vector encoding the TCR of the present disclosure so that the T cell expresses an additional TCR in addition to the native TCR. Further, or alternatively, it may be modified so as not to be able to present the native TCR. There are numerous methods suitable for transfecting T cells with DNA or RNA encoding the TCR of the present disclosure. By way of non-limiting example, the transfection method may include a rapid RNA-based transfection system. T cells expressing the TCR of the present disclosure are suitable for use in adoptive cell therapy-based treatment of diseases such as cancer. There are numerous suitable methods by which adoptive cell therapy can be carried out. For example, adoptive cell therapy (ACT) may include the use of autologous tumor-infiltrating lymphocytes and may include a lymphocyte depletion preparative regimen prior to ACT. In some embodiments, a virus encoding a TCR, such as a retrovirus, can be used to genetically modify lymphocytes to convert normal lymphocytes into lymphocytes with anti-cancer activity. Tumor regression can be mediated by adoptively transferring lymphocytes with anti-cancer activity to a patient in need of treatment for metastatic melanoma, for example. In some embodiments, ACT may include treatment of patients with cancer expressing viral antigens or allogeneic antigens, treatment of patients with cancer expressing viral antigens, and / or ACT using genetically modified lymphocytes.In some embodiments, the ACT methods include, for example, genetic modification of lymphocytes to introduce new recognition specificities, such as using one or more αβ TCRs and / or one or more chimeric TCRs; genetic modification of lymphocytes to alter T cell function, such as using costimulatory molecules (e.g., CD28, 41BB), cytokines (e.g., IL2, IL15), homing molecules (e.g., CD62L, CCR7), and / or molecules that can prevent apoptosis (BCL2); modification of host lymphocyte depletion, such as using selective depletion of CD4+ cells or T regulatory cells; blocking of inhibitory signals to reactive lymphocytes, such as using antibodies against CTLA4 and / or PD-1; administration of a vaccine to stimulate the transplanted cells, such as using a recombinant virus encoding one or more antigens; administration of alternative cytokines to support cell growth, such as using IL15 and / or IL21; stimulation of APCs, such as using Toll-like receptor agonists; generation of less differentiated lymphocytes, such as using alternative culture conditions and growth-promoting cytokines in vitro; and overcoming of antigen escape variants, such as using natural killer cells.

[0332] The TCRs of the disclosure for use in adoptive therapy are generally glycosylated when expressed by the transfected T cells. The glycosylation pattern of the transfected TCR can be altered by mutations in the gene being transfected.

[0333] In some embodiments, the peptide-binding substructure may be a chimeric antigen receptor (CAR). A CAR is a genetically engineered receptor. A CAR that binds to the peptides or pMHC complexes of the present disclosure can be generated by incorporating an antigen-binding domain that specifically binds to the peptide or pMHC complex into the extracellular domain of the CAR. The CAR can be introduced into immune cells such as T cells, NK cells, or macrophages and expressed by them. The CAR can be programmed to recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell presenting the antigen. When these antigens are present on tumor cells, immune cells expressing the CAR can target and kill the tumor cells.

[0334] The overall structure of a CAR typically includes an extracellular domain that binds to an antigen (e.g., the peptides or pMHC complexes of the present disclosure), a hinge, a transmembrane domain, and an intracellular domain, which includes a signaling domain and optionally one or more co-stimulatory domains.

[0335] The extracellular domain of the CAR may include any polypeptide that specifically binds to a desired antigen (e.g., the peptides or pMHC complexes of the present disclosure). For example, this extracellular domain may include antibody fragments such as scFv or VHH. The CAR can also be engineered to bind to two or more desired antigens, which may be arranged in tandem and separated by a linker sequence. For example, bispecific or multispecificity can be provided to the CAR by arranging one or more domain antibodies, scFv, llama VHH antibodies, or other VH-only antibody fragments in tandem via a linker.

[0336] The hinge domain may be present between the extracellular domain and the transmembrane domain of the CAR, thereby providing flexibility, for example, to enable effective binding of the extracellular domain to the intended target. The hinge domain may be a polypeptide having a length of about 2 to 100 amino acids. The hinge may contain or consist of flexible residues such as Gly and Ser, whereby adjacent protein domains can move freely relative to each other. A relatively long hinge may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. The hinge may be derived from the hinge region or a portion of the hinge region of any immunoglobulin. Non-limiting examples of linkers include a portion of the human CD8α chain; the extracellular domain of CD28; the Ig hinge from IgG, IgM, IgA, IgD, or IgE; the FcyRllla receptor; or functional fragments thereof.

[0337] The transmembrane domain of CAR may be derived from a transmembrane protein such as the α-chain, β-chain, or ζ-chain 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), 4-1BB (CD137), 4-1BBL, 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 a functional fragment thereof.

[0338] The intracellular signaling domain of a CAR is involved in inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity (including the release of cytotoxic factors to target cells to which the CAR binds), or other cellular responses induced after antigen binding to the extracellular CAR domain, by transmitting the signal of effective binding of the CAR to the target antigen into the interior of immune effector cells. Non-limiting examples of intracellular signaling domains of a CAR include those derived from CD3ζ, CD3ε, CD3δ, CD3γ, CD5, CD22, CD39, CD79A, CD79B, CD66d, CD226, DAP10, DAP12, the gamma chain of the Fcε receptor I (FCER1G), or FcRβ.

[0339] The intracellular co-stimulatory domain of a CAR provides a second signal necessary to efficiently activate and function T lymphocytes when bound to an antigen. Such co-stimulatory domains may 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.

[0340] CARs can be generated by standard molecular biology techniques. The extracellular domain that binds to the desired antigen may be derived from an antibody or antigen-binding fragment thereof described herein.

[0341] In another aspect, the disclosure further provides cells comprising the peptide binding moiety structures (e.g., TCRs and CARs) of the disclosure. In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell, NK cell, or macrophage. The host cell may be an autologous cell or an allogeneic cell to the subject to whom the cell is (therapeutically) administered.

[0342] In some embodiments, the TCRs of the present disclosure are provided as TCR-T cells. In some embodiments, the CARs of the present disclosure are provided as CAR-T cells. The TCR-T or CAR-T cells of the present disclosure can be generated using any method known in the art to modify T cells to express a TCR or a CAR.

[0343] Cells expressing the peptide binding moiety structures (e.g., TCRs and CARs) of the present disclosure may also contain one or more additional genes. The additional genes can be used to enhance the effector functions of cells expressing the peptide binding moiety structures (e.g., TCRs and CARs). Non-limiting examples of classes of additional genes include: (a) antibodies (fragments thereof, and second targeting moiety structures such as bispecific antibodies (including, e.g., bispecific T cell engagers (BiTE))), (b) secreted 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), (e) constitutively active cytokine receptors (e.g., C7R), (f) dominant negative receptors (DNR; e.g., TGFRII DNR), (g) ligands of co-stimulatory molecules (e.g., CD80, 4-1BBL), (h) nuclear factors of activated T cells (NFAT) (e.g., NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5), or (j) suicide genes (e.g., CD20, truncated EGFR or HER2, inducible caspase 9 molecule). In some embodiments, cells expressing the peptide binding moiety structures (e.g., TCRs and CARs) of the present disclosure may express a second targeting moiety structure that targets a liver or another known liver cancer antigen.

[0344] Pharmaceutical Compositions, Dosage Forms, and Administration In a further aspect, the present disclosure provides a pharmaceutical composition comprising a peptide, peptide-based molecule (such as a complex containing the peptide (e.g., peptide-MHC (pMHC) complex), fusion protein, or conjugate, etc.), nucleic acid molecule, vector, cell, or peptide-binding moiety, together with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition of the present disclosure may be in any suitable form (depending on the desired mode of administration to the patient). Suitable compositions and modes of administration are known to those skilled in the art; see, for example, Johnson et al., Blood. 2009; 114(3):535-46.

[0345] The pharmaceutical composition may contain the peptide or peptide-based molecule of the present disclosure in either free form or in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of a peptide of the present disclosure, in which the peptide is modified by creating an acidic or basic salt of the agent. For example, an acidic salt is prepared from the free base with a suitable acid (where typically the neutral form of the agent has a neutral -NH2 group). Suitable acids for preparing acidic salts include both organic acids such as 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 such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. In contrast, basic salts of acid moieties that may be present on the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc.

[0346] The compositions of the present disclosure may comprise a plurality of peptides described herein, such as 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides (e.g., SEQ ID NOs: 1-54 and 110-112). In some embodiments, the compositions of the present disclosure 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, 51, 52, 53, or 54 amino acid sequences selected from SEQ ID NOs: 1-54 and 110-112, or derivatives thereof, or pharmaceutically acceptable salts thereof.

[0347] In some embodiments, the peptide or peptide-based molecule may be present in solution at a concentration of about 1 μg / mL to 50 mg / mL, such as 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, 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.

[0348] The pharmaceutical composition can be adapted for administration by any suitable route, such as parenteral (including subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation, or intranasal route.

[0349] Such compositions can be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with one or more carriers or one or more excipients under sterile conditions.

[0350] Furthermore, disclosed herein is a pharmaceutical formulation comprising a peptide, peptide-based molecule (such as a complex comprising one or more of the above peptides (e.g., a peptide-MHC (pMHC) complex), a fusion protein, or a conjugate, etc.), a nucleic acid molecule, a vector, a cell, or a binding moiety structure.

[0351] The pharmaceutical compositions based on the peptides, peptide-based molecules (such as a complex comprising one or more of the above peptides (e.g., a peptide-MHC (pMHC) complex), a fusion protein, or a conjugate, etc.), nucleic acid molecules, vectors, cells, or binding moiety structures disclosed herein can be formulated by any conventional method using one or more physiologically acceptable carriers and / or excipients. The peptides, peptide-based molecules (such as a complex comprising one or more of the above peptides (e.g., a peptide-MHC (pMHC) complex), a fusion protein, or a conjugate, etc.), nucleic acid molecules, vectors, cells, or binding moiety structures can be formulated for administration by, for example, injection, inhalation, or insufflation (through the mouth or nose), or for oral, buccal, parenteral or rectal administration, or for direct administration to an organ or tissue.

[0352] The pharmaceutical composition can be formulated for a variety of modes of administration, including systemic administration, topical administration, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co. (Easton, Pennsylvania). For systemic administration, injection including intramuscular, intravenous, intraperitoneal, and subcutaneous is preferred. For injection, the pharmaceutical composition can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. Additionally, the pharmaceutical composition can be formulated in a solid form that is redissolved or suspended immediately before use. The lyophilized form of the pharmaceutical composition is also suitable.

[0353] In some embodiments, the pharmaceutical composition of the present disclosure may be lyophilized. As a non-limiting example, the resulting lyophilized product can be reconstituted into a hydrous composition by the addition of a hydrous solvent. In some embodiments, this hydrous composition may be suitable for direct parenteral administration to a patient. Accordingly, a further embodiment of the present disclosure is a hydrous pharmaceutical composition obtainable by reconstitution of a lyophilized product using a hydrous solvent.

[0354] In some embodiments, the compositions disclosed herein may include a lyophilized formulation. As a non-limiting example, the lyophilized formulation may include a peptide, mannitol, and / or Tween® 80 of the present disclosure. As another non-limiting example, the lyophilized formulation may include a peptide, mannitol, and poloxamer 188 disclosed herein. In some embodiments, the pharmaceutical composition may include a lyophilized formulation comprising a reconstitution liquid composition.

[0355] In some embodiments, the pharmaceutical compositions of the present disclosure can provide formulations with enhanced solubility and / or wettability compared to known compositions. As a non-limiting example, enhancement of the solubility and / or wettability of the lyophilized product can be achieved by using an appropriate composition of excipients. Thereby, the pharmaceutical compositions of the present disclosure, including the peptides of SEQ ID NOs: 1 to 54 and variants thereof, can be developed to exhibit desired storage stability, for example, at -20°C, +5°C, or +25°C, and can be readily redissolvable such that they can be completely dissolved within seconds to over 2 minutes using a buffer or other excipients, with or without using an ultrasonic homogenizer. Further, this composition can be readily provided to a patient 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 pH value of the resulting solution may be from pH 2.7 to pH 9.

[0356] For oral administration, the pharmaceutical composition can be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as, for example, binders (such as pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); fillers (such as lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, or silica); disintegrants (such as potato starch or sodium starch glycolate); or wetting agents (such as sodium lauryl sulfate). Tablets can also be coated by methods known in the art. Liquid formulations for oral administration can be in the form of, for example, solutions, syrups, or suspensions, or can be provided as dry products for reconstitution before use with water or other suitable vehicles. Such liquid formulations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (such as sorbitol syrup, cellulose derivatives, or hydrogenated edible oils); emulsifying agents (such as lecithin or gum arabic); non-aqueous vehicles (such as almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (such as p-hydroxybenzoic acid or methyl or propyl sorbate). The formulations can optionally also contain buffering salts, flavoring agents, coloring agents, and sweetening agents.

[0357] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, optionally with the addition of a preservative. The pharmaceutical composition can further be formulated as a suspension, solution, or emulsion in an oily or aqueous vehicle and may contain other agents including suspending agents, stabilizers, and / or dispersing agents.

[0358] Furthermore, the pharmaceutical composition can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated using a suitable polymeric material, or a hydrophobic material (e.g., as an emulsion in an acceptable oil), or an ion exchange resin, or as a poorly soluble derivative, e.g., as a poorly soluble salt. Other suitable delivery systems include microspheres that offer the possibility of long-term local non-invasive delivery of the drug. This technology can include microspheres having precapillary size, which can be injected via a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The administered therapeutic agent is then slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.

[0359] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, suitable permeation enhancers are used in the formulation for the barrier to be permeated. Such permeation enhancers are common in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. Further, detergents may be used to enhance permeation. Transmucosal administration can be effected using a nasal spray or a suppository. For local administration, the vector particles described herein can be formulated as an ointment, salve, gel, or cream, as is generally known in the art. A lavage solution can also be used locally for the treatment of wounds or inflammation to accelerate healing.

[0360] Suitable pharmaceutical forms for use by injection include: sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. This form must be stable under the manufacturing conditions and certain storage parameters (e.g., refrigeration and freezing), and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0361] When the formulations disclosed herein are used as therapeutic agents for enhancing the immune response of a subject, the therapeutic agent can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed using the free amino groups of the protein), which can be formed using, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed using free carboxyl groups are derived from, for example, inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0362] 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, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin to maintain the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it is preferable to include isotonic agents such as, for example, sugars or sodium chloride. Sustained absorption of injectable compositions can be achieved by using agents in the composition that delay absorption, such as aluminum monostearate and gelatin.

[0363] A sterile injectable solution can be prepared by incorporating the required amount of the active compound or active construct, optionally with various ones of the other ingredients enumerated above, into a suitable solvent and then carrying out sterile filtration.

[0364] After formulation, the solution can be administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulations are readily administered in a variety of dosage forms such as injectable solutions of the type described above, but sustained release capsules or microparticles and microspheres etc. can also be employed.

[0365] For example, for parenteral administration in an aqueous solution, the solution needs to be buffered appropriately if necessary and the liquid diluent needs to be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be used will be apparent to those skilled in the art in light of the present disclosure. For example, a single dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected into the proposed injection site.

[0366] The person responsible for administration will in any case determine the appropriate dosage for the individual subject. For example, a subject can be administered a peptide, peptide-based molecule (a complex comprising one or more of the above peptides (e.g., a peptide-MHC (pMHC) complex), a fusion protein, or a conjugate etc.), a nucleic acid molecule, a vector, a cell, or a binding moiety structure described herein, as necessary, or in response to exposure to a pathogen (e.g., HBV), or in response to the subject's condition (e.g., cancer), daily or weekly, or monthly, biennially, or annually, over a period of time.

[0367] In addition to compounds formulated 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; liposomal formulations; sustained release capsules; biodegradable forms and any other forms currently in use.

[0368] Alternatively, solutions, sprays, aerosols, or inhalants for nasal or inhalation use may be employed. The nasal drops can be an aqueous solution designed to be administered as droplets or sprays via the nasal route. The nasal drops can be prepared to be similar to nasal mucus in many respects. Thus, the aqueous nasal drops are isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. Further, antibacterial preservatives and suitable pharmaceutical stabilizers similar to those used in ophthalmic preparations may be included in the formulation as needed. Various commercially available nasal preparations are known, and these can contain, for example, antibiotics and antihistamines and are used for the prevention of asthma.

[0369] Oral formulations can contain excipients such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders. In certain defined embodiments, the oral pharmaceutical composition can contain an inert diluent or assimilable edible carrier, or can be enclosed in hard or soft gelatin capsules, or can be compressed into tablets, or can be directly incorporated into food. For oral therapeutic administration, the active compound can be combined with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0370] Tablets, troches, pills, capsules, etc. may contain the following: binders such as tragacanth, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, wintergreen oil, or strawberry flavor. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the materials of the above types. Various other materials may be present as a coating or for other modification of the physical shape of the unit dosage form. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes, and flavoring agents such as strawberry or orange flavor.

[0371] Further embodiments disclosed herein may relate to kits for use with methods and compositions. The kits can also include suitable containers such as, for example, vials, tubes, micro or microcentrifuge tubes, test tubes, flasks, bottles, syringes, or other containers. When providing additional components or agents, the kits can enclose one or more additional containers into which the agent or component can be placed. The kits herein include means for enclosing peptides, peptide-based molecules (complexes containing one or more of the above peptides such as peptide-MHC (pMHC) complexes, fusion proteins, or conjugates, etc.), nucleic acid molecules, vectors, cells, or binding moieties, and any other reagent containers sealed for commercialization. Such containers may include injection-molded or blow-molded plastic containers in which the desired vials are held. Optionally, one or more additional active agents such as, for example, anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents may be required for the described compositions.

[0372] The dosage range and frequency of administration can vary depending on the nature of the composition, the medical condition, and the parameters of the particular patient and the route of administration used. The dosage can also depend on the subject receiving the administration. For example, a relatively low dosage may be required if the subject is a young person, while a relatively high dosage may be required if the subject is an adult. In certain embodiments, the more accurate dosage can depend on the weight of the subject. Suitable, but non-limiting, examples of dosages of pharmaceutical compositions, including those disclosed herein, can vary depending on the age and build of the subject receiving the administration, the target disease, the purpose of the treatment, the physical condition, the route of administration, etc. Non-limiting examples of suitable dosages include, for example, 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. Depending on the severity of the condition, the frequency and duration of treatment can be adjusted. In certain embodiments, after the initial dose, administration of a second dose or multiple subsequent doses that can be substantially the same as or less than the initial dose can be continued, where the subsequent doses are separated by at least 1 day to 3 days; at least 1 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.

[0373] The composition may include intravenous administration, intratumoral administration, intradermal administration, intraarterial administration, intraperitoneal administration, intralesional administration, intracranial administration, intraarticular administration, intraprostatic administration, intrapleural administration, intratracheal administration, intranasal administration, intravitreal administration, intravaginal administration, rectal administration, topical administration, intratumoral administration, intramuscular administration, intrathecal administration, subcutaneous administration, subconjunctival administration, intravesicular administration, mucosal administration, pericardial administration, intraumbilical administration, intraocular administration, oral administration, local administration, administration by inhalation, administration by injection, administration by infusion, administration by continuous infusion, administration by local perfusion, administration via catheter, administration via lavage, administration with a cream, or administration with a liquid composition to the subject.

[0374] The specific additional agent used in combination therapy can be formulated and administered by any means known in the art.

[0375] The compositions disclosed herein can include adjuvants such as aluminum salts and other mineral adjuvants, surfactants, bacterial derivatives, vehicles, and cytokines. The adjuvants can also have antagonistic immunomodulatory properties. For example, the adjuvant can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein can also include adjuvant therapy.

[0376] The peptides or peptide-based molecules of the present disclosure can be provided in the form of a vaccine composition. The vaccine composition can be useful for the treatment or prevention of HBV infection and / or HBV-induced diseases or disorders. As is understood, vaccines can take multiple forms (see, e.g., 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 can include additional peptides or peptide-based molecules, whereby the peptides or peptide-based molecules of the present disclosure can be one of a mixture of multiple peptides or peptide-based molecules. To enhance the immune response, an adjuvant can be added to the vaccine composition. Particularly with respect to the peptide-containing vaccine compositions of the present disclosure, pharmaceutically acceptable adjuvants include, but are not limited to, aluminum salts, Amplivax, AS15, QS21 stimulon from Aquila, AsA404 (DMXAA), beta-glucan, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1P321, IS Patch, ISS, 1018 ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid 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, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, and / or badimezan.

[0377] Alternatively, the vaccine composition may take the form of an APC that displays the peptides of the present disclosure as a complex with MHC. Preferably, the APC is an immune cell, more preferably a dendritic cell or a B cell. The peptides may be pulsed onto the surface of the cells (Thurner, J Exp Med. 1999; 190(11):1669-78), or nucleic acids encoding the peptides of the present disclosure may be introduced into dendritic cells or B cells (e.g., by electroporation) (Van Tendeloo, Blood. 2001; 98(1):49-56).

[0378] The pharmaceutical compositions of the present disclosure can be administered directly i.d., i.m., s.c., i.p., and i.v. into a patient's body, into an infected organ, or systemically, or can be applied ex vivo to cells derived from a patient or a human cell line that will later be administered to the patient, or can be used in vitro for the selection of a subset of immune cells derived from a patient that will later be readministered to the patient. When administering nucleic acids to cells in vitro, it may be useful to transfect the cells to co-express an immune-stimulatory cytokine such as interleukin-2. Peptides or peptide-based molecules may be substantially pure, or may be combined with an immune-stimulatory adjuvant, or may be administered using a suitable delivery system such as liposomes, virus particles, VLPs. Peptides or peptide-based molecules can also be conjugated to a suitable carrier such as keyhole limpet hemocyanin (KLH) or mannan (see, e.g., WO 95 / 18145 and Longenecker et al., 1993).

[0379] In some embodiments, the peptide-containing compositions described herein further comprise an accessory molecule that can regulate the survival or activity of TCR-expressing cells.

[0380] Non-limiting examples of useful accessory molecules include, for example, anti-CD28 antibody, anti-CD80 (B7.1) antibody, anti-CD86 (B7.2) antibody, anti-CD3 antibody, anti-CD2 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CD47 antibody, and functional derivatives, variants, and fragments thereof.

[0381] Accessory molecules used in the peptide-containing compositions described herein include, for example, molecules that provide signals that mediate T cell responses including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by the binding of the TCR / CD3 complex to the pMHC complex.

[0382] Accessory molecules can be, for example, inhibitory or stimulatory antibodies, peptide ligands, costimulatory peptides, cytokines, etc. Non-limiting examples of accessory 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 costimulatory ligand (ICOS-L); intercellular adhesion molecule (ICAM); CD30L; CD40; CD70; CD83; HLA-G; MICA; MICB; FIVEM; lymphotoxin β receptor; 3 / TR6; ILT3; ILT4; HVEM; an agonist or antibody that binds to a ligand that specifically binds to the Toll ligand receptor and B7-H3; and antibodies that specifically bind to ligands that specifically bind to CD27, CD28, B7.1 (CD80), B7.2 (CD86), 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD3, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0383] Further non-limiting examples of co-stimulatory 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, CD70); members of the immunoglobulin superfamily (e.g., VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chains, ICOS, ICOS ligand, HHLA2, butyrophilin, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMS (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin like receptor (LILR), killer cell immunoglobulin like receptor (KIR)), members of the lectin superfamily, selectins, cytokines / chemokines and cytokine / chemokine receptors, growth factors and growth factor receptors, adhesion molecules (integrins, fibronectin, cadherin), or the extracellular domain of a multi-span transmembrane protein, or an antibody targeting any of these molecules.

[0384] In some embodiments, the peptide-containing compositions described herein further comprise a cytotoxic agent. In certain specific embodiments, the cytotoxic agent is a toxin, or a radioisotope (e.g., a radioactive conjugate), 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 derived from bacteria, fungi, plants, or animals, or fragments, mutants, or derivatives thereof. Examples of enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. Non-limiting examples of suicide genes include, for example, thymidine kinase, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, β-galactosidase, hepatic cytochrome P450-2B1, linamarase, horseradish peroxidase, and carboxypeptidase.

[0385] Examples of methods for introducing the polypeptide or polynucleotide of the present disclosure into a cell or a subject include 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, the nucleic acid or protein can be introduced into a cell or a subject in a carrier such as poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid coacervates, or lipid nanotubes.

[0386] The use of nanoparticles for the delivery of the polypeptide or polynucleotide compositions of the present disclosure is contemplated herein. Exemplary nanoparticles include, but are not limited to, polymeric nanoparticles, inorganic nanoparticles, liposomes, lipid nanoparticles (LNP), immune stimulating complexes (ISCOM), virus-like particles (VLP), or self-assembling proteins. The nanoparticles may be calcium phosphate nanoparticles, silicon nanoparticles, or gold nanoparticles. For example, the polymeric nanoparticles may include one or more synthetic polymers such as poly(d,l-lactide-co-glycolide) (PLG), poly(d,l-lactic acid-co-glycolic acid) (PLGA), poly(g-glutamic acid) (g-PGA), poly(ethylene glycol) (PEG), or polystyrene, or one or more natural polymers such as polysaccharides exemplified by pullulan, alginic acid, inulin, and chitosan. The use of polymeric nanoparticles can be advantageous due to the properties of the polymers that can be included in the nanoparticles. For example, the natural and synthetic polymers described above can have good biocompatibility and biodegradability, non-toxicity, and / or operability to a desired shape and size. The polymeric nanoparticles can also form hydrogel nanoparticles, i.e., hydrophilic three-dimensional polymer networks with favorable properties including a flexible mesh size, a large surface area for multivalent conjugation, a high water content, and a high antigen loading capacity. Poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for the formation of hydrogel nanoparticles. Inorganic nanoparticles typically have a robust structure and comprise a shell that encapsulates the antigen or a core to which the antigen can be covalently attached. The core may include one or more atoms such as gold (Au) atoms, silver (Ag) atoms, copper (Cu) atoms, Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd, or Au / Ag / Cu / Pd, or calcium phosphate (CaP).

[0387] Other molecules suitable for complexing with the polypeptide or polynucleotide of the present disclosure include cationic molecules such as polyamidoamine, dendritic polylysine, polyethyleneimine or polypropyleneimine, polylysine, chitosan, DNA- gelatin coacervate, DEAE dextran, dendrimer, or polyethyleneimine (PEI).

[0388] In some embodiments, the antibodies of the present disclosure can be conjugated to nanoparticles. Nanoparticles that can be used for conjugation with the antibodies of the present disclosure include, but are not limited to, PEGylated liposomes, poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles (PLGA / MMT NP), poly(lactide-co-glycolide) (PLGA) nanoparticles, poly-(malic acid)-based nanoparticles, chitosan shell nanoparticles, carbon nanotubes, and other inorganic nanoparticles (nanoparticles made of magnesium-aluminum layered double hydroxide containing succinimidyl carbonate (DSC), and TiO2 nanoparticles, etc.). Nanoparticles can be developed to target virus-infected cells and conjugated with the antibodies contained in pharmaceutical compositions.

[0389] Therapeutic methods The compositions of the present disclosure, including the peptides, peptide-based molecules (complexes containing one or more of the above peptides (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates, etc.), nucleic acid molecules, vectors, cells, or binding moiety structures of the present disclosure, can be used for the prevention and / or treatment of viral infections (e.g., HBV infection) and / or diseases or disorders caused by viral infections (e.g., HBV infection).

[0390] In one aspect, disclosed herein is a method for modulating the activity, proliferation, or survival of cells comprising a TCR, the method comprising contacting the cells with a composition of the present disclosure (e.g., a peptide, a complex (e.g., a pMHC complex), a fusion protein, or a conjugate).

[0391] In some embodiments, the cells are lymphocytes such as, for example, T cells (e.g., CD4+ T cells or CD8+ T cells). In some embodiments, the target T cells are CD4+ T cells such as, for example, 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, for example, cytotoxic T cells. In some cases, the target T cells are memory T cells, which can be CD4+ T cells or CD8+ T cells, and generally memory T cells are CD45RO+. In some cases, the target T cells are NK-T cells.

[0392] In some embodiments, the contacting step is ex vivo. In some embodiments, the contacting step is in vivo in a subject (e.g., a human).

[0393] In some embodiments, the cells are mammalian cells (e.g., human cells).

[0394] In some embodiments, for example, when the target T cells are CD8+ T cells, the peptide is presented by class I MHC polypeptides. In some embodiments, for example, when the target T cells are CD4+ T cells, the peptide is presented by class II MHC polypeptides.

[0395] The interaction of T cells with the peptides described herein can result in activation, induction of anergy, or death of the T cells, which occurs when a TCR-binding molecule (e.g., pMHC complex) binds to the TCR of the T cell. "Activation of a T cell" refers to the induction of signal transduction within the T cell that results in the production of cell products (e.g., interleukin 2) by the T cell. "Anergy" refers to a decrease in the responsiveness of T cells to an antigen. Activation and anergy can be measured, for example, by measuring the amount of IL-2 produced by the T cell after the pMHC complex binds to the TCR. Anergic cells will have a decrease in IL-2 production when compared to stimulated T cells. Another method for measuring the reduced activity of anergic T cells is to measure calcium mobilization, intracellularly and / or extracellularly, by the T cell upon binding of its TCR. "T cell death" refers to the permanent cessation of substantially all functions of the T cell.

[0396] In another aspect, provided herein is a method of inducing an immune response against HBV infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, or binding substructures).

[0397] In certain embodiments, the generation of an immune response comprises an increase of about 1.5-fold to 20-fold, or greater than 20-fold, when compared to a control, in the activity of target antigen-specific cytotoxic T lymphocytes (CTLs) in a subject administered a composition of the present disclosure. In certain embodiments, the generation of an immune response comprises an increase of about 1.5-fold to 20-fold, or greater than 20-fold, when compared to a control, in the activity of target-specific CTLs in a subject administered a composition of the present disclosure. In a further embodiment, the generation of an immune response comprises an increase of about 1.5-fold to 20-fold, or greater than 20-fold, when compared to a control, in target antigen-specific cell-mediated immune activity, as measured by an ELISpot assay that measures the secretion of cytokines such as interferon gamma (IFN-γ), interleukin 2 (IL-2), tumor necrosis factor-alpha (TNF-α), or other cytokines.

[0398] In a further embodiment, the generation of an immune response comprises an increase of 1.5-fold to 5-fold when compared to a suitable control, in the production of target-specific antibodies in a subject administered a composition of the present disclosure. In another embodiment, the generation of an immune response comprises an increase of about 1.5-fold to 20-fold, or greater than 20-fold, when compared to a control, in the production of target-specific antibodies in a subject administered a composition of the present disclosure.

[0399] Activation of T cells can be determined by measuring changes in the levels of cytokine and / or T cell activation marker expression, and / or induction of antigen-specific proliferating cells. Expression of cytokines and T cell activation markers can be measured using techniques known to those skilled in the art, including but not limited to immunoprecipitation followed by Western blot analysis, ELISA, flow cytometry, Northern blot analysis, and RT-PCR. Cytokine release can be measured 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.

[0400] Regulation of T cells can be evaluated, for example 3 by measuring proliferation, for example, by incorporation of H-thymidine, counting of cells with trypan blue, and fluorescence-activated cell sorting (FACS).

[0401] The anti-tumor response of T cells can be determined in a xenograft tumor model. The tumor can be established using any human cancer cell line that expresses the relevant tumor-associated antigen. To establish a xenograft tumor model, about 5×10 6 viable cells can be subcutaneously injected, for example, into nude athymic mice using, for example, Matrigel (Becton Dickinson). The endpoints of the xenograft model can be determined using methods known to those skilled in the art based on tumor size, animal body weight, cancer survival time, and histochemical and histopathological examinations.

[0402] In related aspects, disclosed herein is a method of treating HBV infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition of the present disclosure (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, binding moiety structures).

[0403] In related aspects, disclosed herein is a method of preventing or reducing the likelihood of a disease or disorder induced by HBV in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition of the present disclosure (e.g., one or more peptides, complexes (e.g., pMHC complexes), fusion proteins, conjugates, nucleic acid molecules, vectors, cells, binding moiety structures).

[0404] A disease or disorder induced by HBV may be, for example, hepatitis (chronic or acute), liver fibrosis, cirrhosis, liver failure, or liver cancer. Liver cancer may be hepatocellular carcinoma (HCC), or intrahepatic cholangiocarcinoma and hepatoblastoma. In one embodiment, the liver cancer is hepatocellular carcinoma (HCC).

[0405] Hepatitis B virus infection may be acute (self-limiting) or chronic (long-lasting). In the case of self-limiting infection, the infection naturally resolves within a few weeks to a few months. In some cases, "chronic hepatitis" may result. Chronic hepatitis occurs when the body cannot completely eliminate the virus even when symptoms may not persist.

[0406] Acute infection by the hepatitis B virus is associated with acute viral hepatitis, a disease that begins with general malaise, loss of appetite, nausea, vomiting, body aches, low-grade fever, and dark urine and progresses to the development of jaundice. Skin itching has been pointed out as an indicator of symptoms that can occur in any hepatitis virus type. After lasting for several weeks, this disease gradually improves in most infected individuals. A small number of patients may develop more severe liver diseases (such as fulminant liver failure) and as a result may die. This infection may be completely asymptomatic and may go undetected. The persistent presence of the virus over several years may lead to cirrhosis. This type of infection dramatically increases the incidence of liver cancer (such as hepatocellular carcinoma).

[0407] Carriers of HBV can be identified by detecting the presence of HBV viral antigens or antibodies produced by the host in serum or blood. For screening for the presence of this infection, hepatitis B surface antigen (HBsAg) is most commonly used. PCR tests are also used for the detection and measurement of viral nucleic acids in clinical specimens.

[0408] When the disease to be treated is cancer, the cancer may specifically be of the following histological types, but is not limited thereto: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; co-occurrence of hepatocellular carcinoma and cholangiocarcinoma; cord adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; anaplastic carcinoma; eosinophilic carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; follicular papillary adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; serous papillary cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory breast cancer; Paget's disease of the breast; pancreatic acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant blastoma; Sertoli cell tumor; malignant Leydig cell tumor; malignant liposarcoma; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomus tumor; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma of giant congenital nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; fetal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; Müllerian duct mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal cell tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; undifferentiated embryonal cell tumor; embryonal cell tumor; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant angioendothelioma;Kaposi's sarcoma; malignant hemangioendothelioma; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontogenic sarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; epithelioma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; anaplastic oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; Hodgkin's paragranuloma; malignant small lymphocyte lymphoma; diffuse large cell lymphoma; malignant follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasmacytic leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastoid leukemia; myelosarcoma; and hairy cell leukemia.;

[0409] When used in the treatment of various diseases, it is contemplated that the above-described compositions and methods can be used in combination with other therapeutic agents suitable for that disease or similar diseases. Also, by administering two or more of the embodiments described herein simultaneously, additive or synergistic effects can be produced. When co-administered with a second therapeutic agent, the embodiments described herein and the second therapeutic agent may be administered simultaneously or sequentially (in any order). The preferred therapeutically effective dosage for each agent can be reduced by additive or synergistic effects.

[0410] In some embodiments, the compositions and methods disclosed herein can be used to enhance the efficacy of vaccines targeting HBV infection or HBV-induced diseases (such as liver cancer like HCC). Thus, the compositions and methods described herein can be administered to a subject simultaneously with, or prior to (e.g., 1 to 30 days before), administering to the subject a reagent (small molecule, antibody, or cellular reagent) that acts to induce an immune response (e.g., for the treatment of HBV infection or liver cancer).

[0411] The compositions and methods described herein can also be administered in combination with an anti-tumor antibody, or an antibody targeting a pathogenic antigen (such as HBV) or an allergen.

[0412] The compositions and methods described herein can be combined with other immunomodulatory therapies such as, for example, 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 combined with other therapies having the ability to modulate the function or stability of NKT, which includes, but is not limited to, CD1d, CD1d-fusion proteins, CD1d dimers, or even larger polymers of CD1d, which may or may not be loaded with an antigen, a CD1d-chimeric antigen receptor (CD1d-CAR), or any of the other five known CD1 isomers present in the human body (CD1a, CD1b, CD1c, CD1e) in any of the above forms or formulations, alone or in combination with each other or with other agents.

[0413] The treatment methods described herein can be combined with additional immunotherapies and therapies. For example, when used in the treatment of 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 certain embodiments, other therapeutic agents that can be used in combination cancer therapy with the inhibitors described herein include anti-angiogenic agents. For example, a number of anti-angiogenic agents including the following have been identified and are known in the art: TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloprotease (TIMP1 and TIMP2), prolactin (16Kd fragment), angiostatin (38Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor β, interferon α, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and those listed in Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein can be used in combination with VEGF antagonists or VEGFR antagonists such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers that can block VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinase, and any combination thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab, or ranibizumab).

[0414] Non-limiting examples of chemotherapeutic compounds that can be used in combination therapy include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronic acid, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, irinotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronic acid, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0415] These chemotherapeutic compounds can be categorized, for example, into the following groups according to their mechanism of action: antimetabolite / anticancer agents such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), taxanes (paclitaxel, docetaxel), microtubule disruptors such as vincristin, vinblastin, nocodazole, epothilone, and navelbine, epipodophyllotoxin (etoposide, teniposide), DNA-damaging agents (actinomycin, amsacrine, anthracycline, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, iphosphamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16)), including antiproliferative / antimitotic agents; antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracycline, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine of L-asparagine); antiplatelet agents;Antiproliferative / antimitotic alkylating agents such as nitrogen mustard (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonic acid - busulfan, nitrosourea (carmustine (BCNU) and analogs, streptozocin), trazenes - dacarbazine (DTIC); antiproliferative / antimetabolic antagonists such as folic acid analogs (methotrexate); platinum complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytic agents (tissue plasminogen activator, streptokinase, and urokinase, etc.), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti - migratory agents; anti - secretory agents (breveldin); immunosuppressive agents (cyclosporine, tacrolimus (FK - 506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti - angiogenic 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 (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, and caspase activators;and chromatin disruptors;

[0416] For the treatment of viral infections, the combination therapies described herein can include co - administration of the compositions and methods described herein with antiviral agents. Non - limiting examples of antiviral agents that can be used include adefovir and entecavir, telbivudine, immune system modulators (such as interferon α, β, or γ), didanosine, lamivudine, zanamavir, lopanivir, nelfinavir, efavirenz, indinavir, valacyclovir, zidovudine, amantadine, rimantidine, ribavirin, ganciclovir, foscarnet, and acyclovir, or any salts or variants thereof. See also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20.sup.th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0417] Kit The present disclosure further includes a kit that may include any of the various compositions of the present disclosure including the peptides, peptide-based molecules (complexes including one or more of the above peptides, such as peptide-MHC (pMHC) complexes, fusion proteins, or conjugates, etc.), nucleic acid molecules, vectors, cells, or binding moiety structures of the present disclosure.

[0418] In one aspect, the present disclosure may include, for example: (a) a container containing a pharmaceutical composition disclosed herein, such as a pharmaceutical composition in solution or lyophilized form; (b) an optional second container containing a diluent or reconstitution solution for the lyophilized formulation; and / or (c) an optional instruction manual for (i) the use of the solution or (ii) the reconstitution and / or use of the lyophilized formulation.

[0419] In some embodiments, the kit may further include one or more of, for example but not limited to, (i) a buffer, (ii) a diluent, (iii) a filter, (iv) a needle, and / or (v) a syringe. As a non-limiting example, the container may be a bottle, vial, syringe, or test tube. In some embodiments, the container may be a reusable container. In some embodiments, the pharmaceutical composition may be lyophilized.

[0420] The kit of the present disclosure may include the lyophilized formulation of the present disclosure contained in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (such as dual-chamber vials), syringes (such as dual-chamber syringes), and test tubes. The container may be formed from various materials such as glass or plastic. The kit and / or the container may contain instructions on the container or attached to the container indicating the reconstitution of the lyophilized formulation and / or the instructions for using the kit. For example, it may be indicated on the label that the lyophilized formulation must be reconstituted to an appropriate peptide concentration. The label may also indicate that the formulation can be used for any route of administration disclosed herein, such as the parenteral route of administration disclosed herein, or is intended for such a route of administration.

[0421] The container holding the formulation may be a reusable vial, which can enable repeated administration (e.g., 2 to 6 administrations) of the reconstituted formulation. The kit may further include a second container containing a suitable diluent (such as a sodium bicarbonate solution).

[0422] When the diluent and the lyophilized formulation are mixed, the final peptide concentration is achieved in the reconstituted formulation. The kit may further include other materials desirable from a commercial and / or user perspective, which may include, for example, but not limited to, other buffers, diluents, filters, needles, syringes, and / or package inserts that may include instructions for use.

[0423] The kit of the present disclosure may have a single container containing a formulation of the pharmaceutical composition according to the present disclosure, with or without other components (such as other compounds or pharmaceutical compositions of these other compounds), or may have separate containers for each component.

[0424] In some embodiments, the kits of the present disclosure may include the formulations of the present disclosure, packaged for use in combination with the co - administration of a second compound (adjuvant (e.g., GM - CSF, chemotherapeutic agent, natural product, hormone or antagonist, anti - angiogenesis agent or inhibitor, apoptosis inducer or chelating agent) or a pharmaceutical composition thereof, etc.). The components of the kit may be pre - combined, or each component may be placed in a separate container prior to administration to the patient. The components of the kit may be provided as one or more liquid solutions. The liquid solutions described herein may be aqueous solutions, e.g., sterile aqueous solutions. The components of the kit can also be provided as solids, which can be converted to liquids by adding a suitable solvent that can be provided in a separate container, etc.

[0425] The containers of the therapeutic kit may be vials, test tubes, flasks, bottles, syringes, or any other means for enclosing solids or liquids. If there are two or more components, the kit may include a second vial or other container, thereby enabling separate dosing. The kit may also include another container for a pharmaceutically acceptable liquid. In some embodiments, the kit may include an apparatus (e.g., one or more needles, syringes, eye - dropping devices, pipettes, etc.) that enables the administration of the agent of the present disclosure, which is a component of the present kit.

[0426] Methods and systems for identifying immunogenic virus - derived peptides The present disclosure further includes methods and systems for identifying immunogenic virus - derived peptides. Some or all of the steps of this method can be performed by a computing device. In some aspects, some or all of the steps of this method can be stored as computer - readable instructions in a memory, and thus, by executing these instructions on one or more processors, the functions related to this method can be performed. As a non - limiting example, methods and systems for identifying immunogenic virus - derived peptides can include the aspects of the HBV genome reconstruction flow shown in FIG. 1A.

[0427] In one aspect, the present disclosure may include a method for identifying an immunogenic virus-derived peptide, the method comprising the following steps: (a) obtaining a plurality of RNA contig sequences derived from an infected subject infected with a virus, the plurality of RNA contig sequences including a plurality of virus-derived RNA contig sequences and a plurality of endogenous RNA contig sequences of the infected subject; (b) identifying the plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences; (c) performing an assembly of the viral RNA sequence based on the plurality of virus-derived RNA contig sequences; (d) identifying a protein sequence based on the viral RNA sequence; and (e) identifying an immunogenic virus-derived peptide based at least in part on the identified protein sequence.

[0428] In one aspect, a system configured to perform functions related to the above-described method 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 include instructions that, when executed by one or more processors, cause the computing device to: (a) receive, as an input, a plurality of RNA contig sequences derived from an infected subject infected with a virus, the plurality of RNA contig sequences including a plurality of virus-derived RNA contig sequences and a plurality of endogenous RNA contig sequences of the infected subject; (b) identify the plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences; (c) perform an assembly of the viral RNA sequence based on the plurality of virus-derived RNA contig sequences; (d) identify a protein sequence based on the viral RNA sequence; (e) identify an immunogenic virus-derived peptide based at least in part on the identified protein sequence; (f) provide the immunogenic virus-derived peptide as an output.

[0429] In some embodiments, the plurality of RNA contig sequences may be from a single infected subject.

[0430] In some embodiments, the infected subject may be human.

[0431] In some embodiments, the plurality of virus-derived RNA contig sequences are from the virus that the infected subject is infected with.

[0432] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are from the endogenous RNA of the infected subject.

[0433] In some embodiments, the step of identifying a plurality of virus-derived RNA contig sequences from among the plurality of RNA contig sequences (step b) may include: comparing at least a portion of the contig sequences of the plurality of RNA contig sequences with a reference virus sequence; and further identifying the plurality of virus-derived RNA contig sequences such that each contig sequence of the plurality of virus-derived RNA contig sequences includes at least a portion corresponding to the reference virus sequence.

[0434] In some embodiments, each contig sequence of the plurality of virus-derived RNA contigs may be different from the plurality of infected subject endogenous RNA contig sequences.

[0435] In some embodiments, each contig sequence of the plurality of virus-derived RNA contig sequences may not include an infected subject endogenous RNA contig sequence.

[0436] In some embodiments, the reference virus sequence may include a reference genome.

[0437] In some embodiments, the reference genome may include a hepatitis B virus genome.

[0438] In some embodiments, the step of performing assembly of the viral RNA sequence based on a plurality of virus-derived RNA contig sequences (step c) may include: performing assembly of the viral RNA sequence by overlapping common sequence portions at the ends of at least a part of the plurality of virus-derived RNA contig sequences such that at least a part of the plurality of virus-derived RNA contig sequences overlap linearly.

[0439] In some embodiments, the step of identifying a protein sequence based on the viral RNA sequence such that the protein sequence includes translation of the viral RNA sequence (step d) may include: identifying the protein sequence without the need for comparison with a database of viral proteins.

[0440] In some embodiments, the step of identifying a protein sequence based on the viral RNA sequence such that the protein sequence includes translation of the viral RNA sequence (step d) may include: identifying a plurality of protein sequences based on the viral RNA sequence such that each of the plurality of protein sequences includes translation of the viral RNA sequence; and identifying the protein sequence as a protein sequence that frequently occurs within the plurality of protein sequences.

[0441] In some embodiments, the protein sequence can be identified based on the viral RNA sequence associated with a single infected subject.

[0442] In some embodiments, the step of identifying an immunogenic virus-derived peptide based at least in part on the protein sequence (step e) may include: identifying MHC molecules 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 an MHC-peptide complex with the MHC molecules; and identifying an immunogenic virus-derived peptide based on the one or more peptides.

[0443] Methods and systems for identifying integration sites of viral genes within a subject's genome The present disclosure further includes methods and systems for identifying integration sites of viral genes within a subject's genome. Some or all of the steps of this method can be performed by a computing device. In some embodiments, some or all of the steps of this method can be stored as computer-readable instructions in a memory, and thus by executing these instructions on one or more processors, the functions associated with this method can be performed. As a non-limiting example, methods and systems for identifying integration sites of viral genes within a subject's genome can include the embodiment of the integration flow of the HBV integration site shown in FIG. 1A.

[0444] In one aspect, the present disclosure may include a method for identifying integration sites of viral genes within a subject's genome, the method comprising the following steps: (a) obtaining a plurality of RNA contig sequences from an infected subject infected with a virus, such that the plurality of RNA contig sequences include a plurality of virus-derived RNA contig sequences, a plurality of infected subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences including virus and infected subject endogenous portions; (b) identifying the plurality of hybrid RNA contig sequences from within the plurality of RNA contig sequences; (c) comparing the infected subject endogenous portion with respect to at least a portion of the plurality of hybrid RNA contig sequences to a subject reference genome; and (d) identifying an integration site including the subject's gene based at least in part on the comparison of the infected subject endogenous portion to the subject reference genome.

[0445] In one aspect, a system configured to perform functions related to the above-described method can include a non-transitory computer-readable medium configured to communicate with one or more processors of a computing device. This non-transitory computer-readable medium may include instructions that, when executed by the one or more processors, cause the computing device to: (a) obtain, as input, a plurality of RNA contig sequences derived from an infected subject infected with a virus, such that the plurality of RNA contig sequences include a plurality of virus-derived RNA contig sequences, a plurality of subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences including virus and subject endogenous portions; (b) identify the plurality of hybrid RNA contig sequences from within the plurality of RNA contig sequences; (c) compare the subject endogenous portion with respect to at least a portion of the plurality of hybrid RNA contig sequences to a subject reference genome; (d) identify an integration site containing a gene of the subject based at least in part on a comparison of the subject endogenous portion to the subject reference genome; (e) provide the integration site as output.

[0446] In some embodiments, the plurality of RNA contig sequences may be derived from a single infected subject.

[0447] In some embodiments, the infected subject may be human.

[0448] In some embodiments, the plurality of virus-derived RNA contig sequences may be derived from the virus with which the infected subject is infected.

[0449] In some embodiments, the virus can include hepatitis B virus.

[0450] In some embodiments, the plurality of subject endogenous RNA contig sequences may be derived from the endogenous RNA of the infected subject.

[0451] In some embodiments, the subject reference genome can include the human genome.

[0452] Certain embodiments and implementations of the technology of the present disclosure have been described above with reference to systems and methods, and / or computer program products, according to exemplary embodiments or implementations of the technology of the present disclosure. It will be understood that the method steps can be implemented by computer-readable program instructions. Similarly, according to some embodiments or implementations of the technology of the present disclosure, some method steps need not be performed in the order presented, may be repeated, or may not necessarily be performed all at once.

[0453] By loading these computer-readable program instructions into a general-purpose computer, a special-purpose computer, a processor, or other programmable data processing apparatus, a particular machine can be created, whereby the instructions executed on the computer, processor, or other programmable data processing apparatus generate means for implementing one or more functions associated with the method steps. These computer program instructions may be stored in a computer-readable memory capable of causing a computer or other programmable data processing apparatus to function in a particular manner, and thus, these instructions stored in the computer-readable memory generate a manufactured article including instruction means for implementing one or more functions associated with the method steps.

[0454] Non-transitory computer-readable media include, but are not limited to, random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), flash memory or other memory technologies, compact disc ROM (CD-ROM), digital versatile disk (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or magnetic storage devices, or any other tangible physical medium capable of storing computer-readable information.

[0455] Embodiments or implementations of the techniques of the present disclosure can provide a computer program product including a computer-usable medium having computer-readable program code or program instructions recorded thereon, the computer-readable program code being adapted to be executed to implement one or more functions associated with the exemplary methods presented herein. Similarly, by loading computer program instructions onto a computer or other programmable data processing apparatus, a series of operational elements or steps can be executed on the computer or other programmable data processing apparatus to generate a computer-implemented process, whereby the instructions executed on the computer or other programmable apparatus provide elements or steps for implementing one or more functions associated with the exemplary methods presented herein.

[0456] Accordingly, the illustrations and descriptions of the methods support combinations of means for performing the specified functions, combinations of elements or steps for performing the specified functions, and combinations of program instruction means for performing the specified functions. It will also be understood that at least some method steps and combinations of method steps can be implemented by a special-purpose hardware-based computer system for performing the specified functions, elements, or steps, or a combination of special-purpose hardware and computer instructions.

[0457] Certain implementations of the technology of the present disclosure can be utilized in customer devices that may include mobile computing devices. Those skilled in the art will recognize that there are multiple categories of mobile devices, which are generally known as portable computing devices that can be battery-powered but are not usually classified as laptops. For example, mobile devices include, but are not limited to, portable computers, tablet PCs, Internet tablets, PDAs, ultra-mobile PCs (UMPCs), wearable devices, and smartphones.

[0458] Certain implementations of the technology of the present disclosure can be utilized in medical equipment, medical devices, and / or related peripheral devices.

Examples

[0459] The present disclosure will also be described and demonstrated by the following examples. However, the use of these examples and any other examples elsewhere in this specification are merely illustrative and do not limit in any way the scope and meaning of the present disclosure or any of the terms exemplified. Similarly, the present disclosure is not limited to any particular preferred implementation described herein. In fact, numerous modifications and variations of the present disclosure may become apparent to those skilled in the art upon reading this specification, and these variations can be implemented without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is to be limited only by the terms of the appended claims and the full scope of the equivalents to which these claims are entitled.

[0460] Example 1. Generation of an RNAseq database from samples of HBV-infected patients. Comparative genomic analysis is often guided by a small number of "reference" genomes. As of January 2022, the database of the National Center for Biotechnology and Information (NCBI) contains a repository of over 13,000 HBV genomic sequences classified into at least 10 different phylogenetic lineages (A - J). Each lineage is represented by one HBV genome, but these reference sequences do not capture the full range of natural genomic diversity. Such lack of coverage can have an adverse effect on the identification of new viral peptides, because the sequences of patient isolates may be significantly different from the reference genome sequences used in comparative analysis.

[0461] To circumvent this problem, an RNAseq-based approach was developed to generate a database of patient-specific HBV genomic sequences directly reconstructed from RNA reads amplified from liver samples of patients from Asian populations (Figure 1A). Using a de novo sequencing strategy, patient-specific HBV genomes were reconstructed and HBV integration sites within the human chromosome were identified. All human RNA reads were converted into large contigs, and contigs with loose homology to the HBV reference genome were marked as HBV-specific sequences. Next, HBV contigs were grouped into two categories based on the percentage of the contig sequences covering the HBV reference. Contigs that mapped completely to the HBV reference were reassembled without reference to assemble patient-specific HBV genomic sequences. Specific coding sequences from each reconstructed genome were stored in a large-scale patient-specific HBV database. Contigs that hybrid-match to the HBV reference were mapped against the human genome, and hybrid contigs that match both HBV and human sequences were selected. Specifically, hybrid contigs that match both human and HBV references were filtered to identify integration sites within the human genome using a homology sequence alignment approach. Subsequently, all HBV coding sequences were extracted and translated using patient-specific viral sequences. Coverage of all RNA reads from bulk RNA sequencing of all human liver samples against the HBV reference genome is shown in Figure 1B. Using this approach, a total of 80 unique coding sequences (large S = 14, middle S = 14, small S = 16, pre-capsid = 6, capsid = 12, X = 16, and polymerase = 2) were identified from virus isolates representing four phylogenetic lineages (A, B, C, D) (Figure 1C). The phylogenetic relationship between the reconstructed HBV genomes and reference HBV sequences representing eight HBV lineages (A, B, C, D, E, F, G, H) is shown in Figure 1C. Here, the tree was generated using a phylogenetic approach by the neighbor-joining method with the midpoint rooted.By lead mapping analysis, it was shown that the depth of coverage of viral reads was high and consistent in the genomic regions encoding the S and X proteins, while there were significant gaps in the coverage of the rest of the genome (Figure 1B). These gaps in the genome suggested that the reads might be derived from partially integrated HBV DNA sequences. The N-terminal region of the polymerase gene across the whole genome was not well covered, while the C-terminal part of the polymerase gene was well detected. For the majority of HBV+ samples, hybrid contigs combining both HBV and human sequences were also identified. These sequences were likely the result of the integration of HBV DNA into the contig sequences of human genes. The genomic positions of the HBV sequences at these junctions were called "breakpoints". Mapping the HBV breakpoints seen in RNAseq reads with HBV / human junctions, as shown in Figure 1D, it was found that the majority of HBV breakpoints occurred at nucleotide positions 1601 - 1900 of the HBV genome. In this figure, the frequency of the genomic positions of the breakpoints is shown at 100-nucleotide intervals across all samples studied, but no clear pattern was observed for the host genome (Table 3). These data suggest that at least some of the detected HBV sequences are derived from integrated DNA rather than cccDNA. Using the reconstruction of the HBV genome and HBV sequences from patient liver samples, HBV peptides presented on the surface of HCC tumors were discovered.

[0462]

Table 3-1

Table 3-2

Table 3-3

[0463] Example 2. Characterization of the immunopeptidome of HBV-infected patient samples.Using the anti-HLA class I antibody (W6 / 32), HLA class I complexes were immunopurified from homogenized hepatocellular carcinoma (HCC) liver tissue samples of 48 HBV-seropositive patients (ProteoGenex and BioIVT, Tables 4-5). Specifically, tissue lysates in NP-40 were incubated with W6 / 32-conjugated sepharose beads in column format, after which the beads were washed and HLA was eluted in glycine under acidic conditions. Peptides and HLA molecules were further concentrated with C18 Sep-Pak and eluted sequentially with 30% acetonitrile, 0.1% TFA and 70% acetonitrile, and 0.1% TFA, respectively. Proteins were separated under non-reducing conditions. Only 10% of the 70% acetonitrile eluate (1 / 10 of the HLA concentrate) was loaded onto the gel. Immunoblotting was performed with anti-HLA-I W6 / 32 and goat anti-mouse HRP. Western blotting of liver lysates, flow-through, and concentrated eluates demonstrated that W6 / 32-conjugated sepharose beads captured HLA-I almost completely (Figure 5). Following elution of the complex, HLA-I-bound peptides were identified using an Orbitrap Fusion Lumos mass spectrometer coupled to a nanoLC system. Raw mass spectrometry (MS) data files were searched against a composite database of human UniProt and HBV sequences obtained from RNA HBV reads from patient samples. On average, a total of approximately 8,500 peptides were detected from liver samples of patients of various ages (Figure 6). Figure 2A shows the peptide length distribution by mass spectrometry for liver tissues of all HBV-positive patients serologically. To generate these data, raw files were searched using PEAKSX with a 5% false discovery rate (FDR) at the peptide level. The length distribution of peptides isolated from most liver tissue samples was consistent with that of the class I HLA concentrate, with peptides 9 residues in length (9-mer) being the most abundant, followed by peptides 10 residues in length (10-mer) and peptides 11 residues in length (11-mer). Thus, most of the 9-mers were detected from the HLA class I concentrate from a high proportion of patient liver samples.Peptides of 9 to 12 residues accounted for more than 50% of the total isolated peptides (Figure 6).

[0464] Table 4 shows the patient sample ID, diagnosis (from the vendor), and HBV serological test results (i.e., HBV status, from the vendor) for all patient samples analyzed, the number of HBV unique peptides detected by mass spectrometry (MS), the detected HBV POL 606-616 variants, the detection status of the peptides STLPETTVVRR (SEQ ID NO: 43), FLLTRILTI (SEQ ID NO: 3), and SAISSTFSK (SEQ ID NO: 41), and the HBV genotypes identified from the RNAseq results. HCC represents Hepatocellular Carcinoma; CCA represents Cholangiocarcinoma; QC represents Quality Control.

[0465]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

[0466] Tables 5A and 5B show the patient and sample information provided by the vendor. All samples were flash-frozen (FF), and the specimen type for all samples was liver. The HBV diagnosis corresponding to all samples was HBV positive. One (Sample ID: ILS24966D3-DS2) was obtained from a postmenopausal patient. For all samples except the first six samples (Sample IDs: 09945T1(3), 0000032532, 0000040312, 0000031295, 0000049186, and 0000049219) for which Proteogenex was used as the vendor, the vendor used was BioIVT. The column for Country (Ctry) indicates the country where the sample was collected. Smoking years and alcohol years refer to the smoking period and alcohol use period, respectively. M is male; F is female; API is Asian Pacific Islander; Dx is diagnosis; Histo is histological; Grp is group; Wt is weight; Ht is height; Cigs is cigarettes; Ctry is country; VN is Vietnam; n / a represents not applicable.

[0467]

Table 5A-1

Table 5A-2

Table 5A-3

[0468]

Table 5B-1

Table 5B-2

[0469] Example 3. High proportion of the identified peptides predicted to bind to HLA using NetMHCpan 4.0 analysis. The polymorphism of HLA-I has a great impact on the epitopes presented on the tissue surface. Therefore, DNA sequencing of liver tissue samples was performed to determine the HLA genotype (Table 6), and this was used to predict the binding of the detected peptides by NetMHCpan 4.0.

[0470] [Table 6-1] [Table 6-2]

[0471] A high proportion (50 - 100% for most of the samples) of the detected 9-mer peptides were predicted binders of the HLA-I identified in the corresponding patient samples (Figure 2B and Table 7). In particular, Figure 2B shows a plot indicating the percentage of the 9-mer peptides identified by mass spectrometry that are binders to HLA-A, HLA-B, and HLA-C alleles. The HLA alleles were identified from the DNA sequencing of each patient sample. Most of the nanomer peptides detected by mass spectrometry were predicted binders to the patient HLA alleles predicted by NetMHCpan 4.0. Table 7 shows the number of nanomers (peptides 9 residues in length, 9-mer) and the predicted binding rate of these nanomers to each patient allele by NetMHC Pan4.0. Strong binders and weak binders are defined by their rank according to the NetMHC prediction, with a rank less than 0.5 considered a strong binder and 0.5 - 2 considered a weak binder. In some samples, the total percentage of binders to HLA-A, HLA-B, and HLA-C alleles exceeded 100%.

[0472] [Table 7-1]

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

[0473] Next, Seq2Logo was used to perform motif analysis of the detected peptides to identify residues overexpressed at various peptide positions (Figs. 7A - 7X). The anchor residues at positions 2 and 9 that identify HLA-I binding were significantly overexpressed in all tissue samples, and the residue identities corresponded to the HLA-I genotypes present. For example, the hydrophobic anchor residues (i.e., Leu and Val), which are characteristic of HLA-A02 conjugates, were enriched at positions 2 and 9 of the peptides isolated from HLA-A02+ patient samples. The high proportion of predicted HLA conjugates and the overexpression of characteristic anchor residues of the isolated peptides provided high reliability in the enrichment of HLA-I and the identification of peptides.

[0474] Example 4. Identification of unique HLA-related HBV peptides using an HBV genotype-specific database. A major challenge in the development of immunotherapies against HBV is the genomic variability among HBV strains. RNA sequencing was used to identify the HBV genotypes present in each patient sample (Figure 1C). According to the reported prevalence of HBV genotypes in the Asian population, HBV genotypes B and C were most frequently identified, found in 24 of 48 samples (12 each). HBV genotypes A and D were detected in only one patient sample each (Table 4). RNA sequencing data for 18 of the samples had low HBV coverage, suggesting that these samples likely did not contain HBV virus, perhaps due to viral latency or clearance. No HBV peptides were detected from any of these 18 tissues, further confirming the effectiveness of the proteogenomic approach described in this example and highlighting the good correlation between the two complementary methods. HBV peptides were detected in a total of 30 liver samples, but the HBV genotypes present in 4 of these samples could not be determined by RNA sequencing attempts.

[0475] The number of HBV peptides presented by HLA-I is likely to depend on the degree of HBV infection, proteasomal processing of the antigen, and the quality of the tissue sample. Indeed, the number of HBV-specific peptide sequences identified from infected livers varied from sample to sample, and overall, the sequence coverage of the indicated HBV proteins was low (Table 4 and Figure 1B). Using a PEAKS ion cut-off score = 20, a total of 49 unique HBV peptides 9 - 12 residues in length were identified (Table 8). Table 8 shows the HBV peptides and proteins detected by searching against a patient-specific database. Peptides were first filtered at a 5% false discovery rate (FDR), followed by filtering with a minimum cut-off score of -logP = 20.

[0476]

Table 8-1

Table 8-2

Table 8-3

Table 8-4

[0477] Most of the detected viral peptides were predicted by NetMHC pan 4.0 to be binders of the HLA-I alleles present in the corresponding patient samples (Figure 3B and Table 9). The top four HLA genotypes from HLA-A and HLA-B with the largest number of detected HBV peptides are shown in Figure 3A. Since the source of the samples obtained was the Asian population, a high proportion of the patient samples were of the HLA-02 and HLA-A11 genotypes. In particular, Figure 3B shows the HBV peptide sequences identified from the immune peptidome of the patient's liver samples and the predicted binding of these HBV peptide sequences to the patient's HLA alleles predicted by Pan NetMHC 4.0. Strong binders were ranked below 0.5 and weak binders were ranked between 0.5 and 2 according to the NetMHC prediction. For HBV peptides predicted to bind to more than two alleles, the affinity rank for the strongest binding is shown. The target HBV peptides and the frequency of detection of the target HBV peptides in the HBV+ samples are shown in Figure 3C. Table 9 shows a complete list of the HBV peptides together with the predicted binding affinities of the HBV peptides to the patient's HLA alleles predicted by Pan NetMHC 4.0.

[0478]

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

[0479] The large surface antigen protein is the source of several strong HLA-A02 predicted binders (NetMHC pan 4.0): GLSPTVWLSV (S 348-357 )(SEQ ID NO: 8), GMLPVCPLL (S 276-284 )(SEQ ID NO: 10), FLLTRILTI (S 194-202 )(SEQ ID NO: 3), and GMLPVCPLI (S 276-284 )(SEQ ID NO: 9), as well as the weak binder NILSPFMPLL (S 370-379 )(SEQ ID NO: 37), all of which were detected at relatively low frequencies (Table 8). In contrast, several HLA-A11-related peptides: STLPETTVVRR from the capsid protein (C 141-151 )(SEQ ID NO: 43), and SAISSTFSK from the polymerase (SEQ ID NO: 41) were found at relatively high frequencies (30 - 50%). HLA-A11-restricted HBV genotype-specific polymerase peptides, GSLPQEHIVQK from genotype B (P 606-616 )(SEQ ID NO: 12), and GTLPQEHIVHK from genotype C (P 606-616 )(SEQ ID NO: 13) and GTLPQEHIVQK (P 606-616)(SEQ ID NO:14) was detected. By NetMHC pan 4.0, the first peptide variant was predicted to be a weak HLA-A11 binder, while the latter two peptides were predicted to be strong binders of HLA-A11 (Figure 3B). The total frequency of this polymerase peptide and its variants (GSLPQEHIVQK (SEQ ID NO:12), GTLPQEHIVHK (SEQ ID NO:13), and GTLPQEHIVQK (SEQ ID NO:14)) in HLA-A11 tissue samples was over about 76% (13 out of 17 samples).

[0480] Subsequently, an analysis using synthetic peptide analogs was performed to verify the peptides identified above. FLLTRILTI(S 194-202 )(SEQ ID NO:3), STLPETTVVRR(C 141-151 )(SEQ ID NO:43), GSLPQEHIVQK(P 606-616)(SEQ ID NO: 12), and variants, of HBV peptides predicted to bind to HLA-A02 and HLA-A11 by NetMHC, were verified by aligning retention times and matching the fragmentation patterns of the peptides to their heavy synthetic versions (Figure 4 and Tables 10 - 13). Table 10 shows MS2 ions from the fragmentation of GSLPQEHIVQK (SEQ ID NO: 12) and its heavy analog. Table 11 shows MS2 ions from the fragmentation of STLPETTVVRR (SEQ ID NO: 43) and its heavy analog. Table 12 shows MS2 ions from the fragmentation of GTLPQEHIVHK (SEQ ID NO: 13) and its heavy analog. Table 13 shows MS2 ions from the fragmentation of FLLTRILTI (SEQ ID NO: 3) and its heavy analog. Heavy synthetic peptides with sequences similar to the target HBV peptides, and 50 fmol of modified heavy leucine (Thermo), were spiked into HLA eluted peptides and separated on nano-LC connected online to a mass spectrometer. The raw MS files were searched against a composite database of human Uniprot and HBV containing heavy leucine (+7.02) as a variable modification using PEAKSX. The fragmentation patterns (b ions and y ions) of the HBV peptides and their heavy analogs, and their co-elution and identification in the same MS1 scan, were used for peptide verification. The ion charge of each peptide was 1, except for the peptides marked with an asterisk (*) which had an ion charge of 2. FR represents fraction; Mod represents modification.

[0481]

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

[0482]

Table 11-1

Table 11-2

Table 11-3

[0483]

Table 12-1

Table 12-2

Table 12-3

Table 12-4

Table 12-5

[0484]

Table 13-1

Table 13-2

Table 13-3

Table 13-4

Table 13-5

[0485] The synthetic peptides used in this analysis are heavy leucine with the HBV peptide ( 13 C(6)15 It differed by the amount of N(1), +7.0172), and was mixed with HLA eluted peptides (50 fmol) before LC-MS / MS analysis. All peptides tested eluted with the same retention time and the same MS1 scan as their heavy analogs during the LC-MS / MS run and also exhibited similar fragmentation profiles (Figures 9A - 9H). The extracted ion chromatograms were also used to estimate the expression levels or copy numbers of some of the target peptides, including polymerase peptide variants (copy number approximately 100 - 500 / cell), from the intensity of the parent ions of the corresponding heavy peptide analogs. Even with relatively low surface presentation, the 3 polymerase peptide P 606-616 variants could be distinguished based on fragment ions (Figure 8).

[0486] This disclosure describes the identification of unique HBV peptides. Of a total of 49 HLA-I restricted HBV-specific peptides derived from patient liver samples, 34 unique peptides were detected from the envelope protein. The detection of STLPETTVVRR (C 141-151 )(SEQ ID NO: 43) from the HBV capsid correlates with previous studies. By leveraging the patient-specific database generated by RNAseq analysis, the peptide GSLPQEHIVQK (P 606-616 )(SEQ ID NO: 12) from the HBV polymerase and its variants were identified as the most frequent HBV epitopes in the analyzed population. This peptide is a predicted HLA-A11 binder and appeared in more than 80% of the HLA-A11+ patient population described herein.

[0487] The reconstruction of the HBV genome present in each sample of this disclosure provided insights into regions of the HBV genome that may be integrated into hepatocytes during the development of HCC. Regions of the genome encompassing the small surface antigen protein, the X protein, and the second half of the polymerase protein were frequently detected. Regions of the genome encompassing the capsid protein and the first half of the polymerase protein were not detected at all or were only partially detected.

[0488] When the RNAseq data and LC / MS-MS data reported in this specification are used in combination, an approach for the discovery and selection of peptide targets suitable for T cell-based therapeutic agents is demonstrated. As a non-limiting example, an ideal peptide target can be one that is extremely abundant on the cell surface, found in common among multiple patients, conserved across multiple HBV genotypes, and falls within a region of the HBV genome that is commonly integrated in HCC. Using the methods reported in this specification, peptides that meet all or specific aspects of the above criteria can be identified. Peptide GSLPQEHIVQK(P 606-616 )(SEQ ID NO: 12) was frequently found in HLA-A11 patient samples and fell within a region of the HBV genome that is commonly integrated. T cell-based therapeutic agents can be designed, for example, around the region of the conserved peptide GSLPQEHIVQK(P 606-616 )(SEQ ID NO: 12). By investigating more patient samples expressing various HLA alleles using the approach described in this specification, peptides that meet all or specific aspects of the above criteria can be identified.

[0489] The following are the methods used in the above examples.

[0490] Obtaining HBV-positive HCC tissues. All patient tissues were purchased in cryopreserved form from BioIVT. Detailed patient information, including age, gender, and diagnostic tests, is provided in Tables 4 and 5A - 5B. The tissues were cryopreserved until sample preparation.

[0491] Tissue lysis and HLA affinity enrichment. The tissue was ground in liquid nitrogen using a SPEX SamplePrep Freezer / Mill dual-chamber cryogenic grinder and then lysed by sonication in ice-cold lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), and 10 mM EDTA (pH 8.0)) supplemented with HALT protease and phosphatase inhibitors on ice. The volume of the lysis buffer was determined by the weight of the tissue (e.g., 5 ml of lysis buffer for 0.5 g of tissue).

[0492] Anti-HLA class I (W6 / 32) was conjugated to NHS Sepharose beads by incubating overnight in coupling buffer (0.2 M NaHCO3 + 0.5 M NaCl (pH 8.3)) at 4°C. The reaction was stopped with 0.1 M Tris-HCl (pH 8.5), and the beads were washed with the same Tris-HCl solution and 0.1 M acetate buffer.

[0493] The pre-clarified tissue lysate was passed through a column packed with 1 ml of HLA class I bead bed under gravity. The column was then washed with Seppro dilution buffer and 20 mM Tris-HCl (pH 8), and HLA-peptides were eluted with 0.1 M glycine (pH 2.7).

[0494] Sample preparation for mass spectrometry. The glycine eluate was loaded onto a C18 Sep-Pak, and the peptides were then selectively eluted with 30% ACN / 0.1% TFA. The peptides were further purified and analyzed by nano-LC-MS / MS.

[0495] Liquid chromatography-tandem mass spectrometry (LC-MS / MS). The above HLA peptides were loaded onto a nanoViper Acclaim PepMap100 C18 trap column (inner diameter 75 μm × 2 cm, 3 μm, 100 angstroms (Å)), heated to 40 °C, and separated using a nanoViper Acclaim PepMap RSLC C18 column (inner diameter 75 μm × 25 cm, 2 μm, 100 Å) with a New Objective SilicaTip (7 cm) having a distal conductive coating attached to the inlet side end of the emitter. The gradient was delivered at 300 nL / min by an EASY-nLC 1200 HPLC system. Subsequently, a 120-minute elution gradient with mobile phases A (water / 0.1% formic acid) and B (80% acetonitrile / 0.1% formic acid) was used: 3% B at 3 minutes, linearly to 35% B at 100 minutes, and then linearly to 45% B at 123 minutes. Peptides eluted from the column were ionized at 1.9 kV by a Flex ion source and analyzed using a Thermo Fusion Lumos Tribrid mass spectrometer with Xcalibur 4.1.31.9. Data acquisition was performed in data-dependent mode by running a survey scan on a high-field Orbitrap analyzer (m / z range 300 - 1500, resolution 60,000) with an automatic gain control target of 4.0E5 and a maximum ion fill time of 100 milliseconds. MS / MS analysis was performed by separation of precursor ions at 1.2 m / z in the quadrupole, application of 32% normalized HCD (higher-energy collisional dissociation) collision energy, and analysis of fragment ions in the Orbitrap at a resolution of 15,000. The dynamic exclusion window was set to 6 seconds, monoisotopic precursor selection (MIPS) was set for peptides, the maximum injection time was set to 100 milliseconds, and the charge state was unknown. Charge states +1 to +4 were included, and advanced peak determination was switched on.

[0496] In the FAIMS-compatible experiments, the settings were the same except that the FAIMS device was placed between the nanoelectrospray source and the mass spectrometer. FAIMS separation was carried out with the following settings: the temperature of the inner and outer electrodes was set to 100 °C (unless otherwise stated), FAIMS carrier gas flow 5.0 L / min, asymmetric waveform with a DV of -5000 V, inlet plate voltage 250 V, and CV setting time 25 milliseconds. The FAIMS carrier gas was N2 and the ion separation gap was 1.5 mm. The described CVs were applied to the FAIMS electrodes. For experiments with external stepping or a single CV, the selected CV was applied to all scans of the entire analysis. For experiments with internal CV stepping, each selected CV was applied to consecutive survey scans and MS / MS cycles (1 s); the CV for MS / MS was always paired with the appropriate CV from the corresponding survey scan.

[0497] Determination of the HLA genotype of patient samples. For sample preparation and sequencing, the amount of DNA sample was determined by fluorescence and the quality was evaluated by running 25 ng of the sample on a 1% precast agarose gel. Samples containing high molecular weight gDNA with the majority of DNA fragments exceeding 20 kb and having a concentration of 10 ng / ul or higher passed the quality assessment. The DNA samples were normalized to 10 ng / ul and 50 ng was used for amplification of full-length HLA amplicons. The target was amplified with three pools of variant tolerant primers optimized for the same annealing temperature and PCR conditions using LA Taq DNA polymerase. The resulting amplicon pools were combined equimolarly as determined by automated capillary electrophoresis and fluorescence. A DNA library was prepared for Illumina-based sequencing using a custom NEB kit. The amplicons were enzymatically fragmented to an average insert size of 250 bp and universal adapters were ligated to the DNA fragments. To facilitate highly multiplexed sequencing, unique 10-base pair barcode sequences were added to the DNA fragments during PCR with NEBNext Ultra II Q5 Master Mix. The samples were pooled and sequenced using 150-base pair paired-end sequencing on an Illumina Nextseq 500.

[0498] For data analysis, after completion of sequencing, the raw data from each Illumina Nextseq run was collected in local buffered storage and uploaded to a local high-performance computing platform for automated analysis. Conversion from BCL files to FASTQ-formatted reads was performed using bcl2fastq conversion software (Illumina Inc., San Diego, CA) and assigned to samples identified by specific barcodes. An updated version of the PHLAT program was used with a reference sequence consisting of the GRCh38 genome sequence and HLA type reference sequences within the IPD-IMGT / HLA database v3.30.0 to subject all reads within the sample-specific FASTQ files to HLA typing analysis.

[0499] RNA sequencing. Total RNA was extracted from human liver tissues using the MagMAX kit. Strand-specific RNA-seq libraries were prepared from 1 μg of RNA using the KAPA stranded mRNA-Seq kit (KAPA Biosystems). The libraries were amplified by performing 12 cycles of PCR. The amplified libraries were size-selected at 400 - 600 bp using PippinHT. Sequencing was performed on an Illumina HiSeq (registered trademark) 2500 (Illumina) by 2 × 100-cycle multiplexed paired-read run.

[0500] Mapping of the patient's liver RNA sequences to the HBV reference genome. Bulk RNA-seq reads were aligned to the reference genome ayw (NC_003977.2) using minimap2 (v2.17). The alignment was then sorted by coordinates and quality-controlled by inspection with samtools flagstats (v1.9) and bedtools genomeCoverageBed (v2.17.0). Subsequently, duplicates were marked and removed with the Picard toolkit (v2.18.2).

[0501] Workflow for reconstructing the HBV genome from the patient's liver RNAs...

Claims

1. An isolated peptide, or a pharmaceutically acceptable salt thereof, comprising one or more amino acid sequences selected from SEQ ID NOs: 14, 112, 11-13, and 111.

2. The isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising any one amino acid sequence from SEQ ID NOs: 14, 112, 11-13, and 111.

3. The isolated peptide is the isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising any one amino acid sequence from SEQ ID NOs: 14, 112, 11-13, and 111.

4. The isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising two or more amino acid sequences selected from SEQ ID NOs: 14, 112, 11-13, and 111.

5. The isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the length of the isolated peptide is 11 to 30 amino acids.

6. The isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising 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 is produced by expression in a heterologous host cell, or The isolated peptide is produced by synthesis. An isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof.

8. An isolated peptide according to claim 1, or a pharmaceutically acceptable salt thereof, which, when presented as a complex with a major histocompatibility complex (MHC) molecule on the surface of an antigen-presenting cell (APC), induces a hepatitis B virus (HBV)-specific immune response in the body of a target.

9. A fusion protein comprising one or more isolated peptides according to claim 1 or pharmaceutically acceptable salts thereof fused to one or more heterologous molecules.

10. The aforementioned one or more heterologous molecules enhance the peptide-specific immune response in the target body, or The fusion protein according to claim 9, wherein the one or more heterologous molecules mediate peptide delivery to a specific site within the body of the target.

11. The fusion protein according to claim 9, wherein the one or more heterologous molecules are MHC molecules, or fragments or derivatives thereof.

12. A fusion protein comprising an isolated peptide or a pharmaceutically acceptable salt thereof as described in claim 1, and an MHC molecule or a fragment or derivative thereof.

13. The fusion protein according to claim 12, wherein the MHC molecule or fragment thereof is an MHC class I molecule.

14. The fusion protein according to claim 13, wherein the MHC class I molecule is a human leukocyte antigen (HLA) class I molecule.

15. The fusion protein according to claim 12, wherein the MHC molecule or fragment thereof is an MHC class II molecule.

16. The fusion protein according to claim 15, wherein the MHC class II molecule is an HLA class II molecule.

17. The fusion protein according to claim 12, wherein the MHC molecule or fragment thereof is in a soluble form.

18. A conjugate comprising one or more isolated peptides according to claim 1 or pharmaceutically acceptable salts thereof, conjugated to one or more heterologous molecules.

19. The aforementioned one or more heterologous molecules enhance the peptide-specific immune response in the target body, or The conjugate according to claim 18, wherein the one or more heterologous molecules mediate the delivery of peptides to a specific site within the body of the target.

20. The conjugate according to claim 18, wherein the one or more heterogeneous molecules are MHC molecules, or fragments or derivatives thereof.

21. The conjugate according to claim 18, wherein the one or more isolated peptides or pharmaceutically acceptable salts thereof are conjugated to particles.

22. A conjugate comprising an isolated peptide or a pharmaceutically acceptable salt thereof as described in claim 1, and an MHC molecule or a fragment or derivative thereof.

23. The conjugate according to claim 22, wherein the MHC molecule or fragment thereof is an MHC class I molecule.

24. The conjugate according to claim 23, wherein the MHC class I molecule is a human leukocyte antigen (HLA) class I molecule.

25. The conjugate according to claim 22, wherein the MHC molecule or fragment thereof is an MHC class II molecule.

26. The conjugate according to claim 25, wherein the MHC class II molecule is an HLA class II molecule.

27. ​​The conjugate according to claim 22, wherein the MHC molecule or fragment thereof is in a soluble form.

28. An oligomeric complex comprising two or more isolated peptides or pharmaceutically acceptable salts thereof according to claim 1.

29. A non-covalent complex comprising an isolated peptide or a pharmaceutically acceptable salt thereof as described in claim 1, and an MHC molecule or a fragment or derivative thereof.

30. The non-covalent complex according to claim 29, wherein the MHC molecule or fragment thereof is an MHC class I molecule.

31. The non-covalent complex according to claim 30, wherein the MHC class I molecule is a human leukocyte antigen (HLA) class I molecule.

32. The non-covalent complex according to claim 29, wherein the MHC molecule or fragment thereof is an MHC class II molecule.

33. The non-covalent complex according to claim 32, wherein the MHC class II molecule is an HLA class II molecule.

34. The non-covalent complex according to claim 29, wherein the MHC molecule or fragment thereof is in a soluble form.

35. (i) one or more isolated peptides or pharmaceutically acceptable salts thereof according to any one of claims 1 to 8, one or more fusion proteins according to any one of claims 9 to 17, one or more conjugates according to any one of claims 18 to 27, one or more oligomeric complexes according to claim 28, or one or more non-covalent complexes according to any one of claims 29 to 34, or any combination thereof; and (ii) a pharmaceutically acceptable carrier or excipient, comprising a pharmaceutical composition.

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

37. An isolated polynucleotide comprising one or more isolated peptides as described in claim 1, or a nucleotide sequence encoding a fusion protein comprising the one or more isolated peptides and an MHC molecule or a fragment or derivative thereof.

38. The isolated polynucleotide according to claim 37, wherein the nucleotide sequence is operably linked to a promoter.

39. The isolated polynucleotide according to claim 37, wherein the isolated polynucleotide comprises DNA.

40. The isolated polynucleotide according to claim 37, wherein the isolated polynucleotide comprises RNA.

41. The RNA is mRNA, and / or The isolated polynucleotide according to claim 40, wherein the RNA is self-replicating RNA.

42. A vector comprising the isolated polynucleotide described in claim 37.

43. The vector is an expression vector, and / or The vector according to claim 42, wherein the vector is a viral vector.

44. A host cell comprising an isolated polynucleotide according to claim 37 or a vector containing the isolated polynucleotide.

45. The host cell according to claim 44, wherein the host cell is a prokaryotic cell.

46. The host cell according to claim 44, wherein the host cell is a eukaryotic cell.

47. The host cell according to claim 46, wherein the host cell is an APC.

48. (i) an isolated polynucleotide according to any one of claims 37 to 41, or a vector according to claim 42 or 43; and (ii) a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

49. The pharmaceutical composition according to claim 48, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle carrier.

50. A composition for inducing an immune response to hepatitis B virus (HBV) infection in the body of a subject who needs to have an immune response to HBV infection induced, a) One or more isolated peptides or pharmaceutically acceptable salts thereof according to any one of claims 1 to 8; b) The fusion protein according to any one of claims 9 to 17; c) The conjugate according to any one of claims 18 to 27; d) The oligomer complex according to claim 28; e) The non-covalent complex according to any one of claims 29 to 34; f) an isolated polynucleotide according to any one of claims 37 to 41; g) The vector according to claim 42 or 43; or h) The host cell according to any one of claims 44 to 47; A composition containing the following:

51. A composition for inducing an immune response to hepatitis B virus (HBV) infection in the body of a subject who needs to have an immune response to HBV infection induced, comprising one or more isolated peptides or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 8.

52. A composition for inducing an immune response to hepatitis B virus (HBV) infection in the body of a subject who needs to have an immune response to HBV infection induced, comprising the fusion protein described in any one of claims 9 to 17.

53. The composition according to claim 52, wherein the immune response comprises an increase in the production of target-specific antibodies in the subject.

54. A composition for inducing an immune response to HBV infection in the body of a subject who needs to have an immune response to HBV infection induced, comprising activated T cells produced by contacting T cells with an APC that presents an isolated peptide according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof as a complex with an MHC molecule.

55. A composition for treating, preventing, or reducing the likelihood of HBV-induced diseases or disorders in the target population, a) One or more isolated peptides or pharmaceutically acceptable salts thereof according to any one of claims 1 to 8; b) The fusion protein according to any one of claims 9 to 17; c) The conjugate according to any one of claims 18 to 27; d) The oligomer complex according to claim 28; e) The non-covalent complex according to any one of claims 29 to 34; f) an isolated polynucleotide according to any one of claims 37 to 41; g) The vector according to claim 42 or 43; or h) The host cell according to any one of claims 44 to 47; A composition containing the following:

56. A composition for treating, preventing, or reducing the likelihood of HBV-induced diseases or disorders in a subject in need, comprising one or more isolated peptides or pharmaceutically acceptable salts thereof as described in any one of claims 1 to 8.

57. The composition according to claim 55, wherein the disease or disorder induced by HBV is hepatitis, hepatic fibrosis, cirrhosis, or liver cancer.

58. The composition according to claim 57, wherein the liver cancer is hepatocellular carcinoma (HCC).

59. It's a kit: (i) a) One or more isolated peptides or pharmaceutically acceptable salts thereof according to any one of claims 1 to 8; b) The fusion protein according to any one of claims 9 to 17; c) The conjugate according to any one of claims 18 to 27; d) The oligomer complex according to claim 28; e) The non-covalent complex according to any one of claims 29 to 34; f) an isolated polynucleotide according to any one of claims 37 to 41; g) The vector according to claim 42 or 43; or h) The host cell according to any one of claims 44 to 47; and (ii) Packaging and / or instructions for use of the kit A kit that includes this.

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