Virus peptides and uses thereof

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 the peptides or peptide-based molecules, pharmaceutical compositions (e.g., vaccine compositions), and their use for the treatment or prevention of HBV infection and / or HBV-induced diseases. The disclosure also provides binding moieties that bind to the peptides or peptide-based molecules disclosed herein and their use for the treatment or prevention of HBV infections and / or HBV-induced diseases. The present disclosure also provides methods and systems for identifying immunogenic virus-derived peptides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Application No. 63 / 398,305, filed on August 16, 2022, and U.S. Provisional Application No. 63 / 349,804, filed on June 7, 2022, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML file format, the entire contents of which are incorporated herein by reference. The XML copy was created on May 30, 2023, named 250298_000482_SL.xml, and is 98,432 bytes in size. Technical Field

[0005] The present disclosure relates to methods and compositions involving isolated peptides derived from hepatitis B virus (HBV), and the use of such methods and compositions for treating or preventing HBV infection and / or HBV-induced diseases. Background Art

[0006] Chronic hepatitis B virus (HBV) infection affects ~250 million people worldwide and can lead to serious complications, including acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV vaccination is effective in preventing infection, but there is currently no cure for patients with chronic HBV infection.

[0007] The persistence or control of HBV infection depends on the host immune response, which is mainly mediated by cytotoxic T cells, which mediate adaptive immunity by recognizing HLA-peptide complexes presented on the surface of infected cells through T cell receptor (TCR) interactions. Although proteogenomics can be used to identify HLA-associated mutant peptides in cancer, HBV-HCC tumor cells may contain HBV DNA fragments that have been inserted into the host genome, ultimately posing a challenge to the identification of HBV epitopes. Further complicating this effort is the existence of thousands of HBV strains distributed across 10 different HBV genotypes that differ from each other by at least 8% at the nucleotide level. Therefore, the development of methods to identify HBV epitopes may be helpful to HBV-infected patients. Summary of the invention

[0008] As described in the Background section above, there is a great need in the art to develop methods for identifying HBV epitopes that may be helpful to HBV infected patients. The present application meets these and other needs.

[0009] In one aspect, provided herein is an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112, wherein the isolated peptide is 8-12 amino acids in length.

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

[0011] In some embodiments, the isolated peptide consists essentially of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

[0012] In some embodiments, the isolated peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

[0013] In another aspect, provided herein is an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide comprises two or more amino acid sequences selected from any one of SEQ ID NOs: 1-54 and 110-112.

[0014] In some embodiments, the isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide consists of the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), wherein X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H or L.

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

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

[0017] In some embodiments, the isolated peptide is produced synthetically.

[0018] In some embodiments, the isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof induces a hepatitis B virus (HBV)-specific immune response in a subject when presented on the surface of an antigen presenting cell (APC) in a complex with a major histocompatibility complex (MHC) molecule.

[0019] In another aspect, provided herein is a fusion protein comprising one or more isolated peptides disclosed herein fused to one or more heterologous molecules.

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

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

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

[0023] In another aspect, provided herein is a conjugate comprising one or more isolated peptides disclosed herein conjugated to one or more heterologous molecules.

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

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

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

[0027] In some embodiments, the one or more peptides are conjugated to a particle.

[0028] In another aspect, provided herein is an oligomeric complex comprising two or more isolated peptides disclosed herein.

[0029] 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.

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

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

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

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

[0034] 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.

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

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

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

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

[0039] In another aspect, provided herein is a conjugate comprising: an isolated peptide disclosed herein, and an MHC molecule or a fragment or derivative thereof.

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

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

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

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

[0044] 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.

[0045] In some embodiments, the pharmaceutical compositions disclosed herein further comprise an adjuvant.

[0046] In another aspect, provided herein is an isolated molecule that binds to an isolated peptide disclosed herein, a fusion protein disclosed herein, a conjugate disclosed herein, an oligomeric complex disclosed herein, or a non-covalent complex disclosed herein.

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

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

[0049] In some embodiments, the molecule is a surrogate scaffold.

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

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

[0052] In another aspect, provided herein is an isolated cell comprising a CAR disclosed herein.

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

[0054] In some embodiments, the immune cell is a T cell, a NK cell, or a macrophage.

[0055] In another aspect, provided herein is an isolated cell comprising a TCR disclosed herein.

[0056] In some embodiments, the isolated cell is an immune cell.

[0057] In some embodiments, the immune cell is a T cell, a NK cell, or a macrophage.

[0058] 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.

[0059] In another aspect, provided herein is an isolated polynucleotide comprising: a nucleotide sequence encoding one or more isolated peptides disclosed herein, or a nucleotide sequence encoding a fusion protein disclosed herein.

[0060] In some embodiments, the nucleotide sequence is operably linked to a promoter.

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

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

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

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

[0065] In another aspect, provided herein is a vector comprising the isolated polynucleotide disclosed herein.

[0066] In some embodiments, the vector is an expression vector.

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

[0068] In another aspect, provided herein is a host cell comprising an isolated polynucleotide disclosed herein or a vector disclosed herein.

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

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

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

[0072] 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.

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

[0074] 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:

[0075] a) one or more isolated peptides disclosed herein;

[0076] b) a fusion protein disclosed herein;

[0077] c) the conjugates disclosed herein;

[0078] d) an oligomeric complex as disclosed herein;

[0079] e) a non-covalent complex as disclosed herein;

[0080] f) the pharmaceutical compositions disclosed herein;

[0081] g) a molecule disclosed herein;

[0082] h) an isolated cell as disclosed herein;

[0083] i) an isolated polynucleotide disclosed herein; or

[0084] j) A vector disclosed herein.

[0085] 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.

[0086] In another aspect, provided herein is a method of inducing an immune response against HBV infection in a subject in need thereof, comprising administering to the subject activated T cells generated by contacting the T cells with APCs that present an isolated peptide disclosed herein in complex with an MHC molecule.

[0087] In another aspect, provided herein is a method of treating an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of:

[0088] a) one or more isolated peptides disclosed herein;

[0089] b) a fusion protein disclosed herein;

[0090] c) the conjugates disclosed herein;

[0091] d) an oligomeric complex as disclosed herein;

[0092] e) a non-covalent complex as disclosed herein;

[0093] f) the pharmaceutical compositions disclosed herein;

[0094] g) a molecule disclosed herein;

[0095] h) an isolated cell as disclosed herein;

[0096] i) an isolated polynucleotide disclosed herein; or

[0097] j) A vector disclosed herein.

[0098] In another aspect, provided herein is a method of preventing or reducing the likelihood of an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of:

[0099] a) one or more isolated peptides disclosed herein;

[0100] b) a fusion protein disclosed herein;

[0101] c) the conjugates disclosed herein;

[0102] d) an oligomeric complex as disclosed herein;

[0103] e) a non-covalent complex as disclosed herein;

[0104] f) the pharmaceutical compositions disclosed herein;

[0105] g) a molecule disclosed herein;

[0106] h) an isolated cell as disclosed herein;

[0107] i) a polynucleotide disclosed herein; or

[0108] j) A vector disclosed herein.

[0109] In another aspect, provided herein is a method of treating an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides disclosed herein.

[0110] In another aspect, provided herein is a method of preventing or reducing the likelihood of an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides disclosed herein.

[0111] In some embodiments, the HBV-induced disease or condition is liver inflammation, liver fibrosis, cirrhosis, or liver cancer.

[0112] In some embodiments, the liver cancer is hepatocellular carcinoma (HCC).

[0113] In another aspect, there is provided a kit comprising:

[0114] (i) a) one or more isolated peptides disclosed herein;

[0115] b) a fusion protein disclosed herein;

[0116] c) the conjugates disclosed herein;

[0117] d) an oligomeric complex as disclosed herein;

[0118] e) a non-covalent complex as disclosed herein;

[0119] f) the pharmaceutical compositions disclosed herein;

[0120] g) a molecule disclosed herein;

[0121] h) an isolated cell as disclosed herein;

[0122] i) an isolated polynucleotide disclosed herein; or

[0123] j) a vector disclosed herein; and

[0124] (ii) Instructions for use of the packaging and / or kit.

[0125] In another aspect, provided herein is a method for identifying an immunogenic virus-derived peptide, the method comprising:

[0126] a) obtaining a plurality of RNA contig sequences derived from an infected subject infected with a virus, wherein the plurality of RNA contig sequences comprises a plurality of virus-derived RNA contig sequences and a plurality of RNA contig sequences endogenous to the infected subject;

[0127] b) identifying a plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences;

[0128] c) assembling viral RNA sequences based on the plurality of virus-derived RNA contig sequences;

[0129] d) identifying a protein sequence based on the viral RNA sequence; and

[0130] e) identifying immunogenic virus-derived peptides based at least in part on the identified protein sequences.

[0131] In some embodiments, the plurality of RNA contig sequences are derived from an infected subject.

[0132] In some embodiments, the infected subject is a human.

[0133] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

[0134] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

[0135] In some embodiments, identifying a plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences comprises:

[0136] comparing at least a portion of the contig sequences of the plurality of RNA contig sequences to a reference viral sequence; and

[0137] The plurality of virus-derived RNA contig sequences are identified such that each contig sequence in the plurality of virus-derived RNA contig sequences comprises at least a portion of a sequence corresponding to the reference viral sequence.

[0138] In some embodiments, each contig sequence in the plurality of virus-derived RNA contigs is different from the plurality of infected subject endogenous RNA contig sequences.

[0139] In some embodiments, each contig sequence in the plurality of virus-derived RNA contig sequences lacks an RNA contig sequence endogenous to an infected subject.

[0140] In some embodiments, the reference viral sequence comprises a reference genome.

[0141] In some embodiments, the reference genome comprises a hepatitis B virus genome.

[0142] In some embodiments, assembling a viral RNA sequence based on the plurality of virus-derived RNA contig sequences comprises:

[0143] The common sequence portions overlap at the ends of at least a portion of the plurality of virus-derived RNA contig sequences so that at least a portion of the plurality of virus-derived RNA contig sequences are linearly overlapped to assemble the viral RNA sequence.

[0144] In some embodiments, identifying a protein sequence based on the viral RNA sequence such that the identified protein sequence includes a translation of the viral RNA sequence comprises:

[0145] Protein sequences are identified without comparison to viral protein databases.

[0146] In some embodiments, identifying the protein sequence based on the viral RNA sequence such that the identified protein sequence includes a translation of the viral RNA sequence, further comprising:

[0147] identifying a plurality of protein sequences, each based on the viral RNA sequence, such that each of the plurality of protein sequences comprises a translation of the viral RNA sequence, and

[0148] The protein sequence is identified as a frequently occurring protein sequence among the plurality of protein sequences.

[0149] In some embodiments, the protein sequence identified based on the viral RNA sequence is associated with a single infected subject.

[0150] In some embodiments, identifying the immunogenic virus-derived peptide based at least in part on the protein sequence comprises:

[0151] Identification of MHC molecules associated with individual infected subjects;

[0152] identifying one or more peptides based at least in part on the protein sequence, such that each of the one or more peptides forms a corresponding MHC peptide complex with the MHC molecule; and

[0153] The immunogenic virus-derived peptide is identified based on the one or more peptides.

[0154] In another aspect, a non-transitory computer-readable medium is provided herein, which is configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium including instructions thereon, which when executed by the one or more processors, cause the computing device to:

[0155] a) receiving 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 and a plurality of RNA contig sequences endogenous to the infected subject, and wherein the infected subject is infected with a virus;

[0156] b) identifying the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences;

[0157] c) assembling viral RNA sequences based on the plurality of virus-derived RNA contig sequences;

[0158] d) identifying protein sequences based on the viral RNA sequences;

[0159] e) identifying immunogenic virus-derived peptides based at least in part on the protein sequence; and

[0160] f) providing said immunogenic virus-derived peptide as an output.

[0161] In some embodiments, the plurality of RNA contig sequences are derived from only one infected subject.

[0162] In some embodiments, the infected subject comprises a human.

[0163] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

[0164] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

[0165] In some embodiments, the instructions that cause the computing device to identify the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences further comprise instructions that, when executed by the one or more processors, cause the computing device to:

[0166] comparing at least a portion of the contig sequences of the plurality of RNA contig sequences to a reference viral sequence; and

[0167] The plurality of virus-derived RNA contig sequences are identified such that each contig sequence in the plurality of virus-derived RNA contig sequences comprises at least a portion of a sequence corresponding to the reference viral sequence.

[0168] In some embodiments, each contig sequence in the plurality of virus-derived RNA contigs is different from the plurality of infected subject endogenous RNA contig sequences.

[0169] In some embodiments, each contig sequence in the plurality of virus-derived RNA contig sequences lacks a portion of an RNA contig sequence endogenous to an infected subject.

[0170] In some embodiments, the reference viral sequence comprises a reference genome.

[0171] In some embodiments, the reference genome comprises a hepatitis B virus genome.

[0172] In some embodiments, the instructions causing the computing device to assemble the viral RNA sequence based on the plurality of virus-derived RNA contig sequences further include instructions that, when executed by the one or more processors, cause the computing device to:

[0173] The common sequence portions overlap at the ends of at least a portion of the plurality of virus-derived RNA contig sequences so that at least a portion of the plurality of virus-derived RNA contig sequences are linearly overlapped to assemble the viral RNA sequence.

[0174] In some embodiments, the instructions that cause the computing device to identify a protein sequence based on the viral RNA sequence such that the protein sequence includes a translation of the viral RNA sequence further include instructions that, when executed by the processor, cause the computing device to:

[0175] The protein sequence was identified without comparison to a viral protein database.

[0176] In some embodiments, the protein sequence identified based on the viral RNA sequence is a novel protein.

[0177] In some embodiments, the protein sequence identified based on the viral RNA sequence is associated with a single infected subject.

[0178] In some embodiments, the instructions that cause the computing device to identify a protein sequence based on the viral RNA sequence such that the protein sequence includes a translation of the viral RNA sequence further include instructions that, when executed by the one or more processors, cause the computing device to:

[0179] identifying a plurality of protein sequences, each based on the viral RNA sequence, such that each of the plurality of protein sequences, respectively, comprises a translation of the viral RNA sequence; and

[0180] The protein sequence is identified as a frequently occurring protein sequence among the plurality of protein sequences.

[0181] In some embodiments, the instructions that cause the computing device to identify the immunogenic virus-derived peptide based at least in part on the protein sequence further comprise instructions that, when executed by the one or more processors, cause the computing device to:

[0182] identifying a major histocompatibility complex (MHC) molecule associated with the single infected subject;

[0183] identifying 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 a corresponding MHC peptide complex with the MHC molecule; and

[0184] The immunogenic virus-derived peptide is identified based on the one or more peptides.

[0185] In some embodiments, the instructions, when executed by the one or more processors, further cause the computing device to:

[0186] The protein sequence is stored in a database such that the protein sequence is associated with an infected subject in the database.

[0187] In another aspect, the present invention provides a method for identifying an integration site of a viral gene within a gene of a subject, the method comprising:

[0188] a) obtaining 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 RNA contig sequences endogenous to the infected subject, and a plurality of hybrid RNA contig sequences comprising portions endogenous to the virus and to the infected subject;

[0189] b) identifying the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences;

[0190] c) comparing, for at least a portion of the plurality of hybrid RNA contig sequences, an endogenous portion of the infected subject to a reference genome of the subject; and

[0191] d) identifying an integration site comprising a gene of the subject based at least in part on a comparison of an endogenous portion of the infected subject to a reference genome of the subject.

[0192] In some embodiments, the plurality of RNA contig sequences are derived from an infected subject.

[0193] In some embodiments, the infected subject is a human.

[0194] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

[0195] In some embodiments, the virus is hepatitis B virus.

[0196] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

[0197] In some embodiments, the subject reference genome comprises the human genome.

[0198] In another aspect, a non-transitory computer-readable medium is provided herein, which is configured to communicate with one or more processors of a computing device, the non-transitory computer-readable medium including instructions thereon, which when executed by the one or more processors cause the computing device to:

[0199] a) receiving 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 RNA contig sequences endogenous to the infected subject, and a plurality of hybrid RNA contig sequences comprising portions endogenous to the virus and to the infected subject;

[0200] b) identifying the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences;

[0201] c) comparing, for at least a portion of the plurality of hybrid RNA contig sequences, an endogenous portion of the infected subject to a reference genome of the subject;

[0202] d) identifying an integration site comprising a gene of the subject based at least in part on a comparison of an endogenous portion of the infected subject to a reference genome of the subject; and

[0203] e) providing said integration site as output.

[0204] In some embodiments, the plurality of RNA contig sequences are derived from only one infected subject.

[0205] In some embodiments, the infected subject comprises a human.

[0206] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

[0207] In some embodiments, the virus comprises hepatitis B virus.

[0208] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

[0209] In some embodiments, the subject reference genome comprises the human genome.

[0210] These and other aspects of the present invention will be apparent to those of ordinary skill in the art from the following description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0211] Figures 1A-1D A method for identifying HBV-specific signatures using bulk RNA sequencing (RNAseq) of human liver samples is shown. RNA contigs that fully match the HBV reference are used to reconstruct the genome of HBV strains from infected patients. Heterozygous contigs that match both human and HBV references are filtered to identify integration sites within the human genome ( Figure 1A ). Coverage of total RNA reads from bulk RNA sequencing of whole human liver samples against the HBV reference genome ( Figure 1B Phylogenetic relationships between the reconstructed HBV genome and reference HBV sequences representing eight HBV lineages (A, B, C, D, E, F, G, H) ( Figure 1C ). Location of genomic breakpoints of HBV integration in all samples ( Figure 1D ).

[0212] Figure 2A-2B Peptide length distribution and percentage of conjugates from identified peptides with 9 amino acid residues (9-mer) are shown. Peptide length distribution of liver tissues of all HBV-positive patients was determined by serological mass spectrometry ( Figure 2A ). The figure shows the percentage (%) of binders from 9-mer peptides against HLA-A, HLA-B and HLA-C alleles identified by mass spectrometry ( Figure 2B ).

[0213] Figures 3A-3CHLA genotypes from HLA-A and HLA-B with the highest number of detected HBV peptides, predicted HLA allele binders, and a graph of target peptides with corresponding detection frequencies in HBV positive (+) samples are shown. The four most common HLA alleles from HLA-A, HLA-B, and HLA-C in the analyzed patient samples, and the number of samples that were HBV positive (detected HBV peptides) by mass spectrometry ( Figure 3A ). HBV peptide sequences identified from immunopeptidomics of patient liver samples and their predicted binding to patient HLA alleles as predicted by Pan NetMHC 4.0 ( Figure 3B ). Detection frequency of target HBV peptides and target HBV peptides in HBV+ samples ( Figure 3C ).

[0214] Figures 4A-4D Validation was demonstrated for four HBV peptides, including two polymerase variants ( Figure 4A and Figure 4C ) and two other peptides ( Figure 4B and Figure 4D ).

[0215] Figure 5 An example of an immunoblot is shown, demonstrating HLA enrichment by the HLA-I specific W6 / 32 antibody conjugated to agarose beads.

[0216] Figure 6 Bar graphs showing total peptide counts and peptides with 9–12 residues identified by mass spectrometry are shown.

[0217] Figures 7A-7X Motif analysis of all 9-mer peptides identified by PEAKSX search of mass spectrometry raw files is shown.

[0218] Figure 8 MS2 fragment ion spectra showing three polymerase peptide variants showed extensive fragmentation for confident peptide identification.

[0219] Figures 9A-9H Depicted is an MS1 ​​analysis showing the co-elution of endogenous peptides and corresponding synthetic heavy analogs of validated HBV peptides in a mass spectrometry run. Figures 9A-9G Retention time and estimated copy number calculated using peptide intensities of heavy and endogenous peptides of HLA-associated HBV peptides in patient samples ( Figure 9H ).

[0220] Figures 10A-10B The pan-HLA-A antibody was shown to be 100 μg / ml peptide ( Fig. 10A ) and 33 μg / ml peptide ( Fig. 10B) 606-616 Cell binding curves of peptide-pulsed K562 cells. DETAILED DESCRIPTION

[0221] Chronic HBV infection causes liver inflammation, fibrosis, and cirrhosis, which severely impair liver function and eventually lead to hepatocellular carcinoma (HCC); nearly 25-40% of HBV carriers will develop HCC. Vaccination and antiviral drugs (such as reverse transcriptase inhibitors and interferon therapy) are the preferred preventive and therapeutic options for the management of HBV infection. However, once the infection is established, antiviral drugs can only control viral transcription and are largely ineffective in hepatitis B surface antigen (HBsAg) seroclearance. In addition, the integration of HBV DNA fragments into the host genome introduces genetic damage and chromosomal instability, which can further lead to the development of HCC. Globally, it is estimated that chronic HBV infection accounts for ~50% of primary liver cancer.

[0222] The persistence or control of HBV infection depends on the host immune response, which is mainly mediated by the adaptive immune system after HBV escapes the innate immune system. + T cells protect the host from intracellular pathogens and tumors. + T cells play a key role in the defense against HBV infection and are primarily responsible for clearing the virus in acute infection and preventing the development of HCC. + T cells are often undetectable, which is thought to be a result of chronic exposure of the liver to high antigen loads and tolerogenic hepatocytes.

[0223] Since CD8 + The key role of T cells in defending against viral infections, using CD8 + T cells have become an important aspect of immunotherapy. Cytotoxic T cells mediate adaptive immunity by interacting with T cell receptors (TCRs) expressed on the surface of infected cells and recognizing HLA-peptide complexes presented on the surface of infected cells. These HLA-peptide complexes can be used as targets to develop different immunotherapeutic drugs, including, for example, bispecific antibodies and CAR-T therapy.

[0224] Proteogenomics can be used to identify HLA-associated mutant peptides in cancer. HBV-HCC tissues may lack HBV covalently closed circular DNA (cccDNA), which serves as a template for viral protein synthesis. In contrast, tumor cells can contain HBV DNA fragments that have been inserted into the host genome, which can be transcribed and ultimately lead to the expression of viral antigens on the cell surface. This poses a challenge to the identification of HBV epitopes, as the exact regions of HBV genome integration can vary from patient to patient. Further complicating this effort is the existence of thousands of HBV strains, distributed across 10 different HBV genotypes (genotypes A to J), which differ from each other by at least 8% at the nucleotide level.

[0225] Among other things, the present disclosure provides peptides derived from separations of hepatitis B virus (HBV) and fragments or derivatives thereof. Various peptide-based molecules are also provided, including complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, and conjugates comprising peptides. Polynucleotides and vectors encoding peptides or peptide-based molecules described herein are further provided herein. Binding moieties (e.g., antibodies, alternative skeletons, T cell receptors (TCRs), or chimeric antigen receptors (CARs)) bound to peptides or peptide-based molecules are also provided. The compositions of the present disclosure can be used to induce an immune response to HBV infection and / or for treating or preventing HBV-induced diseases or conditions. In one aspect, the present disclosure also provides a method for identifying immunogenic virus-derived peptides.

[0226] Lymphocytes (such as T cells) play an important role in adaptive anti-infection, anti-tumor, autoimmunity and transplant rejection. In general, T cell-mediated immune responses involve close contact between T cells and antigen presenting cells (APCs), such as immune synapses. The pairing of several molecules is involved in the formation of immune synapses, including but not limited to: (a) T cell receptors (TCRs) on T cells, which specifically bind to peptides present in the peptide binding grooves of major histocompatibility complex (MHC) molecules on APCs; and (b) CD28 (on T cells), which is paired with B7 molecules on APCs. TCR forms a TCR complex with CD3 molecules, and when TCR is paired with a peptide-MHC (pMHC) complex, the signal is sent through CD3. Signal transduction by TCR complexes and CD28 on T cells leads to T cell activation.

[0227] 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 chromosome 14 in humans or mice), and also includes the entire δ locus encoding the δ chain, while the β chain is encoded by a nucleic acid sequence located within the β locus (on chromosome 6 in mice or chromosome 7 in humans). Most T cells have αβTCRs, while a few T cells carry γδTCRs. The T cell receptor α and β polypeptides (as well as similar γ and δ polypeptides) are linked to each other by disulfide bonds. Each of the two polypeptides that make up the TCR contains an extracellular domain that contains a constant region and a variable region, a transmembrane domain, and a cytoplasmic tail region (the transmembrane domain and cytoplasmic tail region are also part of the constant region).

[0228] The variable region of each TCR contains a unique characteristic structure, the idiotype or idiotype, which determines the specificity of the TCR. Generally, a TCR will bind to a pMHC complex only if it contains an idiotype that recognizes a peptide presented in the context of MHC (e.g., a unique conformation of a particular pMHC complex).

[0229] Immunotherapeutic approaches to treat disease act to modulate T cell activity in vivo, e.g., to enhance anti-infective and anti-tumor responses, or, e.g., to downregulate autoimmunity and transplant rejection. However, this approach can lack specificity, as immunotherapies can target signaling of the TCR complex by binding to CD3 and / or co-stimulatory molecule pairing. This approach can result in adverse side effects, e.g., an overactive immune response or systemic immunosuppression. Therefore, therapies that exploit the unique, specific interactions between the TCR and pMHC complexes could offer the ability to specifically modulate the activity of specific T cells in vivo and provide therapies based on T cell modulation.

[0230] MHC molecules present virus-derived peptides on the surface of infected cells, and recognition of these pMHC complexes by CD8+ cytotoxic T cells, followed by activation, provides an important mechanism for immune-based viral protection. Cells infected with HBV, including those that develop into cancer cells, can express a variety of HBV-related antigens. Peptides derived from these antigens may form complexes with MHC molecules and be displayed on the cell surface. HBV-derived peptides presented by MHC are detected by T cells carrying the corresponding TCR, resulting in targeted killing of infected cells. However, due to the selection process that occurs during T cell maturation in the thymus, T cells that recognize HBV-derived peptides with sufficiently high affinity are often lacking in the circulating pool. Therefore, infected cells often escape elimination by the immune system.

[0231] Identification of HBV-derived peptides presented on infected cells (e.g., HBV-induced cancer cells) can allow the development of immunotherapeutic agents designed to specifically target and destroy HBV-infected cells (e.g., HBV-induced cancer cells). Such agents can be moieties that bind to HBV-derived peptides and / or pMHC complexes, or agents that, for example, can act by inducing a T cell response. For example, such agents may be based on antibodies, TCRs, and / or CARs.

[0232] The present disclosure is based in part on a proteomic approach that detects MHC-associated HBV peptides from HBV-infected cells. The HLA-I-HBV peptide repertoire expressed in the liver of infected patients characterized herein can provide an accurate representation of HBV epitopes in the population.

[0233] HLA-restricted viral peptides can be used as potential targets to deliver immunotherapeutic drugs (such as antibodies (e.g., bispecific antibodies), engineered TCR- or CAR-based cell therapies) to infected tissues. However, genomic variability between viral (such as HBV) strains, as well as differences in patient HLA alleles, can be a challenge to developing therapies for these peptide targets. To address this challenge, the present disclosure provides, in one aspect, a protein genomics method for generating a patient-specific database that allows comprehensive identification of viral peptides, such as HBV-derived peptides, based on viral transcriptomes sequenced from individual patient samples. The HBV-HLA-associated peptides disclosed herein can be used to develop immunotherapeutic drugs (such as antibodies (e.g., bispecific antibodies), engineered TCR- or CAR-based cell therapies) for the treatment of HBV-related diseases or conditions, including hepatocellular carcinoma (HCC), and to improve clinical monitoring to monitor patient-specific HBV diversity and provide information for vaccine development. The protein genomics discovery platform described herein provides a method for identifying virus-derived peptides as antiviral-related immunotherapy targets.

[0234] definition

[0235] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0236] 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 which will become apparent to those skilled in the art upon reading this disclosure.

[0237] The term "about" or "approximately" includes within a statistically significant range of values. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable deviations encompassed by the term "about" or "approximately" depend on the specific system under study and can be readily understood by one of ordinary skill in the art.

[0238] The term "antigen" encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, part thereof or combination thereof), which, when introduced into an immunocompetent host (directly introduced, or expressed in, for example, a DNA or RNA vaccine), is recognized by the host's immune system and is capable of inducing an immune response in the host. The T cell receptor (TCR) recognizes peptides presented in the context of a major histocompatibility complex (MHC) (as part of an immune synapse). The peptide-MHC (pMHC) complex can be recognized by the TCR, and the peptide (antigenic determinant) and the TCR idiotype provide the specificity of the interaction. Therefore, the term "antigen" encompasses peptides presented in the context of MHC, for example, pMHC complexes. Peptides displayed on MHC may also be referred to as "epitopes" or "antigenic determinants". The terms "peptide", "antigenic determinant", "epitope", etc. not only encompass peptides naturally presented by antigen presenting cells (APCs), but may also be any desired peptide, as long as it is recognized by immune cells, for example, 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.

[0239] A single antigen (such as an antigenic polypeptide) may have more than one epitope. An epitope may be defined as a structural epitope or a functional epitope. Functional epitopes are typically a subset of structural epitopes and are defined as those residues that directly contribute to the affinity of the interaction between the MHC molecule and the antigen. An epitope may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, an epitope may include determinants, which are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics. Epitopes formed by continuous amino acids are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents.

[0240] The terms "major histocompatibility complex" and "MHC" encompass the terms "human leukocyte antigen" or "HLA" (the latter two are generally reserved for human MHC molecules), naturally occurring MHC molecules (e.g., MHC class I molecules comprising an MHC class I α (heavy) chain and a β2 microglobulin; MHC class II molecules comprising an MHC class II α chain and an 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 such chains of MHC molecules (e.g., α1, α2 and / or α3 subunits of MHC class I α chain, α1-α2 subunits of MHC class II α chain, β1-β2 subunits of MHC class II β chain), as well as portions thereof (e.g., peptide binding portions, e.g., peptide binding groove), mutants and various derivatives thereof (including fusion proteins), wherein such portions, mutants and derivatives retain the ability to display antigenic peptides for recognition by a TCR (e.g., an antigen-specific TCR). MHC class I molecules contain a peptide binding groove formed by the α1 and α2 domains of the heavy α chain, which can accommodate peptides of about 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of about 9 amino acids (e.g., 5 to 17 amino acids) in the peptide, the openness of the MHC class II peptide binding groove (the α1 domain of the class II MHC α polypeptide associates with the β1 domain of the class II MHC β polypeptide) allows a wider range of peptide lengths. The length of the peptide bound to MHC class II usually varies between 13 and 17 amino acids, but shorter or longer lengths are not uncommon. Therefore, the peptide can move within the MHC class II peptide binding groove, thereby changing the 9-mer directly located in the groove at any given time. In some embodiments, the peptide-MHC complex described herein can be a peptide-MHC complex from a non-human animal. In other embodiments, the peptide-MHC complex described herein can include a peptide-HLA complex, i.e., a peptide-MHC complex from a human. Conventional identification of specific MHC variants is used herein. For example, HLA-A11 refers to the human leukocyte antigen from the gene position (called locus) number 11 of the A genome (thus MHC class I); gene HLA-DR11 refers to the human leukocyte antigen encoded by the gene in the DR region (thus MHC class II) locus number 11.

[0241] "MHC-peptide complex", "peptide-MHC complex", "pMHC complex", "peptide in groove" and the like include: (i) MHC molecules, e.g., MHC molecules of humans and / or non-human animals, or parts thereof (e.g., peptide binding grooves thereof, and, e.g., extracellular parts thereof), and (ii) antigenic peptides (e.g., HBV-derived peptides), wherein the MHC molecules and antigenic peptides are complexed in such a way that the pMHC complex can specifically bind to a T cell receptor. The pMHC complex encompasses pMHC complexes expressed on the cell surface and soluble pMHC complexes.

[0242] "HLA-peptide complex," "peptide-HLA complex," "pHLA complex," and the like refer to an MHC-peptide complex wherein the MHC molecule is a human leukocyte antigen (HLA) molecule.

[0243] The term "T cell" or "T lymphocyte" is used in the broadest sense herein to refer to all types of immune cells 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, T-regulatory cells (Treg) and NK-T cells. T cells may 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 "γ-δ T cells (γδ T cells)", which refers to a specialized population, i.e., a small portion of T cells with a unique TCR on their surface, which is different from most T cells (wherein the TCR is composed of two glycoprotein chains, referred to as α chain and β-TCR chain), and the TCR in γδ T cells is composed of a γ chain and a δ chain.

[0244] The term "antigen presenting cell" or "APC" refers to any cell that presents an antigen associated with a major histocompatibility complex molecule (MHC class I or MHC class II molecule, or both) on the cell surface.

[0245] The terms "antibody", "antibodies", "immunoglobulin" and the like refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partially or completely synthetic. These terms include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies for antigen-specific TCRs), as well as epitope-binding fragments of any of the above. The terms "antibody" and "antibodies" also refer to covalent bivalent antibodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, the entire contents of which are incorporated herein by reference, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910, the entire contents of which are incorporated herein by reference. Antibodies useful in the present disclosure include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen binding site. 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.

[0246] The terms "specific binding", "binding in a specific manner", "antigen specificity", etc., indicate that the molecules involved in the specific binding can form relatively stable complexes with each other under physiological conditions, but cannot non-specifically form stable complexes with other molecules outside the specified binding pair. Therefore, a peptide binding portion (e.g., an antibody, a surrogate backbone, a CAR or a TCR) that binds to an HBV-derived peptide or a peptide-based molecule (such as a complex (e.g., a pMHC complex), a fusion protein or a conjugate) in a specific manner indicates that the peptide binding portion forms a stable intermolecular non-covalent bond with the HBV-derived peptide or a peptide-based molecule (such as a complex (e.g., a pMHC complex), a fusion protein or a conjugate) containing the peptide. Specific binding can be measured by an equilibrium dissociation constant (K) in the low micromolar to picomolar range. D ) to characterize (i.e., smaller K D =K indicates tighter binding). High specificity can be in the low nanomolar range and very high specificity can be in the picomolar range. For example, a peptide binding moiety can exhibit binding to an HBV-derived peptide or a peptide-based molecule (such as a complex (e.g., a pMHC complex), a fusion protein, or a conjugate comprising the peptide, K DAbout 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 well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0247] The terms "protein" and "polypeptide" are used interchangeably herein to encompass a variety of naturally occurring proteins and synthetic proteins, including protein fragments of all lengths, fusion proteins, and modified proteins, including but not limited to glycoproteins, and all other types of modified proteins (e.g., proteins produced by 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".

[0248] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and include polymeric forms of nucleotides of any length, including ribonucleotides (RNA), deoxyribonucleotides (DNA), or analogs or modified versions thereof. They include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, complementary DNA (cDNA), DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0249] The term "operably linked" or similar terms refers to a juxtaposition in which the components described are in such a relationship that they are allowed to function in the intended manner. For example, a control sequence "operably linked" to a coding sequence is linked in a manner that achieves expression of the coding sequence under conditions compatible with the control sequence. "Operably linked" sequences include both expression control sequences adjacent to the target gene and expression control sequences that act in trans or remotely to control the target gene (or target sequence). The term "expression control sequence" includes polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are linked. "Expression control sequences" include: appropriate transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance polypeptide stability; and sequences that enhance polypeptide secretion when desired. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0250] The term "isolated" refers to a population of molecules (such as polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system (such as a recombinant cell) that produces the molecules, as well as proteins that have undergone at least one purification or separation step. "Isolated" refers to a molecule that is substantially free of other cellular materials and / or chemicals, and encompasses molecules that have been isolated to a higher degree of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0251] The term "derivative" as used herein refers to a peptide, polypeptide or polynucleotide, or a variant or analog thereof, comprising one or more mutations and / or chemical modifications compared to a reference peptide, polypeptide or polynucleotide. Mutations and / or chemical modifications are described in further detail below, for example, and may include insertions, substitutions, deletions, transversions and / or inversions at one or more positions in an amino acid or nucleotide sequence.

[0252] The term "treating" a state, disorder, disease or condition or "treatment" of a state, disorder, disease or condition includes: (1) preventing, delaying or reducing the incidence and / or likelihood of at least one clinical or subclinical symptom of the state, disorder, disease or condition occurring in a subject who may have or is susceptible to the state, disorder, disease or condition but does not yet experience or show clinical or subclinical symptoms of the state, disorder, disease or condition; or (2) inhibiting the state, disorder, disease or condition, i.e., arresting, reducing or delaying the development of the disease or at least one clinical or subclinical symptom thereof or its recurrence; or (3) alleviating the state, disorder, disease or condition, i.e., causing regression of the state, disorder, disease or condition, or causing regression of at least one clinical or subclinical symptom of the state, disorder, disease or condition. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0253] "Individual" or "subject" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of disease (e.g., mice, rats). In preferred embodiments, the subject is a human.

[0254] The term "effective" as applied to dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered alone. The exact amount required will vary from subject to subject and will depend on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs used, the mode of administration, etc.

[0255] The phrase "pharmaceutically acceptable" used in conjunction with the compositions described herein refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and generally do not produce adverse effects when administered to mammals (e.g., humans). 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 humans.

[0256] The term "administering" and the like refers to and includes administering a composition to a subject or system (e.g., to a cell, organ, tissue, organism, or a related component or a group of components thereof). A skilled artisan will appreciate that the route of administration may vary, for example, depending on the subject or system to which the composition is administered, the nature of the composition, the purpose of administration, and the like. For example, in certain embodiments, administration to an animal subject (e.g., to a human or rodent) may be bronchial (including by bronchial instillation), buccal, enteral, intercutaneous, intraarterial, intracutaneous, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and / or vitreous administration. In some embodiments, administration may involve intermittent administration. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time.

[0257] In light of the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques can be employed within the skill of the art. These techniques are fully explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Vols. 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 [I. R. L. Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989); Cloning (1984); Ausubel, FM et al. (Editors). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994.These techniques include site-directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Pat. 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. 26:680-681 (1996). 2 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14. 3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270 and 6,242,222, Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson, Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996), Ogel and McPherson, 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.

[0258] Peptides disclosed herein

[0259] In one aspect, the present disclosure provides an isolated peptide comprising an amino acid sequence derived from hepatitis B virus (HBV).

[0260] The HBV genome exhibits genetic variability, estimated at 1.4–3.2 × 10 per locus per year. -5 The rate of nucleotide substitutions. A large number of viral variants arise due to nucleotide misincorporation by the viral polymerase in the absence of any proofreading ability. This variability gives rise to subtypes of the virus. HBV is divided into multiple genotypes based on at least 8% intergroup differences in the complete genome sequence, each with a clear geographical distribution. For example, genotype A is common in sub-Saharan Africa, northern Europe, and West Africa; genotypes B and C are widespread in Asia; genotype C is mainly found in Southeast Asia; genotype D is distributed in Africa, Europe, Mediterranean countries, and India; genotype G is observed in France, Germany, and the United States; and 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.

[0261] HBV is an enveloped DNA virus and a member of the Hepadnaviridae family. HBV contains a small, partially double-stranded (DS), relaxed circular DNA (rcDNA) genome that replicates via reverse transcription of pregenomic RNA (pgRNA), an RNA intermediate. The circular DNA genome of HBV is considered atypical because the DNA is not completely double-stranded. One end of the full-length strand is linked to the viral DNA polymerase. The full-length genome is approximately 3020-3320 nucleotides long, while the short-length genome is 1700-2800 nucleotides long. The negative sense (i.e., non-coding) strand is complementary to the viral mRNA.

[0262] The HBV genome encodes four genes: genes C, X, P, and S. The core protein is encoded by gene C (HBcAg), whose start codon is preceded by an upstream in-frame AUG start codon, from which the pre-core protein is produced. HBeAg is produced by proteolytic processing of the pre-core protein. DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg (hepatitis B core antigen)). The HBsAg gene is a long open reading frame containing three in-frame start codons (ATG), which subdivide the gene into three components: pre-S1, pre-S2, and S. Due to the multiple start codons, three different sized proteins are produced, referred to as large (pre-S1 / pre-S2 / S), medium (pre-S2 / 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.

[0263] Viral DNA is localized in the nucleus after cell infection. The partially double-stranded DNA is made completely double-stranded by completing the positive strand and removing the protein molecule from the negative-sense strand and a small piece of RNA from the positive strand. The non-coding bases are removed from the end of the negative-sense strand and the ends are reconnected.

[0264] The HBV life cycle begins when the virus attaches to a host cell and is then internalized. Sodium taurocholate cotransporting polypeptide (NTCP) may be a functional receptor in HBV infection. Virion relaxed circular DNA (rcDNA) is transported to the nucleus, where the virion rcDNA is repaired to form covalently closed circular DNA (cccDNA). Free cccDNA serves as a template for host RNA polymerase II to transcribe pregenomic RNA (pgRNA) as well as other viral mRNAs. The transcript is then exported to the cytoplasm for subsequent translation of viral proteins. Reverse transcriptase (RT) binds to pgRNA and activates core proteins to assemble into immature nucleocapsids containing RNA. When pgRNA is reverse transcribed by RT to produce mature rcDNA, the immature nucleocapsid matures. A notable feature of hepadnavirus reverse transcription is the initiation of negative-strand DNA synthesis initiated by RT, resulting in covalent bonding of RT to the 5' end of the negative-strand DNA.

[0265] The mature rcDNA-containing nucleocapsids are then enveloped by viral surface proteins and secreted as virions via the secretory pathway or recycled back to the nucleus for further amplification of the cccDNA pool via the recycling pathway.

[0266] The amino acid sequence derived from HBV disclosed herein may include naturally occurring protein amino acids as well as non-protein amino acids and non-naturally occurring amino acids, such as amino acid analogs. In some embodiments, the amino acids that can be used in the practice of the present disclosure may include, for example, but are not limited to: naturally occurring protein (L)-amino acids, their optically active (D)-isomers; chemically modified amino acids, including, for example, amino acid analogs, such as selenocysteine ​​(Sec), penicillamine (3-mercapto-D-valine), pyroglutamic acid (5-oxoproline), etc.; naturally occurring non-protein amino acids, such as norleucine; and chemically synthesized amino acids, which have properties known in the art as amino acid characteristics; and amino acid equivalents.

[0267] In some embodiments, the present disclosure provides an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, comprising 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 the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptide of the present disclosure comprises the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptide of the present disclosure consists essentially of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the isolated peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112. In some embodiments, an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof is provided, wherein the isolated peptide comprises two or more sequences selected from any one of SEQ ID NOs: 1-54 and 110-112.

[0268] A non-limiting exemplary list of HBV-derived peptides is provided in Table 1 below.

[0269] Table 1. Examples of HBV-derived peptides

[0270]

[0271]

[0272]

[0273] In some embodiments, the present disclosure provides an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, the isolated peptide comprising the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), wherein X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H or L. In some embodiments, the present disclosure provides an isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, the isolated peptide consisting of the amino acid sequence GX1LPQX2HIX3X4K (SEQ ID NO: 107), wherein X1 is S or T, X2 is E or D, X3 is V or I, and X4 is Q, H or L.

[0274] The peptides of the present disclosure may be produced synthetically or by hydrolysis. Synthetically produced peptides may include randomly generated peptides, specially designed peptides, and peptides in which at least some amino acid positions are conserved and the remaining positions are random between several peptides. Alternatively, the peptides of the present disclosure may be produced by expression in a heterologous host cell.

[0275] In nature, peptides produced by hydrolysis undergo hydrolysis before antigens bind to MHC molecules. Class I MHCs typically present peptides derived from proteins that are actively synthesized in the cytoplasm. In contrast, class II MHCs typically present peptides derived from exogenous proteins that enter the cell's endocytic pathway or from proteins synthesized in the endoplasmic reticulum (ER). Intracellular trafficking allows peptides to associate with MHC molecules.

[0276] The binding of peptides to the MHC peptide binding groove can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR. This spatial control is due in part to hydrogen bonds formed between the peptide and the MHC molecule. Based on the knowledge of how peptides bind to various MHC molecules, it is possible to identify the major MHC anchor amino acids and surface exposed amino acids that vary between different peptides.

[0277] Preferably, the length of the MHC binding peptide is about 5 to about 40 amino acid residues, more preferably about 6 to about 30 amino acid residues, even more preferably about 8 to about 20 amino acid residues, even more preferably between about 9 and 11 amino acid residues, including peptides of any size between 5 and 40 amino acids in length, in whole number increments (i.e., 5, 6, 7, 8, 9 ... 40). Although natural MHC class II binding peptides vary from about 9 to 40 amino acids, in almost all cases, the peptide can be truncated to a core of about 9-11 amino acids without losing MHC binding activity or T cell recognition.

[0278] In some embodiments, the length of the isolated peptides of the present disclosure can be about 8-12 amino acids. For example, the peptides disclosed herein can be 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids in length.

[0279] The peptides of the present disclosure may contain one or more reversed peptide bonds, one or more non-peptide bonds, one or more chemical modifications, one or more D-isomer amino acids, or any combination thereof.

[0280] In some embodiments, the peptide can be modified to include one or more reverse peptide bonds or non-peptide bonds. This modification can improve the stability and / or binding of the peptide to the MHC molecule, thereby inducing a stronger immune response. In a reverse peptide bond, the amino acid residues are not connected by a peptide (—CO—NH—) bond, but the peptide bond is reversed. This reverse-flip peptide mimetic can be prepared using methods known in the art, such as those described in Meziere et al., 1997 (Meziere C. et al., J Immunol 1997). This method involves making pseudopeptides that include changes involving the skeleton (rather than the direction of the side chain). For example, such pseudopeptides can be useful for the desired MHC binding and / or T helper cell response. Reverse-flip peptides contain NH-CO bonds instead of CO-NH peptide bonds, and are much more resistant to proteolysis. Other non-peptide bonds that can be used are, for example, —CH2—NH, —CH2S—, —CH2CH2—, —CH═CH—, —COCH2—, —CH(OH)CH2—, and —CH2SO—.

[0281] 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, pyridoxalation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups and sulfhydryl modification by performing oxidation of cysteine ​​to cysteic acid, formation of mercuric derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation using iodoacetic acid or iodoacetamide, carbamoylation using cyanate at alkaline pH. Chemical modifications may not correspond to chemical modifications that may be present in vivo.

[0282] For example, modification of arginine residues in proteins can be based on the reaction of ortho-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. Disulfide bonds in proteins can also be selectively reduced. Disulfide bonds are formed and oxidized during thermal treatment of biopharmaceuticals. Woodward reagent K can be used to modify specific glutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine residues and glutamic acid residues. For example, diethyl pyrocarbonate and 4-hydroxy-2-nonenal can be used to modify histidine residues in proteins. The reaction of lysine residues and other α-amino groups can be used, for example, for binding of peptides to surfaces or for protein / peptide crosslinking. Lysine is the attachment site for polyethylene glycol and is also the main modification site for protein glycosylation. Methionine residues in proteins can be modified with, for example, iodoacetamide, bromoethylamine and chloramine T. Tetranitromethane and N-acetylimidazole can be used for modification of tyrosyl residues. Cross-linking through the formation of dityrosine can be accomplished with hydrogen peroxide / copper ions. N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole) have been used in recent studies for modification of tryptophan. Successful modification of therapeutic proteins and peptides with PEG has resulted in an increase in circulation half-life, while cross-linking of proteins / peptides with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde can be used to prepare hydrogels. Chemical modification of allergens in immunotherapy can be achieved by carbamylation with potassium cyanate.

[0283] The peptides of the present disclosure may also be synthesized with other chemical groups present at their N-terminus and / or C-terminus to enhance the stability, bioavailability and / or affinity of the peptide.

[0284] N-terminal modifications can include methylation (e.g., —NHCH3 or —N(CH3)2), acetylation (e.g., with 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 the amino terminus with any blocking group containing a carboxylate functional group defined by RCOO— or a sulfonyl functional group defined by R—SO2, wherein R is selected from alkyl, aryl, heteroaryl, alkylaryl, etc., and the like. Desamino acids can also be incorporated at the N-terminus (so that there is no N-terminal amino group) to reduce susceptibility to proteases or to restrict the conformation of the peptide. In addition, hydrophobic groups, such as benzyloxycarbonyl, dansyl or tert-butoxycarbonyl, can be added to the N-terminus. Similarly, an acetyl group or 9-fluorenylmethoxy-carbonyl group can be placed at the N-terminus.

[0285] The C-terminal modification can include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxyl terminal to introduce structural constraints. It is also possible to cyclize the peptide of the present disclosure, or to incorporate a deamination or decarboxylation residue at the end of the peptide so that there is no terminal amino or carboxyl group to reduce the susceptibility to proteases or to limit the conformation of the peptide. The C-terminal functional group of the compound of the present disclosure includes amide, amide lower alkyl, amide di (lower alkyl), lower alkoxy, hydroxyl and carboxyl, and their lower ester derivatives, and pharmaceutically acceptable salts thereof. In addition, a hydrophobic group, tert-butoxycarbonyl or amide group can be added to the C-terminal.

[0286] Other examples of non-natural modifications include non-encoded α-amino acids, photoreactive cross-linking of amino acids, incorporation of N-methylated and β-amino acids, backbone reduction, retro-inversion using D-amino acids, and C-terminal amidation and PEGylation.

[0287] The peptides described herein can include one or more (e.g., 1, 2, 3 or 4) amino acid replacements and / or insertions and / or deletions. Amino acid replacement refers to replacing an alternative amino acid residue with an amino acid residue at the same position. The inserted amino acid residue can be inserted into any position, and can be inserted so that part or all of the inserted amino acid residues are adjacent to each other, or can be inserted so that the inserted amino acid residues are not adjacent to another inserted amino acid residue. One or more (e.g., 1, 2, 3 or 4) amino acids can be replaced and / or inserted and / or deleted from the sequence of any one of SEQ ID NO: 1-54 and 110-112. Each replacement and / or insertion and / or deletion can occur at any position of any one of SEQ ID NO: 1-54 and 110-112.

[0288] In some embodiments, the peptides of the present disclosure may include additional amino acids (e.g., 1, 2, 3, or 4) located at the C-terminus and / or N-terminus of the sequence of any one of SEQ ID NOs: 1-54 and 110-112. The peptides of the present disclosure may include the amino acid sequence of any one 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.

[0289] The inserted amino acids and the replaced amino acids can be naturally occurring amino acids or non-naturally occurring amino acids, for example, can contain non-natural side chains and / or be linked together by non-natural peptide bonds. Such altered peptide ligands are further discussed in Douat-Casassus et al., J. Med. Chem, 2007; 50(7): 1598-609 and Hoppes et al., J. Immunol 2014; 193(10): 4803-13 and references therein. If more than one amino acid residue is replaced and / or inserted, the replaced / inserted amino acid residues can be identical to or different from each other. Each replacement amino acid can have a different side chain from the replaced amino acid.

[0290] D-amino acid can replace the L-amino acid in the antigenic peptide of the present disclosure. In addition, non-standard amino acids (i.e., except common naturally occurring protein amino acids, such as β-γ-δ-amino acids, and many derivatives of L-α-amino acids) can also be used for replacement or addition to produce peptides of the present disclosure.

[0291] Amino acid substitutions can be conservative, meaning that the substituted amino acid has similar chemical properties to the original amino acid. For example, the following groups of amino acids have similar chemical properties, such as size, charge, and polarity: Group 1 - Ala, Ser, Thr, Pro, Gly; Group 2 - Asp, Asn, Glu, Gln; Group 3 - His, Arg, Lys; Group 4 - Met, Leu, Ile, Val, Cys; Group 5 - Phe, Thy, Trp.

[0292] Substantial changes in function (e.g., affinity for MHC molecules and / or TCRs) can be accomplished by selecting substitutions that are less conservative than those described above, in other words, selecting residues that differ more significantly in their effect on maintaining the structure of the peptide backbone in the region of the substitution (e.g., as a sheet or helical conformation), the bulk of the side chains, or the charge or hydrophobicity of the peptide at positions involved in MHC or TCR binding. Substitutions that would generally be expected to produce the greatest changes in peptide properties are: (a) substitutions of (or replacements for) hydrophobic residues (e.g., Leu, Ile, Phe, Val, or Ala) for hydrophilic residues (e.g., Ser); (b) substitutions of (or replacements for) negatively charged residues (e.g., Glu or Asp) for residues with positively charged side chains (e.g., Lys, Arg, or His); or (c) substitutions of (or replacements for) residues without side chains (e.g., Gly) for residues with bulky side chains (e.g., Phe).

[0293] Other side chains may also be used to replace the natural side chains of the 20 genetically encoded amino acids (or stereoisomeric D-amino acids), for example, with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6- to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxyl, carboxyl and lower ester derivatives thereof, and with 4-, 5-, 6- to 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 may be used. The cyclic group may be saturated or unsaturated, and if unsaturated, 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, furanyl, imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g., morpholino), oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidinyl (e.g., 1-piperidinyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrole, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g., thiomorpholino) and triazolyl. These heterocyclic groups may be substituted or unsubstituted. When a group is substituted, the substituent may be alkyl, alkoxy, halogen, oxygen or substituted or unsubstituted phenyl.

[0294] Other examples of amino acid substitutions include stereoisomers (e.g., D-amino acids) and unnatural amino acids, such as, for example, L-ornithine, L-homocysteine, L-homoserine, L-citrulline, 3-sulfo-L-alanine, N-(L-arginine)succinate, 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, 3,5-diiodo-L-tyrosine, triiodothyronine, L-thyroxine, L-selenocysteine, N-(L-arginine)taurine, 4-aminobutyrate, (R,S)-3-amino-2-methylpropionate, a,a-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.

[0295] Amino acid residues that do not contribute substantially to the interaction with the T cell receptor can be modified by substitution with other amino acids whose incorporation does not substantially affect T cell responsiveness or abolish binding to the relevant MHC.

[0296] Peptides may also contain isosteres of two or more residues. As used herein, "isostere" refers to a sequence of two or more residues that can be replaced by a second sequence because the spatial conformation of the first sequence cooperates with a binding site specific for the second sequence. The term specifically includes peptide backbone modifications well known to those skilled in the art. Such modifications include: modifications of amide nitrogen, α-carbon, amide carbonyl, complete replacement of amide bonds, extensions, deletions or backbone crosslinking.

[0297] Combinations of several substitutions / additions / deletions at more than one position can be developed and tested to determine whether the combination produces an additive or synergistic effect on the immunogenicity of the peptide. In some embodiments, no more than 4 positions within the peptide are altered simultaneously.

[0298] Preferably, the peptides disclosed herein bind to the MHC molecules in the peptide binding groove of the MHC molecules. Generally, the above-mentioned amino acid modifications do not impair the ability of the peptide to bind to the MHC molecules. In some embodiments, the amino acid modifications improve the ability of the peptide to bind to the MHC molecules. For example, mutations can occur at the position where the peptide is anchored to the MHC molecule. For peptides that bind to HLA-A*02 in particular, these anchoring positions and preferred residues at these positions can include, for example, amino acid residues at position 2 and / or the C-terminus of the peptide, which can be considered as the main anchoring positions. For each HLA type, the preferred anchoring residues can be different. As a non-limiting example, for HLA-A*02, the preferred amino acid at position 2 is Leu, lie, Val or Met, and at the C-terminus is Val or Leu. For the stable binding of the peptide to HLA-A*02, multiple positions may be important, including positions 2, 3, 5-7 and 9. The anchoring residues at positions 2 and 9 may be crucial for the binding of the peptide to HLA-A2. However, other peptide side chains (e.g., at position 3) may contribute to the stability of the interaction. In some cases, the optimal length for peptide binding may be greater than 9 residues.

[0299] The immunological properties of peptides can be described by their association with MHC molecules (K on and K off The overall effect of modification of the primary MHC anchor residues on binding to the MHC molecule is shown to be highly predictable. Modification of the secondary MHC anchor residues can affect the affinity of the interaction of the MHC-peptide complex with the TCR, as well as the K relative to the peptide-MHC interaction. on and K off .

[0300] When the HBV peptide is a mutant peptide, a T cell line against a natural (non-mutated) epitope is generated and an immunization strategy is used that is effective enough to generate a useful response in transgenic mice carrying human MHC (such as the A2 allele). The HBV peptide is studied in vitro in the presence of competent APCs, and the functional effects on T cells specific for the natural (non-mutated) epitope are measured. The evaluation is performed at different concentrations of HBV peptides because in the case of cross-reactive peptides, the effect is expected to be biphasic (activated at a limited concentration and inhibited at a higher concentration due to antigen-induced cell death [AICD]). Measurement of the following three parameters is used to define the basic and useful properties of HBV peptides:

[0301] 1. The lowest concentration of HBV markers required to induce effects indicative of T cell activation (e.g., cytokine [e.g., IFN-γ] production);

[0302] 2. The maximum (peak) effect (e.g., cytokine [e.g., IFN-γ] production) at any HBV peptide concentration; and

[0303] 3. HBV peptide concentration at the peak of the activation effect (eg, cytokine [eg, IFN-γ] concentration).

[0304] As a non-limiting example, an HBV peptide that results in a decrease in the values ​​associated with parameter number 1 and number 3, but an increase in the value associated with parameter number 2, may be useful. The use of native epitopes and / or unrelated non-cross-reactive peptides as references is valuable for identifying classes of peptides that may be of value. Peptides that have quantitatively comparable or even modestly diminished properties to native epitopes are still considered useful because, while they retain cross-reactivity, they may exhibit immunological properties that differ from the native peptide, e.g., a reduced ability to break tolerance or re-establish reactivity in vivo or a reduced propensity to induce AICD.

[0305] In addition to its practicality and rapidity, other advantages of this screening approach include, but are not limited to, the use of more relevant polyclonal T cell lines rather than potentially biased T cell clones as readouts, and the integration of multiplexers such as K on , K off The assay is a composite of parameters such as MHC and TCR affinity that can be translated into cross-reactivity and functional affinity of peptide-MHC complexes relative to the TCR. These parameters are predictive of in vivo immunological properties and can therefore define useful groups of peptides that are amenable to further evaluation, optimization, and practical application. Peptides that bind to MHC and retain cross-reactivity against a nominally wild-type peptide-specific TCR are expected to elicit a measurable effect in this assay.

[0306] The peptides disclosed herein, or pharmaceutically acceptable salts thereof, or fragments or derivatives thereof, can be used to induce an immune response. If this is the case, it is important that the immune response is specific to the intended target (e.g., HBV) to avoid the risk of undesirable side effects that may be associated with an "off-target" immune response. Therefore, preferably, the amino acid sequence of the peptide disclosed herein does not match the amino acid sequence of a peptide from any other endogenous protein, in particular, does not match the amino acid sequence of another human protein. In addition, when presented on the surface of an antigen presenting cell (APC) in a complex with an MHC molecule, the amino acid modifications described herein should not impair the ability of the peptide to induce an antigen-specific immune response.

[0307] The peptides may also be modified to increase half-life and / or bioavailability, for example, by pegylation, glycosylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, or acylation.

[0308] The peptides disclosed herein can also be used as structural models for non-peptide compounds with similar biological activity. A variety of techniques can be used to construct compounds with the same or similar desired biological activity as the lead peptide compound, but with more favorable activity than the lead in terms of solubility, stability, and susceptibility to hydrolysis and proteolysis. These techniques include replacing the peptide backbone with a backbone composed of amidates, phosphonates, carbamates, sulfonamides, secondary amines, and N-methyl amino acids.

[0309] The various peptides described herein can be operably linked together. Therefore, in one aspect, the present disclosure provides a kind of separated peptide or polypeptide, or its derivative or its pharmaceutically acceptable salt, the separated peptide or polypeptide comprises two or more amino acid sequences selected from SEQ ID NO:1-54 and 110-112. For example, such multi-epitope peptide or polypeptide can comprise 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20 or 10 to 15 single epitope peptides (e.g., SEQ ID NO:1-54 and 110-112) as described herein. Single epitope peptides (e.g., SEQ ID NO:1-54 and 110-112) can be arranged in any order, and can be the same or different.

[0310] In some embodiments, the present disclosure provides an isolated peptide or polypeptide, or a derivative thereof or a pharmaceutically acceptable salt thereof, wherein the isolated peptide or polypeptide comprises 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.

[0311] Single epitope peptides can be connected by a linker. The linker can be composed of relatively small neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The linker can be selected from, for example, neutral spacer sequences of Table 2 or other non-polar amino acids or neutral polar amino acids. It is to be understood that the linker that is optionally present need not be composed of the same residues, and therefore can be a heterologous or homologous oligomer. When present, the linker is typically at least one or two residues, more typically three to six residues.

[0312] The peptides of the present disclosure can be synthesized by, for example, solid phase synthesis. Thus, the peptides can be immobilized, for example, on a solid support (such as magnetic beads). The peptides of the present disclosure can be synthesized by the Fmoc-polyamide mode of solid phase peptide synthesis. The 9-fluorenylmethoxycarbonyl (Fmoc) group provides temporary N-amino protection. Repeated cleavage of this highly alkali-labile protecting group is accomplished using a 20% piperidine solution in N,N-dimethylformamide. The side chain functional groups can be protected as their butyl ethers (in the case of serine, threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butoxycarbonyl derivatives (in the case of lysine and histidine), 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, 4,4′-dimethoxydiphenylmethyl is used to protect the side chain amide functional groups. The solid support is based on a polydimethyl-acrylamide polymer, which is composed of three monomers: dimethylacrylamide (backbone monomer), bisacryloylethylenediamine (crosslinker), and acryloylsarcosine methyl ester (functionalizing agent). The peptide-resin cleavable linker used is an acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives, except asparagine and glutamine, are added as their preformed symmetrical anhydride derivatives, which are added using a reverse N,N-dicyclohexyl-carbodiimide / 1-hydroxybenzotriazole-mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzenesulfonic acid, or isotin test procedures. After the synthesis is complete, the peptide is cleaved from the resin support by treatment with 95% trifluoroacetic acid containing 50% scavenger mixture, simultaneously removing the side chain protecting groups. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, with the exact choice depending on the constituent amino acids of the peptide being synthesized. In addition, peptides can be synthesized by combining solid-phase and liquid-phase methods.

[0313] The crude peptide was obtained by removal of trifluoroacetic acid by evaporation in vacuo followed by trituration with diethyl ether. Any scavenger present was removed by a simple extraction procedure and the crude peptide free of scavenger was obtained upon lyophilization of the aqueous phase.

[0314] Purification can be performed by techniques such as recrystallization, ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and reversed-phase high performance liquid chromatography, for example using acetonitrile / water gradient separation, or combinations thereof.

[0315] Peptides can be analyzed using thin layer chromatography, electrophoresis (particularly capillary electrophoresis), solid phase extraction (CSPE), reversed phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, and fast atom bombardment (FAB) mass spectrometry, as well as MALDI and ESI-Q-TOF mass spectrometry.

[0316] Alternatively, the peptides can be produced by recombinant expression in heterologous host cells. This approach generally involves expressing the polypeptide in vivo using a vector comprising a nucleic acid sequence encoding the peptide to be expressed; for example, in bacteria, yeast, insect or mammalian cells.

[0317] In further embodiments, an in vitro cell-free system may be used. The peptides may be isolated and / or may be provided in a substantially pure form. For example, they may be provided in a form that is substantially free of other peptides or proteins.

[0318] Peptide-MHC (pMHC) complexes disclosed herein

[0319] In another aspect, the disclosure provides a complex of a peptide of the 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 can be covalently linked, for example, via a linker.

[0320] MHC molecules are generally divided into two categories: class I and class II MHC molecules. MHC class I molecules are integral membrane proteins comprising glycoprotein heavy chains, also referred to herein as α chains, which have three extracellular domains (i.e., α1, α2, and α3) and two intracellular domains (i.e., transmembrane domain (TM) and cytoplasmic domain (CYT)). The heavy chain is non-covalently associated with a soluble subunit called β2 microglobulin (β2m or β2M). MHC class II molecules or MHC class II proteins are heterodimeric integral membrane proteins comprising an α chain and a β chain that are non-covalently bonded. The α chain has two extracellular domains (α1 and α2) and two intracellular domains (TM domain and CYT domain). The β chain comprises two extracellular domains (β1 and β2) and two intracellular domains (TM domain and CYT domain).

[0321] The domains of class I and class II MHC molecules are organized to form an antigenic determinant binding site, e.g., the peptide-binding portion or peptide-binding groove of an MHC molecule. The peptide-binding groove refers to the portion of an MHC molecule that forms a cavity into which a peptide (e.g., an antigenic determinant) can bind. The conformation of the peptide-binding groove can change upon peptide binding, thereby allowing the proper alignment of amino acid residues that are important for TCR binding to the peptide-MHC (pMHC) complex.

[0322] In some embodiments, the MHC molecule includes a fragment of an MHC chain sufficient to form a peptide binding groove. For example, the peptide binding groove of a class I protein may include a portion of the α1 and α2 domains of the heavy chain, capable of forming two β-pleated sheets and two α-helices. A portion of the β2 microglobulin chain stabilizes the MHC class I molecule. Although for most versions of the MHC class II molecule, the interaction of the α chain and the β chain can occur without a peptide, the double-chain molecule of the MHC class II is unstable before the binding groove is filled with a peptide. The peptide binding groove of a class II protein may include a portion 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 a first β-pleated sheet, and the second portion of the α1 domain forms a first α-helix. The first portion of the β1 domain forms a second β-pleated sheet, and the second portion of the β1 domain forms a second α-helix. The X-ray crystal structure of a class II protein with a peptide engaged in the protein binding groove shows that one or both ends of the engaged peptide can protrude outside the MHC protein. Thus, the ends of the α1 and β1 α helices of class II form an open cavity so that the end of a peptide that binds to the binding groove is not buried in the cavity. Furthermore, the X-ray crystal structure of class II proteins shows that the N terminus of the MHC β chain apparently protrudes from the side of the MHC protein in an unstructured manner, as the first four amino acid residues of the β chain could not be assigned by X-ray crystallography.

[0323] The peptides disclosed herein can bind to MHC molecules in a manner (preferably in a specific manner) such that the pMHC complex can bind to the TCR. In certain embodiments, the binding of the pMHC complex to the TCR can induce a T cell response.

[0324] Whether a given peptide will form a complex with an MHC molecule can be determined by assessing whether the MHC can be refolded in the presence of the peptide using, for example, the process described in PCT application WO2018 / 083505, the entire contents of which are incorporated herein for all purposes. If the peptide does not form a complex with the MHC, the MHC will not refold. Refolding can be confirmed using antibodies that only recognize the folded state of the MHC. Alternatively, the ability of the peptide to stabilize MHC on the surface of a transporter associated with antigen processing (TAP)-deficient cell lines (such as T2 cells) can be determined, which lack the ability of TAP-mediated cytoplasmic peptide translocation to the endoplasmic reticulum (ER) to load the peptide onto MHC class I molecules, or other biophysical methods for determining interaction parameters.

[0325] Peptides according to the present disclosure can be provided as MHC groove binding peptides. In some embodiments, MHC groove binding peptides can be designed so that the peptide can be varied 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 compiled from published literature and existing databases. The latest version of the database has 25,860 entries, including 20,717 MHC binders and 4,022 MHC non-binders, for more than 450 MHCs. The database has sequence and structural data for (a) peptide-derived proteins and (b) MHCs. MHCBN has many web tools, including: (i) peptide mapping on query sequences; (ii) searching on any field; (iii) creating datasets; and (iv) online data submission.

[0326] In some cases, the peptide binding tool used to predict binding to MHC-I or MHC-II can be, for example, Antibody Epitope Prediction, ANTIGENIC, BepiPred, CTLPred, DiscoTope, EPIPREDICT, Epitope Cluster Analysis, Epitope ConservancyAnalysis, EUiPro, HLA Peptide Binding Predictions, HLABinding, MAPPP, MHCBench, MHC-Iprocessing predictions, Mosaic Vaccine Tool Suite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, Prediction of AntigenicDeterminants, ProPred, ProPred-1, RankPep, SMM, SVMHC, TAPPred, VaxiJen, or a combination thereof. Other example programs are used, such as BIMAS, SYFPEITHI, or Rankpep.

[0327] In a specific embodiment, the library is genetically engineered using polymerase chain reaction (PCR) or any other suitable technique to construct DNA fragments encoding the peptides, thereby generating a library of altered peptides. By using oligonucleotides randomly mutated within specific triplet codons through PCR technology, the resulting fragment library encodes all possible codon combinations at these positions. Preferably, certain amino acid positions remain constant, which are conserved amino acids required for binding to the MHC peptide binding groove and do not contact the T cell receptor (TCR).

[0328] In some embodiments, when a DNA fragment encoding a peptide is constructed by genetically engineering a library using polymerase chain reaction (PCR) or any other suitable technique, the target TCR is a TCR for which a peptide epitope recognized by a receptor needs to be identified. In some embodiments, the target TCR is from a patient with HBV infection and / or HBV-induced disease or condition (e.g., hepatocellular carcinoma (HCC)). In some embodiments, the TCR includes an alpha chain and a beta chain.

[0329] The 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 MHC chains (e.g., α1, α2, and / or α3 subunits of MHC class I α chain, α1 and / or α2 subunits of MHC class II α chain, β1 and / or β2 subunits of MHC class II β chain), as well as fragments, mutants, and various derivatives thereof (including fusion proteins, e.g., fusions with viral envelope proteins or fusion progenitors), wherein such fragments, mutants, and derivatives retain the ability to display antigenic determinants (to be recognized by antigen-specific TCRs). In a specific embodiment, the MHC comprises a transmembrane domain embedded in a liposome lipid envelope, a recombinant viral particle, or a virus-like particle (VLP).

[0330] Naturally occurring MHC molecules are encoded by gene clusters on chromosome 6 in humans or chromosome 17 in mice. MHC is also known as 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), while MHC class II molecules specifically bind to CD4 molecules expressed on helper T lymphocytes (CD4+T cells). MHC includes, but is not limited to, HLA specificity, 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, the 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.

[0331] In some embodiments, the MHC molecule in the pMHC complex of the present disclosure is a human leukocyte antigen (HLA) molecule. The MHC molecule can 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 class II polypeptide can 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.

[0332] 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, HLA-C*07. There are also allelic variants of the above HLA types, all of which are encompassed by the present disclosure. In some embodiments, the MHC molecule can be HLA-A*02 or HLA-A*11.

[0333] The MHC molecules used herein can also be from any other mammalian or avian species, for example, non-human primates, rodents (eg, mice), rabbits, horses, cows, canines, felines, pigs, etc.

[0334] Naturally occurring MHC class I molecules bind peptides derived from proteolytic degradation of proteins (especially proteins synthesized endogenously in cells). The correspondingly obtained small peptides are transported to the endoplasmic reticulum, where they bind to newly formed MHC class I molecules, and then transported via the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.

[0335] Naturally occurring MHC class I molecules consist of an α (heavy) chain associated with a β2-microglobulin. The heavy chain consists of subunits α1-α3. The α3 subunit of the β2-microglobulin and heavy chain associates. In certain embodiments, the β2-microglobulin and α3 subunits are covalently bound. In certain embodiments, the β2-microglobulin and α3 subunits are non-covalently bound. The α1 and α2 subunits of the heavy chain fold to form a groove for a peptide (e.g., an antigenic determinant), which is displayed and recognized by the TCR.

[0336] Class I molecules can bind peptides that are about 8-10 amino acids in length. All people have between 3 and 6 different class I molecules, each of which can bind many different types of peptides.

[0337] In some embodiments, the MHC contained in the pMHC complex of the present disclosure includes: (i) a class I MHC polypeptide or a fragment, mutant or derivative thereof, and optionally (ii) a β2 microglobulin polypeptide or a fragment, mutant or derivative thereof. In a specific embodiment, the class I MHC polypeptide is linked to the β2 microglobulin polypeptide via a peptide linker.

[0338] In a specific embodiment, 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.

[0339] In some embodiments, the peptide disclosed herein forms a complex with one or more MHC class I alpha heavy chains. In some embodiments, the MHC class I alpha heavy chain is completely human. In some embodiments, the MHC class I alpha heavy chain is humanized. For example, humanized MHC class I alpha heavy chains are described in U.S. Patent Publication Nos. 2013 / 0111617, 2013 / 0185819, and 2014 / 0245467. In some embodiments, the MHC class I alpha heavy chain comprises a human extracellular domain (human α1, α2, and / or α3 domain) and a cytoplasmic domain of another species. In some embodiments, the class I alpha heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence can be an HLA-A*0201 sequence. In various aspects, peptide-MHC can include all domains of MHC class I heavy chains.

[0340] 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.

[0341] In some embodiments, the MHC class I molecule comprises a β2-microglobulin (β2m or B2M) polypeptide and a mutation in the heavy chain sequence to generate a disulfide bond between B2M and the heavy chain. In some cases, the heavy chain is HLA, and the disulfide bond connects one of the following residue pairs: B2M residue 12, HLA residue 236; B2M residue 12, HLA residue 237; B2M residue 8, HLA residue 234; B2M residue 10, HLA residue 235; B2M residue 24, HLA residue 236; B2M residue 28, HLA residue 232; B2M residue 98, HLA residue 192; B2M residue 9 9, HLA residue 234; 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 of β2M residue, Arg35 of HLA residue; Trp60 of β2M residue, Gln96 of HLA residue; Trp60 of β2M residue, Asp122 of HLA residue; Tyr63 of β2M residue, Tyr27 of HLA residue; Lys6 of β2M residue, Glu232 of HLA residue; Gln8 of β2M residue, Arg234 of HLA residue; Tyr10 of β2M residue, Pro235 of HLA residue; B2M residue Ser11, HLA residue Gln242; B2M residue Asn24, HLA residue Ala236; B2M residue Ser28, HLA residue Glu232; B2M residue Asp98, HLA residue His192; and B2M residue Met99, HLA residue Arg234, first linker position Gly2, heavy chain (HLA) position Tyr84; light chain (B2M) position Arg12, HLA Ala236; and / or B2M residue Arg12, HLA residue Gly237.

[0342] In some embodiments, the antigenic determinant amino acid sequence can be a sequence of a peptide described herein that can be presented by an MHC class I molecule. In certain embodiments, the sequence can include about 8 to about 15 consecutive amino acids. In certain embodiments, the sequence can include about 8 to about 12 consecutive amino acids.

[0343] In some embodiments, at least one chain of MHC and a peptide are included in the fusion protein. In a specific embodiment, MHC and peptide are separated by a linker sequence. For example, a single-chain molecule may include an antigenic determinant, a β2-microglobulin sequence, and a class I α (heavy) chain sequence from amino to carboxyl terminus. Alternatively, a single-chain molecule may include an antigenic determinant, a class I α (heavy) chain sequence, and a β2-microglobulin sequence from amino to carboxyl terminus. The single-chain molecule may further include an amino-terminal signal peptide sequence. 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 may also include an amino-terminal signal peptide sequence, and 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 may be human, mouse, or porcine.

[0344] In some embodiments, the single-chain molecule may include a first flexible linker between the peptide ligand segment and the β2-microglobulin segment. For example, the linker may extend from the carboxyl terminus of the peptide ligand segment and connect to the amino terminus of the β2-microglobulin segment. Preferably, the linker is constructed to allow the connected peptide ligand to fold into the binding groove, thereby producing a functional MHC-antigen peptide. In some embodiments, the linker may include at least about 10 amino acids and up to about 15 amino acids. In some embodiments, the single-chain molecule may include a second flexible linker inserted between the β2-microglobulin and heavy chain segments. For example, the linker may extend from the carboxyl terminus of the β2-microglobulin segment and connect to the amino terminus of the heavy chain segment. In certain embodiments, the β2-microglobulin and heavy chain can be folded into the binding groove, thereby producing a molecule that can play a role in promoting T cell expansion.

[0345] Suitable linkers for use in MHC can be any of a variety 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 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids. Non-limiting examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 105) and (GGGS)n (SEQ ID NO: 106), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine enters an even larger phi-psi space than alanine and is much less constrained than residues with longer side chains). Exemplary linkers can comprise amino acid sequences, including but not limited to those listed in Table 2. In some embodiments, the linker peptide includes a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in the second polypeptide.

[0346] Table 2. Examples of linker sequences

[0347]

[0348]

[0349] In certain embodiments, the single-chain molecule may comprise a peptide covalently linked to an MHC class I α (heavy) chain by a disulfide bond (i.e., a disulfide bond between two cystines). In certain embodiments, the disulfide bond comprises: a first cysteine, comprising a linker extending from the carboxyl terminus of the antigenic 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 antigenic peptide). In certain embodiments, the second cysteine ​​may be a mutation (addition or substitution) in the MHC class I α (heavy) chain. In certain embodiments, the single-chain molecule may comprise a continuous polypeptide chain and a disulfide bond. In certain embodiments, the single-chain molecule may comprise two continuous polypeptide chains, which are linked by a disulfide bond as the only covalent bond. In some embodiments, in addition to the Cys residue, the linking sequence may also comprise at least one amino acid, including one or more Gly residues, one or more Ala residues, and / or one or more Ser residues.

[0350] In certain embodiments, a disulfide bond can link an antigenic peptide described herein in the class I groove of a pMHC complex if the pMHC complex comprises a first cysteine ​​in a Gly-Ser linker extending between the C-terminus of the peptide and β2-microglobulin, and a second cysteine ​​located at a proximal heavy chain position.

[0351] Connecting the peptide to the MHC class I or MHC class II molecule through a flexible linker helps ensure that the peptide occupies and remains associated with the MHC molecule during biosynthesis, transport and display. However, in some cases, the linker may interfere with the binding of the peptide to the MHC molecule or TCR recognition of the complex. As another approach, in some embodiments, the MHC molecule and the peptide are expressed separately.

[0352] In some embodiments, the β2-microglobulin sequence may include a full-length β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks a leader peptide sequence. Therefore, in certain configurations, the β2-microglobulin sequence may include about 99 amino acids and may be a mouse β2-microglobulin sequence (e.g., GenBank accession number X01838). In some other configurations, the β2-microglobulin sequence may include about 99 amino acids and may be a human β2-microglobulin sequence (e.g., GenBank accession number AF072097.1).

[0353] In some embodiments, the pMHC complex may comprise an MHC sequence disclosed in U.S. Pat. Nos. 4,478,823; 6,011,146; 8,518,697; 8,895,020; 8,992,937; WO 96 / 04314; Mottez et al. J. Exp. Med. 181:493-502, 1995; Madden et al. Cell 70:1035-1048, 1992; Matsumura et al., Science 257:927-934, 1992; Mage et al., Proc. Natl. Acad. Sci. USA 89:10658-10662, 1992; Toshitani et al., Proc. 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, all of which are incorporated by reference in their entirety.

[0354] In some embodiments, the MHC comprises a class II MHC polypeptide or a fragment, mutant or derivative thereof. In a specific embodiment, the MHC comprises an alpha and beta polypeptide of a class II MHC complex or a fragment, mutant or derivative thereof. In a specific embodiment, the alpha and beta polypeptides are connected by a peptide linker. In a specific embodiment, the MHC comprises an alpha and beta polypeptide 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 an alpha and beta polypeptide of a murine H-2A or H-2E class II MHC complex.

[0355] Naturally occurring MHC class II molecules can contain two polypeptide chains, alpha and beta. These chains can be from the DP, DQ or DR genomes. There are about 40 known different human MHC class II molecules. They all have the same basic structure, but there can be slight variations in their molecular structure. MHC class II molecules can bind peptides of 13-18 amino acids in length.

[0356] In some embodiments, the MHC class II alpha chain is completely human. In some embodiments, the MHC class II alpha chain is humanized. For example, humanized MHC class II alpha chains are described in U.S. Patent Nos. 8,847,005, 9,043,996, and 10,154,658, the entire contents of which are incorporated herein by reference. In some embodiments, humanized MHC class II alpha chain polypeptides include a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, class II alpha chains are HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA. In some embodiments, class II alpha chain polypeptides are humanized HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, and / or HLA-DRA.

[0357] In some embodiments, the peptide of the present disclosure forms a complex with one or more MHC class II β chains. In some embodiments, the MHC class II β chains are fully human. In some embodiments, the MHC class II β chain polypeptides are humanized. For example, humanized MHC class II β chain polypeptides are described in U.S. Patent Nos. 8,847,005, 9,043,996, and 10,154,658, the entire contents of which are incorporated herein by reference. In some embodiments, the humanized MHC class II β chain comprises a human extracellular domain and a cytoplasmic domain of another species. In some embodiments, the class II β chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB. In some embodiments, the class II β chain is humanized HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, and / or HLA-DRB.

[0358] The pMHC complexes disclosed herein may be in an isolated and / or substantially pure form. For example, the complexes may be provided in a form substantially free of other peptides or proteins. The MHC molecules disclosed herein may include recombinant MHC molecules, non-naturally occurring MHC molecules, and functionally equivalent fragments of MHC, including derivatives or variants thereof, provided that peptide binding is retained. For example, the MHC molecules may be fused to a therapeutic moiety, attached to a solid support, present in a soluble form, attached to a tag, biotinylated, and / or present in a multimeric form. The peptides disclosed herein may be covalently attached to the MHC.

[0359] Methods for producing soluble recombinant MHC molecules, with which the peptides disclosed herein can form complexes, include but are not limited to expression and purification from E. coli cells or insect cells. Alternatively, the MHC molecules can be synthesized or produced using a cell-free system.

[0360] The peptides disclosed herein can be presented on the cell surface with the MHC complex. Therefore, the present disclosure also provides a cell presenting the pMHC complex disclosed herein on its surface. Such cells can be mammalian cells, preferably immune system cells, or professional antigen presenting cells (APCs), such as dendritic cells or B cells. Other preferred cells include T2 cells. Cells presenting the peptides or pMHC complexes disclosed herein can be isolated, preferably in the form of a homogeneous population, or provided in a substantially pure form. Such cells may not naturally present the complexes disclosed herein, or alternatively the cells may present the complexes at a level higher than they do in nature. Such cells can be obtained by pulsing the cells with one or more peptides of the disclosure (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides), or genetically modifying the cells (by DNA or RNA transfer) to express one or more peptides (e.g., 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides). Pulsing involves the use of typically 10 -5 Up to 10 -12 The cells are incubated with the peptide for several hours at a peptide concentration of 100 M. Such cells can be additionally transduced with HLA molecules (such as HLA-A*02) to further induce presentation of the peptide. The cells can be recombinantly produced. Cells presenting the peptides of the present disclosure can be used to isolate T cells and TCRs activated by or bound to the cells.

[0361] Fusion proteins, conjugates and oligomeric complexes disclosed herein

[0362] The peptides or pMHC complexes disclosed herein can be fused or conjugated with one or more heterologous molecules. The peptides or pMHC complexes disclosed herein can also be in a multimeric form. Therefore, the present disclosure also provides fusion proteins, conjugates and oligomeric complexes comprising the peptides or pMHC complexes disclosed herein.

[0363] In some embodiments, the peptide is fused or conjugated to one or more heterologous molecules, including an MHC molecule (or fragment thereof).

[0364] Heterologous molecules suitable 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, etc. The heterologous molecule can be fused at the N-terminus and / or C-terminus of another polypeptide chain in the peptide and / or pMHC complex.

[0365] Heterologous peptides and polypeptides include, but are not limited to, epitopes (e.g., FLAG) or tag sequences (e.g., His6 (SEQ IDNO: 109), etc.), to allow detection and / or separation of fusion proteins; transmembrane receptor proteins or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof that binds to a transmembrane receptor protein; an enzyme or a portion thereof having catalytic activity; a polypeptide or peptide that promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide that increases stability, such as an immunoglobulin constant region (e.g., an Fc domain); a half-life extension sequence comprising a combination 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), which is designed to form a fusion partner that is primarily hydrophilic or primarily hydrophobic for the fusion protein; a functional or non-functional antibody (e.g., an antibody specific for dendritic cells) or its heavy or light chain; and a polypeptide having an activity (such as a therapeutic activity) different from that of the fusion protein of the present disclosure. In some embodiments, one or more heterologous molecules enhance a peptide-specific immune response in a subject. In some embodiments, the one or more heterologous molecules mediate delivery of the peptide to a specific site within the subject.

[0366] In some embodiments, the fusion protein of the present disclosure may include one or more affinity tags, for example, to allow affinity purification or conjugation to another molecule. Examples of affinity tags include, but are not limited to: His6 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.

[0367] In some embodiments, the fusion proteins of the present disclosure may comprise one or more epitopes not present in the antigen. One such example is the use of a fusion peptide in which a promiscuous helper T cell epitope is covalently linked to the peptide sequence (e.g., via a polypeptide linker or spacer). Non-limiting examples of promiscuous helper T cell epitopes include PADRE peptides, tetanus toxoid peptides (830-843), or influenza hemagglutinin HA (307-319).

[0368] The peptides or pMHC complexes of the present disclosure can be conjugated with additional moieties such as carrier molecules or adjuvants for use as vaccines. Examples of adjuvants used in vaccines include microorganisms such as Bacillus Calmette-Guerin (BCG), and / or substances produced by bacteria such as Detox B (an oil droplet emulsion of monophosphoryl lipid A and the mycobacterial cell wall skeleton). KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), the E2 core protein of the pyruvate dehydrogenase complex are examples of suitable carrier proteins used in vaccine compositions. Other 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 multi-attachment peptide (MAP), albumin, serum albumin, c-reactive protein, conalbumin, lactalbumin, ionophore proteins, acyl carrier proteins, signal transduction adaptor proteins, androgen binding proteins, calcium binding proteins, calmodulin binding proteins, plasma ceruloplasmin, cholesterol ester transfer protein, f-box proteins, fatty acid binding proteins, follistatin, follistatin-related proteins, GTP binding proteins, insulin-like growth factor binding proteins, iron binding proteins, latent TGFβ binding proteins, light-harvesting protein complexes, lymphocyte antigens, membrane transport proteins, neurohypophysial hormone transporters, periplasmic binding proteins, phosphate binding proteins, phosphatidylethanolamine binding proteins, phospholipid transporters, retinol binding proteins, RNA binding proteins, S phase kinase-associated proteins, sex hormone binding globulin, thyroxine binding proteins, transcobalamin, transcortical hormone transporter proteins, transferrin binding proteins, and / or vitamin D binding proteins.

[0369] As a further example, the peptides or pMHC complexes of the present disclosure can be fused to, for example, the 80 N-terminal amino acids of the HLA-DR antigen-associated invariant chain (p33 or Ii) derived from NCBI GenBank Accession No. X00497. The Ii fragment can facilitate the efficient introduction of the peptide or pMHC complex into cells.

[0370] The peptides or pMHC complexes of the present disclosure may also be covalently (e.g., via a linker) or non-covalently linked to a moiety capable of causing a therapeutic effect, such as an antibody, or a cytokine, such as interleukin 2, interferon α, and granulocyte-macrophage colony stimulating factor. Alternatively or additionally, the peptides or pMHC complexes may be encapsulated into liposomes.

[0371] Other suitable heterologous molecules include, but are not limited to, fluorescent or luminescent labels, radioactive labels, nucleic acid probes and contrast agents, antibodies, or enzymes that produce a detectable product. Methods for detecting heterologous molecules may include flow cytometry, microscopy, electrophoresis, or scintillation counting.

[0372] In some embodiments, the peptides or pMHC complexes of the present disclosure may be conjugated to fluorocarbons to increase cellular immunogenicity. When the other polypeptide chain of the peptide or pMHC complex is attached to the fluorocarbon, the ends of the peptide or polypeptide chain, such as the ends not conjugated to the fluorocarbon or other attachments, may be altered, for example by forming micelles to promote the solubility of the fluorocarbon-peptide / polypeptide construct. To facilitate large-scale synthesis of the construct, the N-terminal or C-terminal amino acid residues of the other polypeptide chain of the peptide or pMHC complex may be modified. When the other polypeptide chain of the desired peptide or pMHC complex is particularly sensitive to cleavage by peptidases, the normal peptide bond may be replaced by a non-cleavable peptide mimetic. Such bonds and synthetic methods are well known in the art.

[0373] 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, agarose gels, magnetic beads, plates, tubes, columns. The pMHC complexes may be attached to ELISA plates, magnetic beads, or surface plasmon resonance biosensor chips. Methods for attaching peptides or pMHC complexes to solid supports are well known to those skilled in the art and include, for example, the use of affinity binding pairs, such as biotin and streptavidin, or antibodies and antigens. In some embodiments, the peptides or pMHC complexes are labeled with biotin and attached to a streptavidin-coated surface.

[0374] The peptides or pMHC complexes of the present disclosure may be in the form of polymers, for example, dimers, or tetramers, or pentamers, or octamers or larger polymers. Therefore, in certain aspects, the present disclosure provides oligomeric complexes comprising the peptides or pMHC complexes of the present disclosure. As used herein, the terms "oligomer," "oligomeric," "oligomerization," and "oligomerization" or similar terms encompass dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers or higher types of polymerized monomers comprising the peptide or pMHC complex. Having multiple copies of a peptide or pMHC complex in a large complex may enhance their biological activity, such as immunogenic activity.

[0375] For example, the peptides of the present disclosure can be oligomerized using a biotin / streptavidin system. Biotinylated analogs of peptide monomers can be synthesized by standard techniques. For example, the peptide can be C-terminal biotinylated. Oligomerization is then performed by incubating these biotinylated peptide monomers with streptavidin [e.g., incubating in phosphate buffered saline (PBS) or HEPES-buffered RPMI culture medium at room temperature at a molar ratio of 4:1 for 1 hour]. In a variation of this embodiment, biotinylated peptide monomers can be oligomerized by incubating with anti-biotin antibodies [e.g., goat anti-biotin IgG].

[0376] Typically, oligomeric pMHC complexes can be produced using pMHC labeled with biotin residues and formed into complexes by fluorescently labeled streptavidin. Biotinylation sites can be introduced into pMHC complexes, for example, using BirA enzymes to add biotin to the complex. Alternatively, oligomeric pMHC complexes can be formed by using immunoglobulins as molecular scaffolds. In this system, the extracellular domains of MHC molecules are fused to the constant regions of immunoglobulin heavy chains separated by short amino acid linkers. Oligomeric pMHC complexes are also produced using carrier molecules such as dextran. Oligomeric pMHC complexes can be used to improve the detection of binding moieties (such as T cell receptors) that bind to the complex due to affinity effects.

[0377] 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 moiety can be a peptide linker, such as those described herein (e.g., in Table 2). In some embodiments, polyethylene glycol (PEG) can be used as a linker for oligomerizing peptide monomers. For example, a single PEG moiety can be simultaneously attached to the N-termini of two peptide chains of a peptide dimer.

[0378] Alternatively, the oligomeric peptide or pMHC complex may also contain one or more intramolecular disulfide bonds between the cysteine ​​residues of the peptide or pMHC monomers. Preferably, both monomers contain at least one intramolecular disulfide bond. Most preferably, both monomers contain intramolecular disulfide bonds such that each monomer contains a cyclic group. Such disulfide bonds can be formed by oxidation of the cysteine ​​residues of the peptide core sequence. In one embodiment, the formation of cysteine ​​bonds is controlled by selecting an oxidant of the type and concentration that effectively optimizes the formation of the desired isomer. For example, when the oxidant is DMSO, oxidation of the peptide dimer is preferentially achieved to form two intramolecular disulfide bonds (one on each peptide chain) (prior to the formation of intermolecular disulfide bonds). The formation of cysteine ​​bonds can be controlled by the selective use of sulfhydryl protecting groups during peptide synthesis.

[0379] In some embodiments, peptides described herein or pMHC complexes can be fused or conjugated with dimerization moieties.Dimerization moieties can include, for example, immunoglobulin domains, such as immunoglobulin domains from IgG antibodies (e.g., human IgG), which connect two monomers to produce homodimers or heterodimer molecules.As a non-limiting example, according to the dimerization motif in the protein disclosed herein, it can be constructed to include hinge regions and immunoglobulin domains (e.g., Cy3 domains), for example, carboxyl terminal C domains (CH3 domains), or sequences substantially identical to C domains.Hinge regions can be Ig-derived, and dimerization is promoted by forming interchain covalent bonds (e.g., disulfide bonds).In addition, this homodimer or heterodimer molecule can further include one or more targeting moieties, which are combined with target molecules present in, for example, antigen presenting cells (APCs) (such as dendritic cells or B cells).In this case, hinge regions can serve as flexible spacers between domains, allowing two targeting units to simultaneously bind to two target molecules expressed at variable distances on APCs. Immunoglobulin domains promote dimerization by non-covalent interactions (e.g., hydrophobic interactions). In a preferred embodiment, the CH3 domain is derived from IgG. These dimerization moieties can be exchanged with other multimerization moieties from, for example, other Ig isotypes / subclasses. Preferably, the dimerization motif is derived from natural human proteins, such as human IgG. The example of this homodimeric protein construct is described in US10,590,195, the entire contents of which are incorporated herein by reference.

[0380] Nucleic acids and vectors

[0381] 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 (such as complexes (e.g., pMHC complexes), fusion proteins or conjugates) of the present disclosure. The polynucleotide can be, for example, DNA, cDNA, PNA, RNA or a combination thereof, can be single-stranded and / or double-stranded, can be a native or stabilized form of a polynucleotide, such as, for example, a polynucleotide having a phosphorothioate backbone, and, as long as it encodes a peptide, it can contain or not contain introns.

[0382] In some embodiments, the polynucleotides described herein encode peptides, or fragments or derivatives thereof, comprising 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 the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112. In some embodiments, the polynucleotides described herein encode peptides, or fragments or derivatives thereof, comprising the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

[0383] In some embodiments, the polynucleotides described herein encode more than one peptide selected from any one of SEQ ID NOs: 1-54 and 110-112, or a fragment thereof. For example, the polynucleotides described herein can encode 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides described herein (e.g., SEQ ID NOs: 1-54 and 110-112), or fragments thereof. The peptides can be arranged in any order and can be the same or different.

[0384] In some embodiments, the polynucleotides described herein encode 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 fragments thereof.

[0385] In some embodiments, the polynucleotides described herein are DNA molecules.

[0386] Methods for delivering DNA to a subject include, for example, 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 consisting only of DNA can be administered. Alternatively, the DNA can be adhered to particles, such as gold particles.

[0387] In some embodiments, the polynucleotides described herein are RNA molecules. For example, the RNA molecule can be mRNA or a self-replicating RNA.

[0388] The polynucleotides encoding RNA disclosed herein can be used to make vaccines. RNA cannot be integrated into the genome and therefore has little carcinogenic potential; therefore, RNA can be used to make vaccines. In addition, RNA only needs to enter the cytoplasm, while DNA needs to enter the nucleus. The RNA molecules disclosed herein can be chemically modified and / or incorporated with modified nucleosides to overcome sensitivity to degradation. RNA vaccines can include mRNA and / or self-replicating RNA (also referred to as RNA replicons). The delivery technology of RNA vaccines can also include, for example, condensation with protamine and encapsulation into liposomes or nanoparticles.

[0389] Nucleic acids (DNA or RNA) can also be delivered in complex with cationic compounds (such as cationic lipids). For example, lipid-mediated gene delivery methods are described in WO 91 / 06309, WO 93 / 24640, WO 96 / 18372, U.S. Patent No. 5,279,833, the entire contents of which are incorporated herein by reference.

[0390] Nucleic acid molecules described herein can be produced synthetically. One method is the phosphoramidite method. Without wishing to be bound by theory, in this chemistry, phosphoramidites (a nucleoside with a side protecting group that maintains the integrity of sugar, phosphodiester bonds, and bases in the chain extension step) are coupled to the 5' hydroxyl of a nucleoside fixed on a solid support column through its reactive 3' phosphorus group. The steps of oligonucleotide synthesis may include the following: (1) detritylation, in which the dimethoxytriphenyl (DMT or trityl) group on the 5' hydroxyl group of the support nucleoside is removed by treatment with trichloroacetic acid (TCA); (2) in a coupling step, the phosphoramidite made reactive by tetrazole (a weak acid) is chemically coupled to the last base added to the column support material; (3) in a capping step, any free 5' hydroxyl groups of unreacted column nucleotides are acetylated by treatment with acetic anhydride and N-methylimidazole; (4) in an oxidation step, the unstable internucleotide phosphate bond between the previously coupled base and the most recently added base is oxidized to a more stable phosphotriester bond by treatment with iodine and water. After all bases in the oligonucleotide sequence have been coupled, the intact nucleic acid chain can be cleaved from the column by treatment with ammonium hydroxide, and the base protecting groups are removed by heating in an ammonium hydroxide solution.

[0391] By way of non-limiting example, the synthesis cycle can include the growth of a nucleotide chain from an initially protected nucleoside derived from its terminal 3' hydroxyl group to a solid support. Reagents and solvents can be pumped through the support to induce the continuous removal and addition of sugar protecting groups to separate the reactivity of a specific chemical moiety to a monomer and to allow it to be progressively added to the growing oligonucleotide chain. The assembly of protected oligonucleotide chains can be carried out by a plurality of chemical steps, such as, but not limited to, deblocking, activation / coupling, oxidation, and end-capping. Cleavage and deprotection then expose single-stranded nucleic acids.

[0392] 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 array-derived oligonucleotide libraries, and any one of various error correction and sequence verification steps, or any combination thereof.

[0393] RNA chemical synthesis can be similar to the chemical synthesis for DNA. In some embodiments, the RNA chemical synthesis method can include an additional protecting group at the 2'hydroxyl of ribose. The 2'hydroxyl at the ribose position can be protected with tert-butyldimethylsilyl, which can remain stable throughout the synthesis and can be removed in the final deprotection step by adding alkaline fluoride ions (such as tetrabutylammonium fluoride (TBAF)). The remaining positions on the sugar and base can be protected in the same way as for DNA. By adjusting several parameters in the DNA synthesis protocol, such as, but not limited to, coupling time, monomer delivery rate, washing step frequency, and end-capping agent type, a step-by-step coupling efficiency of up to 99% can be obtained.

[0394] Viral nucleic acid synthesis can be catalyzed by both viral enzymes and host enzymes, and their relative contributions can be determined by the type of virus and specific molecules. In addition to retroviruses, viruses with RNA genomes synthesize mRNA and use virally encoded RNA-dependent RNA polymerases to replicate their genomes. In contrast, retroviruses use RNA-dependent DNA polymerases (reverse transcriptases) encoded by virions to synthesize double-stranded complementary DNA (cDNA) copies of their single-stranded RNA genomes. In subsequent steps, retroviral cDNA can be integrated into host chromosomes and transcribed by host-encoded DNA-dependent RNA polymerase II (pol II) to produce viral information and genomic RNA. In addition to poxviruses, DNA viruses also use host-encoded pol II to transcribe their information. Because poxviruses replicate in the cytoplasm and cannot obtain pol II, poxviruses assemble a new type of transcriptase composed of multiple poxvirus-specific (possibly also derived from one or more hosts) subunits. Most DNA virus families (e.g., Poxviridae, Iridoviridae, Herpesviridae, Adenoviridae) synthesize virally encoded DNA-dependent DNA polymerases. Whereas two families (i.e., Parvoviridae and Papoviridae) utilize host DNA polymerases, the Hepadnaviridae use a virus-encoded reverse transcriptase to replicate viral DNA via an RNA intermediate.

[0395] Due to the degeneracy of the genetic code, nucleic acid molecules of different nucleotide sequences can encode the same amino acid sequence. For expression in various hosts, the polynucleotides can be codon optimized.

[0396] In a further aspect, the disclosure provides a vector comprising a nucleic acid sequence according to the third aspect of the disclosure. In addition to the nucleic acid sequence encoding only the peptide of the present disclosure, the vector may also include one or more additional nucleic acid sequences encoding one or more additional peptides. Once expressed, this additional peptide can be fused to the N-terminus or C-terminus of the peptide of the present disclosure. The above section describes examples of such additional peptides in detail. In one embodiment, the vector includes a nucleic acid sequence encoding a peptide or protein tag, such as, for example, a biotinylation site, a FLAG tag, a MYC tag, a HA tag, a GST tag, a Strep tag, or a polyhistidine tag.

[0397] The vector used in the context of the present disclosure desirably comprises a sequence suitable for introduction into a cell. For example, the vector can be an expression vector, a vector in which the polypeptide encoding sequence is controlled by its own cis-acting regulatory elements, a vector designed to promote gene integration or gene replacement in a host cell, etc.

[0398] In the context of the present disclosure, the term "vector" encompasses a DNA molecule, such as a plasmid, a phage, a phagemid, a virus or other vehicle, which comprises one or more heterologous or recombinant nucleotide sequences (e.g., the above-mentioned nucleic acid molecules of the present disclosure, under the control of a functional promoter and possibly an enhancer) and is capable of functioning as a vector in the sense understood by those of ordinary skill in the art.

[0399] The following vectors are provided by way of example: bacteriophages such as lambda (X) phage, EMBL phage; bacterial vectors such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5; eukaryotic vectors such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG and pSVL; and transposons such as Sleeping Beauty transposon and PiggyBac transposon.

[0400] In some embodiments, the vector is a viral vector. The viral vector can be derived from a naturally occurring viral genome, which is usually modified to be non-replicable, such as non-replicable. Non-replicating viruses need to provide proteins in trans for replication. Typically, these proteins are stably or transiently expressed in a viral production cell line, thereby allowing viral replication. Therefore, viral vectors are generally infectious and non-replicable. The viral vector can be an adenoviral vector, an adeno-associated virus (AAV) vector (e.g., AAV type 5 and type 2), an alphavirus vector (e.g., Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki Forest virus (SFV) and VEE-SIN chimera), a herpesvirus vector (e.g., a vector derived from cytomegalovirus (such as rhesus monkey cytomegalovirus (RhCMV)), an arenavirus vector (e.g., a lymphocytic choriomeningitis virus (LCMV) vector), a measles virus vector, a poxvirus vector (e.g., vaccinia virus, modified Ankara vaccinia virus (M VA), NYVAC (derived from the Copenhagen vaccinia strain) and fowlpox vectors (canarypox virus (ALVAC) and fowlpox virus (FPV) vectors), vesicular stomatitis virus (VSV) vectors, retroviral vectors, lentiviral vectors, simian virus 40 (SV40), bovine papilloma virus, Epstein-Barr virus, Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, Rous sarcoma virus, poxvirus virus-like particles, baculovirus vectors, and bacterial spores.

[0401] As a further example, an adenovirus vector can be derived from a human adenovirus (Ad), but can also be derived from an adenovirus that infects other species, such as a bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), a canine adenovirus (e.g., CAdV2), a porcine adenovirus (e.g., PAdV3 or 5), or apes, such as chimpanzees (Pan), gorillas (Gorilla), orangutans (Pongo), bonobos (Pan paniscus), and common chimpanzees (Pan troglodytes). Poxvirus (Poxviridae) vectors can be derived from variola virus (smallpox), vaccinia virus, cowpox virus, or monkeypox virus. Exemplary vaccinia viruses are Copenhagen vaccinia virus (W), New York attenuated vaccinia virus (NYVAC), ALVAC, TROVAC, and modified Ankara vaccinia virus (MVA).

[0402] Many expression systems are known in the art, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae), filamentous fungi (e.g., Aspergillus species), plant cells, animal cells (e.g., mammalian cells), and insect cells.

[0403] In another aspect, the disclosure provides host cells comprising vectors of the disclosure.Host cells can be prokaryotic cells or eukaryotic cells.In some cases, bacterial cells can be preferred prokaryotic host cells, typically Escherichia coli strains, such as, for example, Escherichia coli strains DH5 and RR1.Non-limiting examples of eukaryotic host cells include yeast, insects 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 embryonic cells NIH / 3T3, COS-1 cells derived from monkey kidneys, and 293 cells for human embryonic kidney cells.The example of insect cells includes Sf9 cells, which can be transfected with baculovirus expression vectors.

[0404] The transformation of a suitable cell host with a DNA construct of the present invention is accomplished by well-known methods, which are generally dependent on the type of vector used. Successfully transformed cells, i.e. cells containing a DNA construct of the present invention, can be identified by, for example, PCR. Alternatively, antibodies can be used to detect the presence of proteins in the supernatant.

[0405] It is to be understood that certain host cells of the present disclosure are useful in preparing peptides or peptide-based molecules of the present disclosure, such as bacteria, yeast, and insect cells. However, other host cells may be useful in certain therapeutic methods. For example, antigen presenting cells (APCs), such as dendritic cells or B cells, may be used to express the peptides of the present disclosure so that the peptides can be loaded into appropriate MHC molecules.

[0406] Another aspect of the present disclosure provides a method of producing 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.

[0407] Peptide and pMHC binding moiety

[0408] The peptides, pMHC complexes or other peptide-based molecules of the present disclosure (such as complexes, fusion proteins or conjugates comprising the peptides disclosed herein) can be used to identify and / or separate binding moieties that specifically bind to the peptides, pMHC complexes or other peptide-based molecules of the present disclosure. Such binding moieties can be used as immunotherapeutic agents and can include antibodies (or antigen-binding fragments thereof), alternative frameworks, TCRs and CARs.

[0409] In one aspect, the disclosure provides peptide binding moieties that bind to the peptides of the disclosure. Preferably, the peptide binding moiety binds to the peptide when the peptide forms a complex with the MHC. In the latter case, the peptide binding moiety may partially bind to the MHC, provided that the peptide binding moiety also binds to the peptide. The peptide binding moiety may only bind to the peptide, and this binding may be specific. The peptide binding moiety may only bind to the pMHC complex, and this binding may be specific.

[0410] The present disclosure also provides a method for identifying a peptide binding moiety that binds to a pMHC complex of the present disclosure, the method comprising contacting a candidate peptide binding moiety with a pMHC complex and determining whether the candidate peptide binding moiety binds to the complex. Methods for determining binding to a pMHC complex include, for example, surface plasmon resonance or any other biosensor technology, ELISA, flow cytometry, chromatography, microscopy. Alternatively, or in addition, binding can be determined by a functional assay in which a biological response is detected upon binding, e.g., cytokine release or apoptosis.

[0411] The candidate peptide binding moiety may be a peptide binding moiety of the type already described, such as an antibody or a TCR.

[0412] For example, antibodies and TCRs can be obtained from display libraries in which the pMHC complexes disclosed herein are used to pan the library. For example, TCRs can be displayed on the surface of phage particles and yeast particles, and such libraries have been used to isolate high-affinity variants of TCRs derived from T cell clones. TCR phage libraries can be used to isolate TCRs with new antigen specificity. Such libraries can be constructed with α-chain and β-chain sequences that correspond to sequences present in natural libraries. However, the random combination of these α-chain and β-chain sequences that occurs during library creation can produce TCR libraries that may not be naturally occurring.

[0413] In some embodiments, the pMHC complex of the present disclosure can be used to screen various TCR libraries displayed on the surface of phage particles. The TCRs displayed by the library may not correspond to those contained in the natural library, for example, they may contain α-chain and β-chain pairings that do not exist in the body, and / or the TCR may contain non-natural mutations and / or the TCR may be in a soluble form. Screening may involve panning the phage library with the pMHC complex of the present disclosure, and then separating the bound phage particles. To this end, the pMHC complex can be connected to a solid phase carrier, such as a magnetic bead, or a column matrix, and the pMHC complex bound to the phage is separated with a magnet or by chromatography. The panning step can be repeated multiple times. The isolated phage can be further amplified in Escherichia coli cells. The isolated phage particles can be tested for specific binding to the pMHC complex of the present disclosure. The binding can be detected using techniques including but not limited to ELISA or SPR, for example using a BiaCore instrument. The DNA sequence of the T cell receptor displayed by the pMHC binding phage can be further identified by the PCR method.

[0414] Alternatively, antigen-binding T cells and TCR can be isolated from fresh blood obtained from patients or healthy donors. This method includes stimulating T cells using autologous dendritic cells (DC), followed by autologous B cells, and then pulsing with peptides disclosed herein. Several rounds of stimulation, such as three or four rounds, can be performed. Then, the specificity of activated T cells can be tested by measuring cytokine release in the presence of T2 cells pulsed with peptides disclosed herein (e.g., using IFNγ ELISpot assay). Activated cells can then be sorted by fluorescence-activated cell sorting (FACS) using labeled antibodies to detect the production of intracellular cytokines (e.g., IFNγ) or the expression of cell surface markers (such as CD137). Sorted cells can be expanded and further verified, for example, by ELISpot assay and / or cytotoxicity for target cells and / or by peptide-MHC tetramer staining. TCR chains from verified clones can then be amplified and sequenced by rapid amplification of cDNA ends (RACE).

[0415] The peptide binding moieties disclosed herein may include, for example, but are not limited to, antibodies, TCRs, or CARs.

[0416] In some embodiments, the peptide binding portion of the present disclosure can be an antibody or an antigen-binding fragment thereof. Antibodies or antigen-binding fragments thereof encompass derivatives, functional equivalents, and homologs of antibodies, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or fully or partially synthesized, and any polypeptide or protein having an antibody binding domain or a binding domain homologous to an antibody binding domain. Therefore, chimeric molecules comprising an immunoglobulin binding domain or an equivalent fused to another polypeptide are included. Humanized antibodies can be modified antibodies having a non-human (e.g., mouse) antibody variable region and a human antibody constant region. Examples of antibodies include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and isotype subclasses thereof; or fragments comprising an antigen-binding domain, such as Fab, scFv, Fv, dAb, Fd; and bivalent antibodies. Antibodies can be polyclonal antibodies or monoclonal antibodies. Monoclonal antibodies may be referred to as "mAbs" herein.

[0417] In some embodiments, the antibody is a multispecific antibody. In some embodiments, the antibody is a bispecific antibody. The bispecific antibody may comprise a second targeting moiety that targets a desired cell or tissue, e.g., liver, or targets another desired antigen (e.g., liver cancer antigen) associated with the same or similar disease or condition.

[0418] Antibodies (e.g., monoclonal antibodies) can be taken and recombinant DNA techniques used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can involve introducing DNA encoding the immunoglobulin variable region or complementary determining region (CDR) of an antibody into the constant region or constant region plus framework region of a different immunoglobulin. Hybridomas (or other antibody-producing cells) can undergo genetic mutations or other changes that may or may not change the binding specificity of the produced antibody.

[0419] It has been shown that fragments of whole antibodies can perform antigen binding function. Examples of binding fragments include: (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of the 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 the VH domain; (v) isolated CDR regions; (vi) a F(ab′)2 fragment, which is 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, allowing the two domains to associate 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) VHH or VNAR antibodies, also known as single domain antibodies or nanobodies (Nb), which can be derived from heavy chain antibodies from, for example, dromedary camels, camels, llamas, alpacas or sharks.

[0420] A bivalent antibody is a multimer of polypeptides, each polypeptide comprising a first domain and a second domain, wherein the first domain comprises an immunoglobulin light chain binding region, and the second domain comprises an immunoglobulin heavy chain binding region, and the two domains are connected (e.g., by a peptide linker) but cannot associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804). If bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be manufactured in a variety of ways, such as chemical preparation or preparation from a hybridoma, or can be any of the above-mentioned bispecific antibody fragments. It is preferred to use scFv dimers or bivalent antibodies rather than whole antibodies. Bivalent antibodies and scFv can be constructed without an Fc region, using only variable domains, thereby potentially reducing the impact of anti-unique reactions. Other forms of bispecific antibodies include single-chain "Janusins". In contrast to bispecific whole antibodies, bispecific bivalent antibodies can also be useful because they can be easily constructed and expressed in E. coli. Bivalent antibodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be easily selected from libraries using phage display. For example, if one arm of the antibody is to remain constant and have specificity for a target antigen, a library can be created where the other arm is varied and antibodies with appropriate specificity selected. An "antigen binding domain" is the part of an antibody that contains the region that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, the antibody may bind only to a specific portion of the antigen, which is called an epitope. The antigen binding domain may be provided by one or more antibody variable domains. The antigen binding domain may contain an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0421] In some embodiments, the peptide binding moiety can 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 moiety can include a TCR mimicking antibody. In some embodiments, such a TCR mimicking antibody can include a high-affinity soluble antibody molecule that confers TCR-like specificity for a tumor or viral epitope, which can target tumor and / or virally infected cells and mediate their specific killing.

[0422] The present disclosure also includes binding moieties based on engineered protein scaffolds or "alternative backbones." Alternative backbones are derived from stable, soluble natural protein structures that have been modified to provide binding sites for target molecules of interest. Examples of alternative backbones include, but are not limited to: affibodies, which are based on the Z domain of Staphylococcal protein A, providing a binding interface on its two alpha helices; anticalins, derived from lipocalins, adding binding sites for small ligands at the open end of the beta barrel fold; monobodies, designed to incorporate fibronectin type III domains (Fn3) of fibronectin or tenascin as protein scaffolds or synthetic FN3 domains (e.g., tenascin); nanobodies and DARPins. Other alternative backbones include Adnectin TM , iMab, EETI-II / AGRP, Kunitz domain, thioredoxin peptide aptamer, Affilin, Tetranectin, Fynomer and Avimer. Alternative backbones usually target the same antigenic protein as antibodies and are potential therapeutic agents. They can act as inhibitors or antagonists, or as delivery vehicles for targeting molecules (such as toxins) to specific tissues in the body. Short peptides can also be used to bind to target proteins. Phylomers are natural structural peptides derived from bacterial genomes. Such peptides represent a variety of protein structural folds and can be used to inhibit / disrupt protein-protein interactions in vivo.

[0423] The alternative backbone is typically a single-chain polypeptide framework comprising a highly structured core associated with a variable domain of high conformational tolerance that allows insertion, deletion or other substitutions within the variable domain. Libraries that introduce diversity into one or more variable domains, and in some cases libraries that introduce structured cores, 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 specificity of the identified binders can be further characterized using known methods. The alternative backbone can be derived from protein A, particularly its Z domain (affine body), ImmE7 (immune protein), BPTI / APPI (Kunitz domain), CTLA-4, charybdotoxin, scorpion toxin, Min-23 (knot peptide), lipocalin (anticarbol), Ras binding protein AF-6 (PDZ domain), neokarzinostatin, fibronectin domain, ankyrin consensus repeat domain or thioredoxin.

[0424] In some embodiments, antibodies described herein or alternative skeletons can be fixed on viral vectors. This modified recombinant viral vector can be used to target the genetic material encoded by the viral vector into cells and / or tissues (e.g., hepatocytes and / or liver tissue). Antibodies or alternative skeletons can be mobilized to viral vectors using a variety of methods, for example, by using affinity binding pairs, such as c-Myc / anti-Myc antibodies, streptavidin / biotin, or by spy-tag / spy-catcher systems. Example vectors that can be modified with antibodies described herein or alternative skeletons 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)).

[0425] In some embodiments, the peptide binding moiety can be a TCR. TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and the IMGT public database of TCR sequences.

[0426] The TCR of the present disclosure can be in any form. For example, the TCR can be an αβ heterodimer, or an αα or ββ homodimer.

[0427] The α / β heterodimeric TCR has an α chain and a β chain. Broadly speaking, each chain contains 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, but this diversity region is usually considered to be part of the joining region. Each variable region includes three hypervariable CDRs (complementarity determining regions) embedded in a framework sequence; CDR3 is considered to be the main mediator of antigen recognition. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, which are distinguished by their framework, CDR1 and CDR2 sequences and partially defined CDR3 sequences.

[0428] The TCRs of the present disclosure may not correspond to TCRs existing in nature. For example, they may contain α-chain and β-chain combinations that are not present in natural repertoires. Alternatively or additionally, the TCRs described herein may be soluble, and / or the α-chain and / or β-chain constant domains may be truncated relative to the native / naturally occurring TRAC / TRBC sequence, such that, for example, the C-terminal transmembrane domain and the intracellular region are absent. Such truncation may result in the removal of cysteine ​​residues that form native interchain disulfide bonds from TRAC / TRBC.

[0429] In addition, the TRAC / TRBC domain may comprise modifications. For example, the α chain extracellular sequence may include modifications relative to natural / naturally occurring TRAC, wherein the amino acid T48 (referenced to IMGT numbering) of TRAC is replaced by C48. Similarly, the β chain extracellular sequence may include modifications relative to natural / naturally occurring TRBC1 or TRBC2, wherein S57 (referenced to IMGT numbering) of TRBC1 or TRBC2 is replaced by C57. These cysteine ​​substitutions relative to the natural α chain and β chain extracellular sequences can form non-natural interchain disulfide bonds, thereby stabilizing the refolded soluble TCR, i.e., the TCR formed by refolding the extracellular α chain and β chain. This non-natural disulfide bond helps to display the correctly folded TCR on the phage. In addition, the use of a stable disulfide-bonded soluble TCR can more conveniently evaluate binding affinity and binding half-life. Alternative positions for forming non-native disulfide bonds include, for example, Thr 45 of TRAC*01 exon 1 and Ser 77 of TRBC1*01 or TRBC2*01 exon 1; Tyr 10 of TRAC*01 exon 1 and Ser 17 of TRBC1*01 or TRBC2*01 exon 1; Thr 45 of TRAC*01 exon 1 and Asp 59 of TRBC1*01 or TRBC2*01 exon 1; Ser 15 of TRAC*01 exon 1 and Glu 15 of TRBC1*01 or TRBC2*01 exon 1. TCRs with non-native disulfide bonds can be full-length or truncated.

[0430] The TCR disclosed herein can be in single-chain form. Single-chain TCR includes the following types of αβTCR polypeptides: Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ or Vα-Cα-L-Vβ-Cβ, optionally reversed, wherein Vα and Vβ are TCRα and β variable regions, respectively, Cα and Cβ are TCRα and β constant regions, respectively, and L is a linker sequence. Single-chain TCR may include non-natural disulfide bonds. TCR may be in soluble form (i.e., without transmembrane or cytoplasmic domains), or may include full-length α chains and β chains. TCR may be provided on a cell surface, such as on a T cell surface.

[0431] The TCRs of the present disclosure may be engineered to include mutations. Methods for generating mutant high-affinity TCR variants, such as phage display and site-directed mutagenesis. Preferably, mutations that increase affinity are made within the variable regions of the α chain and / or β chain. More preferably, mutations that increase affinity are made within the CDRs. There may be 1-15 mutations in the α chain and / or β chain variable regions.

[0432] The TCR of the present disclosure can also be labeled with an imaging compound, such as a label suitable for diagnostic purposes. Such a labeled high-affinity TCR can be used to detect a method for a TCR ligand selected from a CD1 antigen complex, a bacterial superantigen, and an MHC peptide / superantigen complex, the method comprising contacting the TCR ligand with a high-affinity TCR (or a multimeric high-affinity TCR complex) that is specific for the TCR ligand; and detecting binding to the TCR ligand. In a multimeric high-affinity TCR complex (e.g., formed using a biotinylated heterodimer), fluorescent streptavidin can be used to provide a detectable label. Fluorescently labeled polymers are suitable for FACS analysis, such as detecting antigen presenting cells carrying peptides specific to high-affinity TCRs.

[0433] The TCR (or its multivalent complex) of the present disclosure may alternatively or additionally be associated (e.g., covalently or otherwise linked) with a therapeutic agent, which may be, for example, a toxic moiety for cell killing, or an immunostimulant, such as an interleukin or cytokine. Compared to non-polymeric wild-type or high-affinity T cell receptor heterodimers, the multivalent high-affinity TCR complex of the present disclosure may have an enhanced binding ability to TCR ligands. Therefore, the multivalent high-affinity TCR complex according to the present disclosure is particularly suitable for tracking or targeting cells presenting specific antigens in vitro or in vivo, and can also be used as an intermediate for producing more multivalent high-affinity TCR complexes with such uses. Therefore, the high-affinity TCR or multivalent high-affinity TCR complex can be provided in a pharmaceutically acceptable formulation for in vivo use.

[0434] The high affinity TCRs disclosed herein can be used in the production of soluble bispecific agents. A preferred embodiment is an agent comprising a soluble TCR fused to an anti-CD3 specific antibody fragment via a linker.

[0435] In a further aspect, the present disclosure provides nucleic acids encoding TCRs of the present disclosure, TCR expression vectors comprising nucleic acids encoding TCRs of the present disclosure, and cells carrying the vectors. TCRs may be encoded in a single open reading frame or in two different open reading frames. The scope of the present disclosure also includes cells carrying a first expression vector and a second expression vector, wherein the first expression vector comprises nucleic acids encoding the α chains of the TCRs of the present disclosure, and the second expression vector comprises nucleic acids encoding the β chains of the TCRs of the present disclosure. Alternatively, one vector may encode both the α chains and the β chains of the TCRs of the present disclosure.

[0436] Another aspect of the present disclosure provides a cell that displays 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 that it does not correspond to the T cell existing in nature. For example, a vector encoding the TCR of the present disclosure may be used to transfect cells so that the T cell expresses other TCRs in addition to the natural TCR. Alternatively or additionally, the T cell may be modified so that it cannot present the natural TCR. There are many methods suitable for transfecting T cells with DNA or RNA encoding the TCR of the present disclosure. As a non-limiting example, the transfection method may include an RNA-based rapid transfection system. The T cells expressing the TCR of the present disclosure are suitable for treatment based on adoptive therapy for diseases such as cancer. There are many suitable methods for adoptive therapy. For example, adoptive cell therapy (ACT) may include the use of autologous tumor-infiltrating lymphocytes, and may include a lymphocyte depletion preparation scheme before ACT. In some embodiments, a virus encoding TCR (e.g., a retrovirus) may be used for genetic modification of lymphocytes to convert normal lymphocytes into lymphocytes with anti-cancer activity. Adoptive transfer of lymphocytes with anti-cancer activity into patients in need of treatment, such as metastatic melanoma, can mediate tumor regression. In some embodiments, ACT can include treating cancer patients expressing viral or alloantigens, treating cancer patients expressing viral antigens, and / or ACT using genetically modified lymphocytes. In some embodiments, the ACT method can include, for example, using αβTCR and / or chimeric TCR, for example, genetic modification of lymphocytes to introduce new recognition specificity; for example, genetic modification of lymphocytes using co-stimulatory molecules (e.g., CD28, 41BB), cytokines (e.g., IL2, IL15), homing molecules (e.g., CD62L, CCR7) and / or molecules capable of preventing apoptosis (BCL2) to alter T cell function; for example, modifying host lymphocyte depletion using selective depletion of CD4+ cells or T regulatory cells; for example, using CTLA4 and / or PD-1 antibodies to block inhibitory signals on reactive lymphocytes; for example, administering vaccines using recombinant viruses encoding antigens to stimulate transferred cells; for example, administering alternative cytokines using IL15 and / or IL21 to support cell growth; for example, stimulating APCs using toll-like receptor agonists; generating less differentiated lymphocytes in vitro using, for example, alternating culture conditions and growth-promoting cytokines; and, for example, using natural killer cells to overcome antigen escape variants.

[0437] TCRs of the present disclosure intended for adoptive therapy are typically glycosylated when expressed by transfected T cells. The glycosylation pattern of a transfected TCR can be altered by mutations in the transfected gene.

[0438] In some embodiments, the peptide binding moiety can be a chimeric antigen receptor (CAR). CAR is a genetically engineered receptor. By adding an antigen binding domain that specifically binds to a peptide or pMHC complex to the extracellular domain of CAR, a CAR that binds to a peptide or pMHC complex disclosed herein can be produced. CAR can be introduced into immune cells (such as T cells, NK cells or macrophages) and expressed by immune cells. CAR can be programmed to recognize specific antigens and activate immune cells to attack and destroy cells presenting the antigen when bound to the antigen. When these antigens are present on tumor cells, immune cells expressing CAR can target and kill tumor cells.

[0439] The general structure of a CAR generally includes an extracellular domain that binds an antigen (e.g., a peptide or pMHC complex of the disclosure), a hinge, a transmembrane domain, and an intercellular domain comprising a signaling domain and optionally one or more costimulatory domains.

[0440] The extracellular domain of CAR may include any polypeptide (e.g., a peptide or pMHC complex disclosed herein) that specifically binds to a desired antigen. For example, the extracellular domain may include an antibody fragment, such as an scFv or VHH. CAR may also be engineered to bind to two or more desired antigens, which may be arranged in series and separated by a linker sequence. For example, one or more domain antibodies, scFv, llama VHH antibodies or other VH antibody fragments alone may be organized in series by a linker to provide bispecific or multispecific CARs.

[0441] A hinge domain may be present between the extracellular domain and the transmembrane domain of CAR, for example, to provide flexibility to allow the extracellular domain to effectively bind to its intended target. The hinge domain may be a polypeptide having a length of about 2 to 100 amino acids. The hinge may include or consist of flexible residues (such as Gly and Ser) so that adjacent protein domains can move freely relative to each other. When it is necessary to ensure that two adjacent domains do not interfere with each other spatially, a longer hinge may be used. The hinge may be derived from a hinge region or a portion of a hinge region of any immunoglobulin. Non-limiting examples of joints include: a portion of a human CD8 alpha chain, an extracellular domain of CD28, an Ig hinge, a FcγRllla receptor or a functional fragment thereof from IgG, IgM, IgA, IgD or IgE.

[0442] The transmembrane domain of CAR can be a derived transmembrane protein, such as the α, β 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, IT GA6, VLA-6, CD49f, ITGAD, ITGAE, ITGAL, LFA-1, ITGAM, ITGAX, ITGB1, ITGB2, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CEACAM1, CRT AM, 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 functional fragments thereof.

[0443] The intracellular signaling domain of CAR participates in the signal transduction of the effective CAR binding to the target antigen to the inside of the immune effector cell, to trigger effector cell functions, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the target cells bound by CAR, or other cellular responses triggered after the antigen binds to the extracellular CAR domain. Non-limiting examples of the intracellular signaling domain of CAR include those derived from CD3ζ, CD3ε, CD3δ, CD3γ, CD5, CD22, CD39, CD79A, CD79B, CD66d, CD226, DAP10, DAP12, Fcε receptor Iγ chain (FCER1G) or FcRβ.

[0444] The intracellular costimulatory domain of CAR can provide the second signal required for effective activation and function when T lymphocytes bind to antigens. This costimulatory domain can be derived from one or more costimulatory 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, NKD2CSLP76, TRIM, BTLA, GITR, CD226, HVEM and ZAP70.

[0445] CAR can be generated by standard molecular biology techniques. The extracellular domain that binds to the desired antigen can be derived from an antibody or antigen-binding fragment thereof described herein.

[0446] In another aspect, the disclosure also provides cells comprising peptide binding portions of the disclosure (e.g., TCR and CAR). In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is a T cell, a NK cell, or a macrophage. Relative to the subject receiving the cell (as treatment), the host cell can be autologous or allogeneic.

[0447] In some embodiments, the TCR of the present disclosure is provided in the form of TCR-T cells. In some embodiments, the CAR of the present disclosure is provided in the form of CAR-T cells. Any method known in the art for modifying T cells to express TCR or CAR can be used to generate TCR-T or CAR-T cells disclosed herein.

[0448] Cells expressing the peptide binding moieties of the present disclosure (e.g., TCR and CAR) may also include one or more additional genes. The additional genes may be used to increase the effector function of cells expressing peptide binding moieties (e.g., TCR and CAR). Non-limiting examples of additional gene categories include: (a) a second targeting moiety, such as an antibody, including fragments thereof and bispecific antibodies (e.g., bispecific T cell engagers (BiTE)), (b) secretable 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) constitutive Active cytokine receptors (e.g., C7R), (f) dominant negative receptors (DNR; e.g., TGFRIIDNR), (g) ligands of co-stimulatory molecules (e.g., CD80, 4-1BBL), (h) nuclear factor 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 molecules). In some embodiments, cells expressing the peptide binding portions of the present disclosure (e.g., TCR and CAR) may express a second targeting portion targeting the liver or another known liver cancer antigen.

[0449] Pharmaceutical compositions, dosage forms and administration

[0450] In a further aspect, the present disclosure provides a pharmaceutical composition comprising a peptide of the present disclosure, a peptide-based molecule (such as a complex comprising the peptide (e.g., a peptide-MHC (pMHC) complex), a fusion protein or a conjugate), a nucleic acid molecule, a vector, a cell or a peptide binding portion, and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition of the present disclosure can be in any suitable form (depending on the desired method of administration to the patient). Suitable compositions and methods of administration are known to those skilled in the art, for example, see Johnson et al., Blood. 2009; 114(3):535-46.

[0451] Pharmaceutical compositions can include peptides of the present disclosure or peptide-based molecules, which can be in the form of free form or pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of a peptide of the present disclosure, wherein the peptide is modified by an acidic salt or alkaline salt of the manufacturing agent. For example, an acidic salt is prepared by a free alkali (usually wherein the neutral form of the drug has a neutral -NH2 group), involving reaction with a suitable acid. Suitable acids for preparing acidic salts include organic acids and inorganic acids, 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. Conversely, basic salts of acidic 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, and the like.

[0452] The compositions of the present disclosure can comprise a plurality of peptides, for example, 2 to 50, 2 to 40, 2 to 30, 5 to 25, 5 to 20, or 10 to 15 peptides as described herein (eg, 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 a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0453] In some embodiments, the peptide or peptide-based molecule can be present in the solution at a concentration of about 1 μg / mL to 50 mg / mL, for example, a concentration of 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, 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, or about 0.8 mg / mL. g / mL, 0.8mg / mL, 0.9mg / mL, 1mg / mL, 1.25mg / mL, 1.5mg / mL, 1.75mg / mL, 2mg / mL, 2.25mg / mL, 2.5mg / mL, 2.75mg / mL, 3mg / mL, 3.25mg / mL, 3. 5mg / mL, 3.75mg / mL, 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL, 10mg / mL, 11mg / mL, 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL or 20mg / mL.

[0454] The pharmaceutical composition may be suitable for administration by any appropriate route, such as, for example, parenteral (including subcutaneous, intramuscular or intravenous), enteral (including oral or rectal), inhalation or intranasal routes.

[0455] Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier or excipient under sterile conditions.

[0456] In addition, disclosed herein are pharmaceutical dosage forms comprising a peptide of the present disclosure, a peptide-based molecule (such as a complex comprising the peptide (e.g., a peptide-MHC (pMHC) complex), a fusion protein or a conjugate), a nucleic acid molecule, a vector, a cell or a binding moiety.

[0457] Pharmaceutical compositions based on the peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide), nucleic acid molecules, vectors, cells, or binding moieties disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins, or conjugates comprising the peptide), nucleic acid molecules, vectors, cells, or binding moieties can be formulated for administration, for example, by injection, inhalation, or insulation (through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or directly to an organ or tissue.

[0458] The pharmaceutical composition can be formulated for a variety of administrations, including systemic, local or focal administration. Techniques and formulations are available, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous injections. For injection purposes, 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. In addition, the pharmaceutical composition can be formulated in a solid form and redissolved or suspended immediately before use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0459] In some embodiments, the pharmaceutical composition of the present disclosure can be lyophilized. As a non-limiting example, the lyophilized material obtained can be compounded into an aqueous composition by adding to an aqueous solvent. In some embodiments, the aqueous composition can be directly administered to a patient parenterally. Therefore, a further embodiment of the present disclosure is an aqueous pharmaceutical composition that can be obtained by compounding a lyophilized material with an aqueous solvent.

[0460] In some embodiments, the pharmaceutical compositions disclosed herein may include a lyophilized formulation. As a non-limiting example, a lyophilized formulation may include a peptide of the present disclosure, mannitol and / or As another non-limiting example, the lyophilized formulation may comprise a peptide disclosed herein, mannitol, and poloxamer 188. In some embodiments, a pharmaceutical composition may comprise a lyophilized formulation comprising a reconstituted liquid composition.

[0461] In some embodiments, the pharmaceutical composition of the present disclosure can provide a preparation, wherein the lyophilized product of the preparation has enhanced solubility and / or wettability compared to previously known compositions. As a non-limiting example, an appropriate excipient composition can be used to achieve enhanced solubility and / or wettability of the lyophilized product. In this way, the pharmaceutical composition of the present disclosure comprising SEQ ID NO:1-54 peptides and variants thereof can be developed to show desired storage stability at (for example, at -20 ° C, +5 ° C or +25 ° C), and can be easily redissolved so that the lyophilized product can be completely dissolved in a few seconds to two minutes or more minutes by using a buffer or other excipients, with or without an ultrasonic homogenizer. In addition, the composition can be easily provided to patients in need of treatment by any appropriate 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 can be between pH 2.7 and pH 9.

[0462] For oral administration, the pharmaceutical composition can be in the form of tablets or capsules prepared by conventional means, for example, using pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or they can be presented as dry products and compounded with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., ationd oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl paraben or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents and sweeteners, as appropriate.

[0463] The pharmaceutical composition can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. The injection preparation can be presented in unit dosage form, for example in an ampoule or a multi-dose container, optionally with the addition of a preservative. The pharmaceutical composition can be further formulated as a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain other agents, including suspending agents, stabilizers and / or dispersants.

[0464] In addition, pharmaceutical compositions can also be formulated into long-acting preparations. These long-acting preparations can be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Therefore, for example, the compound can be formulated with a suitable polymeric material or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a slightly soluble derivative, for example, as a slightly soluble salt. Other suitable delivery systems include microspheres, which provide the possibility of local non-invasive delivery of drugs over a long period of time. The technology can include microspheres with a pre-capillary size, which can be injected into any selected part of an organ through a coronary catheter without causing inflammation or ischemia. The therapeutic drug administered is slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.

[0465] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to promote penetration. Transmucosal administration can be carried out using nasal sprays or suppositories. For topical administration, the carrier particles described herein can be formulated into ointments, salves, gels or creams commonly known in the art. Cleansing solutions can also be applied topically for the treatment of injuries or inflammations to accelerate healing.

[0466] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be liquid. It must remain stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing), and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0467] If the formulations disclosed herein are used as therapeutic agents to enhance the immune response of a subject, the therapeutic agent can be formulated as a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.).

[0468] 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.), their appropriate mixtures, and vegetable oils. For example, by using a coating (such as lecithin), maintaining the desired particle size in the case of dispersion and using a surfactant, suitable fluidity can be maintained. Preventing the effects of microorganisms can be achieved by various antibacterial and antifungal agents known in the art. In many cases, isotonic agents, such as sugar or sodium chloride, are preferably included. The absorption of the injectable composition can be extended by using an agent that delays absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0469] Sterile injectable solutions can be prepared by incorporating the active compound or construct in the required amount in an appropriate solvent and mixing with various other ingredients as required, followed by filtered sterilization.

[0470] When formulated, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. These preparations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, but sustained release capsules or microparticles and microspheres, etc. can also be used.

[0471] For example, for parenteral administration with an aqueous solution, the solution should be appropriately buffered if necessary, and the liquid diluent is first isotonic with sufficient saline or glucose. These special aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous and intraperitoneal administration. In this context, in view of the present disclosure, those skilled in the art will understand the sterile aqueous media that can be used. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous infusion or injected at the infusion site of the suggestion.

[0472] In any case, the personnel responsible for administration will determine the appropriate dosage for individual subjects. For example, a subject can administer a peptide described herein, a peptide-based molecule (such as a complex comprising the peptide (e.g., peptide-MHC (pMHC) complex), a fusion protein or a conjugate), a nucleic acid molecule, a carrier, a cell or a binding moiety once a day or once a week for a period of time, or monthly, biennially or annually, depending on the need or exposure to a pathogenic organism (e.g., HBV) or the subject's condition (e.g., cancer).

[0473] 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; timed release capsules; biodegradable and any other form currently used.

[0474] Intranasal or inhalable solutions or sprays, aerosols or inhalants may also be used. Nasal solutions may be aqueous solutions designed to be applied to the nasal passages in the form of drops or sprays. Nasal solutions may be prepared to resemble nasal secretions in many respects. Therefore, aqueous nasal solutions are generally isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers (if necessary) may be included in the preparation. Various commercial nasal preparations are known and may include, for example, antibiotics and antihistamines and are used for asthma prevention.

[0475] Oral formulations can include excipients, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, or powders. In certain embodiments, oral pharmaceutical compositions will include inert diluents or assimilable edible carriers, or they can be encapsulated in hard or soft shell gelatin capsules, or they can be compressed into tablets, or they can be directly incorporated into the food of a meal. For oral therapeutic drug administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0476] Tablets, lozenges, pills, capsules, etc. may also contain the following materials: binders, such as gum tragacanth, gum arabic, corn starch or gelatin; excipients, such as calcium hydrogen phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose or saccharin, or flavorings, such as peppermint, wintergreen oil or cherry flavorings, may be added. When the dosage unit form is a capsule, it may also contain a liquid carrier in addition to the above-mentioned types of materials. Various other materials may exist as coatings or otherwise change the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Elixir syrups may contain active compounds, sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes and flavorings, such as cherry or orange flavors.

[0477] Further embodiments disclosed herein may relate to a kit for use with the method and composition. The kit may also include a suitable container, such as a vial, a tube, a micro or microcentrifuge tube, a test tube, a flask, a bottle, a syringe or other container. In the case of providing an additional component or an agent, the kit may include one or more additional containers, into which the agent or component may be placed. The kit herein generally also includes a device for sealingly accommodating peptides, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins or conjugates comprising peptides), nucleic acid molecules, carriers, cells or binding moieties and any other reagent containers for commercial sale. Such containers may include injection molding or blow molding plastic containers, wherein the desired vial is retained. Optionally, for the described composition, one or more additional active agents may be required.

[0478] The dosage range and the frequency of administration can vary according to the properties of the composition and the medical condition and the parameters of the specific patient and the route of administration used. The dosage can also depend on the subject receiving the administration. For example, if the subject is a teenager, a lower dosage may be required, and if the subject is an adult human subject, a higher dosage may be required. In certain embodiments, a more accurate dosage can depend on the subject's weight. Suitable non-limiting examples of the dosage of the pharmaceutical composition comprising those substances disclosed herein can vary according to the age and size of the subject to be administered, the target disease, the purpose of treatment, the 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, an initial dose may be followed by administration of a second or more subsequent doses, which may be approximately the same amount as or less than the initial dose, wherein the subsequent doses are separated by at least 1 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.

[0479] The composition can include administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesically, intramucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by local infusion, by catheter, by lavage, in a cream, or in a lipid composition.

[0480] Certain additional agents used in combination therapy may be formulated and administered by any means known in the art.

[0481] Compositions as disclosed herein may also include adjuvants, such as aluminum salts and other mineral adjuvants, tonicity agents, bacterial derivatives, vehicles, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants may stimulate Th1 or Th2 immunity. Compositions and methods as disclosed herein may also include adjuvant therapy.

[0482] The peptides or peptide-based molecules disclosed herein can be provided in the form of vaccine compositions. Vaccine compositions can be used to treat or prevent HBV infection and / or HBV-induced diseases or conditions. It is to be understood that vaccines can take a variety of 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). Vaccine compositions can include additional peptides or peptide-based molecules so that the peptides or peptide-based molecules disclosed herein are one of a mixture of peptides or peptide-based molecules. Adjuvants can be added to vaccine compositions to enhance immune responses. In particular, for the peptide-containing vaccine compositions of the present disclosure, pharmaceutically acceptable adjuvants include, but are not limited to, aluminum salts, Amplivax, AS15, Aquila QS21stimulon, AsA404 (DMXAA), β-glucan, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact EV1P321, IS Patch, ISS, 1018ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, poly-ICLC, Pam3Cys, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, and / or valdemexin.

[0483] Alternatively, the vaccine composition may be in the form of an APC displaying the peptides of the present disclosure in complex with MHC. Preferably the APC is an immune cell, more preferably a dendritic cell or a B cell. The peptide may be pulsed onto the cell surface (Thurner, J Exp Med. 1999; 190(11): 1669-78), or a nucleic acid encoding the peptides of the present disclosure may be introduced into a dendritic cell or a B cell (e.g., by electroporation. Van Tendeloo, Blood. 2001; 98(1): 49-56).

[0484] The pharmaceutical composition of the present disclosure can be directly administered to patients, affected organs or systemically via intradermal injection (id), intramuscular injection (im), subcutaneous injection (sc), intraperitoneal injection (ip) and intravenous injection (iv), or applied in vitro to cells derived from patients or human cell lines, then applied to patients, or used in vitro to select immune cell subsets derived from patients, and then re-applied to patients. If nucleic acid is applied to cells in vitro, transfected cells can be useful to co-express immunostimulatory cytokines (such as interleukin-2). Peptides or peptide-based molecules can be substantially pure, or combined with immunostimulatory adjuvants or used in conjunction with immunostimulatory cytokines, or administered together with suitable delivery systems (e.g., liposomes, viral particles, VLPs). Peptides or peptide-based molecules can also be conjugated to suitable carriers, such as keyhole limpet hemocyanin (KLH) or mannan (see, e.g., WO 95 / 18145 and Longenecker et al., 1993).

[0485] In some embodiments, the peptide-containing compositions described herein further comprise an accessory molecule that can modulate the survival or activity of cells expressing the TCR.

[0486] Non-limiting examples of useful auxiliary molecules include, for example, anti-CD28 antibodies, anti-CD80 (B7.1) antibodies, anti-CD86 (B7.2) antibodies, anti-CD3 antibodies, anti-CD2 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD47 antibodies, and functional derivatives, mutants and fragments thereof.

[0487] Accessory molecules used in the peptide-containing compositions described herein include 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, for example, the binding of the TCR / CD3 complex to the pMHC complex.

[0488] The accessory molecule can be, for example, an inhibitory or stimulatory antibody, a peptide ligand, a co-stimulatory peptide, a cytokine, 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 co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, FIVEM, lymphotoxin beta receptor, 3 / TR6, ILT 3. ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors and ligands that specifically bind to B7-H3, as well as antibodies 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 ligands that specifically bind to CD83.

[0489] Other non-limiting examples of accessory molecules include, for example, TNF / TNF family members (e.g., OX40L, ICOSL, FASL, LTA, LTB TRAIL, CD153, TNFSF9, RANKL, TWEAK, TNFSF13, TNFSF13b, TNFSF14, TNFSF15, TNFSF18, CD40LG, CD70); immunoglobulin superfamily members (e.g., VISTA, PD1, PD-L1, PD-L2, B71, B72, CTLA4, CD28, TIM3, CD4, CD8, CD19, T cell receptor chain, ICOS, ICOS ligand, HHLA2, butyrophilms, BTLA, B7-H3, B7-H4, CD3, CD79a, CD79b, IgSF CAMs (including CD2, CD58, CD48, CD150, CD229, CD244, ICAM-1), leukocyte immunoglobulin-like receptors (LILRs), killer cell immunoglobulin-like receptors (KIRs)), lectin superfamily members, selectins, cytokines / chemokines and cytokine / chemokine receptors, growth factors and growth factor receptors), adhesion molecules (integrins, fibronectin, cadherins) or the extracellular domains of multi-spanning integral membrane proteins, or antibodies against any of these molecules.

[0490] In some embodiments, the peptide compositions described herein also include a cytotoxic agent. In a specific embodiment, the cytotoxic agent is a toxin or a radioactive isotope (e.g., a radioactive conjugate) or a suicide gene. Non-limiting examples of toxins that can be used in the peptide compositions described herein include, for example, toxins with enzymatic activity from bacteria, fungi, plants or animals, or fragments, mutants or derivatives thereof. 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 (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, PAPI, PAPII and PAP-S, Momordica charantia inhibitor, curcin, crotonin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enonomycin and tricothecene. Non-limiting examples of suicide genes include thymidine kinase, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, β-galactosidase, hepatic cytochrome P450-2B1, linamarase, horseradish peroxidase and carboxypeptidase.

[0491] Methods for introducing polypeptides or polynucleotides of the present disclosure into cells or subjects may include, for example, vector delivery, particle-mediated delivery, exosome-mediated delivery, lipid nanoparticle-mediated delivery, cell-penetrating peptide-mediated delivery, or implant-mediated delivery. In some embodiments, nucleic acids or proteins can be introduced into cells or subjects in carriers such as polylactic acid (PLA) microspheres, poly (D, L-lactic acid-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, or lipid microtubules.

[0492] It is contemplated herein to use nanoparticles to deliver polypeptides or polynucleotide compositions of the present disclosure. Exemplary nanoparticles include, but are not limited to, polymer nanoparticles, inorganic nanoparticles, liposomes, lipid nanoparticles (LNP), immunostimulatory complexes (ISCOM), virus-like particles (VLP) or self-assembling proteins. The nanoparticles can be calcium phosphate nanoparticles, silicon nanoparticles or gold nanoparticles. For example, polymer nanoparticles can include one or more synthetic polymers, such as poly (d, l-lactide-co-glycolide) (PLG), poly (d, l-lactide-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, for example amylopectin, alginate, inulin and chitosan. Due to the characteristics of the polymers that can be included in the nanoparticles, it can be advantageous to use polymer nanoparticles. For example, the natural and synthetic polymers mentioned above can have good biocompatibility and biodegradability, non-toxic properties and / or can be manipulated into desired shapes and sizes. Polymer nanoparticles can also form hydrogel nanoparticles, hydrophilic three-dimensional polymer networks, with good properties, including flexible mesh size, large surface area for multivalent conjugation, high water content and high antigen loading capacity. Polymers such as poly (L-lactic acid) (PLA), PLGA, PEG and polysaccharides are suitable for forming hydrogel nanoparticles. Inorganic nanoparticles usually have a rigid structure and include a shell or a core to which an antigen can be covalently attached, wherein the shell is encapsulated with an antigen. The core can include one or more atoms, such as gold (Au), silver (Au), copper (Cu) atoms, Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd or Au / Ag / Cu / Pd or calcium phosphate (CaP).

[0493] Other molecules suitable for complexing with the polypeptides or polynucleotides of the present disclosure include cationic molecules such as polyamidoamine, dendritic polylysine, polyethylene irinine or polypropylene imine, polylysine, chitosan, DNA-gelatin coarcervate, DEAE dextran, dendrimers or polyethyleneimine (PEI).

[0494] In some embodiments, the antibodies of the present disclosure can be conjugated with nanoparticles. Nanoparticles that can be used to conjugate with 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, nanoparticles based on poly (malic acid), nanoparticles of chitosan shells, carbon nanotubes and other inorganic nanoparticles (such as nanoparticles made of magnesium-aluminum layered double hydroxide and disuccinimidyl carbonate (DSC) and TiO2 nanoparticles). Nanoparticles can be developed and conjugated with antibodies contained in pharmaceutical compositions to target virally infected cells.

[0495] Treatment

[0496] The compositions of the present disclosure, including the peptides of the present disclosure, peptide-based molecules (such as complexes (e.g., peptide-MHC (pMHC) complexes), fusion proteins or conjugates comprising peptides), nucleic acid molecules, vectors, cells or binding portions thereof, can be used to prevent and / or treat viral infections (e.g., HBV infections) and / or diseases or conditions caused by viral infections (e.g., HBV infections).

[0497] In one aspect, disclosed herein is a method of modulating the activity, proliferation or survival of a cell comprising a TCR, comprising contacting the cell with a composition (eg, a peptide, complex (eg, a pMHC complex), fusion protein or conjugate) of the present disclosure.

[0498] In some embodiments, the cell is a lymphocyte, such as, for example, a T cell (e.g., a CD4+T cell or a CD8+T cell). In some embodiments, the target T cell is a CD4+T cell, such as a helper T cell (e.g., a Th1, Th2 or Th17 cell) or a CD4+ / CD25+ / FOXP3+ regulatory T (Treg) cell. In some cases, the target T cell is a CD8+T cell, such as, for example, a cytotoxic T cell. In some cases, the target T cell is a memory T cell, which can be a CD4+T cell or a CD8+T cell, wherein the memory T cell is typically CD45RO+. In some cases, the target T cell is a NK-T cell.

[0499] In some embodiments, the contacting is ex vivo. In some embodiments, the contacting is performed in vivo in a subject (e.g., a human).

[0500] In some embodiments, the cell is a mammalian cell (eg, a human cell).

[0501] In some embodiments, for example, when the target T cell is a CD8+ T cell, the peptide is presented by a class I MHC polypeptide. In some embodiments, for example, when the target T cell is a CD4+ T cell, the peptide is presented by a class II MHC polypeptide.

[0502] The interaction of T cells with peptides described herein can lead to, for example, activation of T cells, induction of anergy or death, which occurs when the TCR of T cells is combined with a TCR binding molecule (e.g., a pMHC complex). "Activation of T cells" refers to inducing a signal transduction pathway in a T cell, causing the T cell to produce a cell product (e.g., interleukin-2). "Anergy" refers to the disappearance of the reactivity of T cells to antigens. Activation and anergy can be measured, for example, by measuring the amount of IL-2 produced by T cells after the pMHC complex is combined with the TCR. Compared with stimulated T cells, the IL-2 produced by anergy cells will be reduced. Another method for measuring the disappearance of the activity of anergy T cells includes measuring intracellular and / or extracellular calcium mobilization of T cells when engaging with their TCR. "T cell death" refers to the permanent cessation of almost all functions of T cells.

[0503] 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 moieties).

[0504] In certain embodiments, generating an immune response includes an increase in target antigen-specific cytotoxic T lymphocyte (CTL) activity by about 1.5 to 20 times, or more, in a subject administering a composition of the present invention compared to a control. In certain embodiments, generating an immune response includes an increase in target-specific CTL activity by about 1.5 to 20 times, or more, in a subject administering a composition of the present invention compared to a control. In a further embodiment, generating an immune response includes an increase in target antigen-specific cell-mediated immune activity by about 1.5 to 20 times, or more, compared to a control, as measured by an ELISpot assay measuring cytokine secretion, such as interferon-γ (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) or other cytokines.

[0505] In a further embodiment, generating an immune response comprises increasing target-specific antibody production by 1.5 to 5 times in a subject administered a composition of the present disclosure compared to an appropriate control. In another embodiment, generating an immune response comprises increasing target-specific antibody production by about 1.5 to 20 times, or more, in a subject administered a composition of the present disclosure compared to a control.

[0506] T cell activation can be determined, for example, by measuring changes in cytokine and / or T cell activation marker expression levels, and / or the induction of antigen-specific proliferating cells. Techniques known to those skilled in the art, including but not limited to, immunoprecipitation, followed by protein immunoblotting analysis, ELISA, flow cytometry, Northern blot analysis and RT-PCR, can be used to measure the expression of cytokines and T cell activation markers. 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-12), 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.

[0507] T cell regulation can also be measured by measuring, for example, proliferation (by, for example, 3 H-thymidine incorporation, trypan blue cell counting, and fluorescence activated cell sorting (FACS) were used to assess the

[0508] The anti-tumor response of T cells can be determined in xenograft tumor models. Any human cancer cell line expressing the relevant tumor-associated antigen can be used to establish the tumor. To establish the xenograft tumor model, approximately 5 × 10 6 The live cells are injected subcutaneously into athymic nude mice, for example, using Matrigel (Becton Dickinson). The endpoints of xenograft tumor models can be determined using methods known to those skilled in the art based on tumor size, animal weight, survival time, and histochemical and histopathological examination of the cancer.

[0509] In a related aspect, 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 moieties).

[0510] In a related aspect, disclosed herein is a method of preventing or reducing the likelihood of developing a HBV-induced disease or condition 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 moieties).

[0511] The disease or condition induced by HBV can be, for example, liver inflammation (chronic or acute), liver fibrosis, cirrhosis, liver failure or liver cancer. Liver cancer can be hepatocellular carcinoma (HCC) or intrahepatic bile duct carcinoma and hepatoblastoma. In one embodiment, liver cancer is hepatocellular carcinoma (HCC).

[0512] Hepatitis B virus infection can be acute (self-limited) or chronic (long-term). People with self-limited infection spontaneously clear the infection within weeks to months. In some cases, "chronic hepatitis" may result. Chronic hepatitis occurs when the body is unable to completely clear the virus, even though symptoms may not persist.

[0513] Acute infection with the hepatitis B virus is associated with acute viral hepatitis, an illness that begins with a general feeling of ill health, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and progresses to jaundice. It has been noted that pruritus is indicative of possible symptoms with all hepatitis virus types. Most affected patients remain ill for several weeks and then gradually improve. A minority of patients may develop more severe liver disease (e.g., fulminant hepatic failure) and may die from it. The infection can be completely asymptomatic and may go unrecognized. Persistence of the virus for several years can lead to cirrhosis. This type of infection significantly increases the incidence of liver cancer (e.g., hepatocellular carcinoma).

[0514] HBV carriers can be identified by testing the serum or blood for the presence of HBV viral antigens or antibodies produced by the host. Hepatitis B surface antigen (HBsAg) is most commonly used to screen for the presence of this infection. PCR testing can also be used to detect and measure the amount of viral nucleic acid in clinical specimens.

[0515] When the disease being treated is cancer, the cancer may specifically be of the following histological types, but is not limited to these: 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; pilomatricoma carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma, familial polyposis coli; solid carcinoma; malignant carcinoid tumor; bronchiolar-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma alveolar adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; skin adnexal carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; cerumenous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma cell tumor; malignant granuloma cell tumor; and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma; non-melanoma melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mixed Müllerian tumor; Wilms tumor; hepatoblastoma; carcinosarcoma; malignant stromal tumor; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian thyroid tumor; choriocarcinoma; malignant mesonephroblastoma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical bone Sarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoderm; cerebellar sarcoma; ganglioblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant small lymphocytic lymphoma; malignant large cell, diffuse lymphoma; malignant follicular lymphoma; mycosis fungoides; other specified non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0516] It is contemplated that when used to treat various diseases, the compositions and methods may be used in conjunction with other therapeutic agents applicable to the same or similar diseases. In addition, two or more embodiments described herein may also be co-administered to produce an additive or synergistic effect. When co-administered with a second therapeutic agent, the embodiments described herein and the second therapeutic agent may be administered simultaneously or sequentially (in any order). Due to additive or synergistic effects, the appropriate therapeutically effective dose of each agent may be reduced.

[0517] In some embodiments, the compositions and methods disclosed herein can be used to enhance the efficacy of vaccines for HBV infection or HBV-induced diseases (e.g., liver cancer, such as HCC). Therefore, the compositions and methods described herein can be administered to a subject simultaneously with an agent (including but not limited to a small molecule, antibody, or cell-acting agent) that acts to cause an immune response (e.g., for treating HBV infection or liver cancer) or before the agent is administered to the subject (e.g., 1-30 days before).

[0518] The compositions and methods described herein can also be administered in conjunction with anti-tumor antibodies or antibodies directed against pathogenic antigens (eg, HBV) or allergens.

[0519] The compositions and methods described herein can be combined with other immunomodulatory treatments, 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 treatments described herein can also be used in combination with other treatments that have the ability to modulate NKT function or stability, including but not limited to CD1d, CD1d fusion proteins, CD1d dimers, or larger polymers of CD1d that are not loaded or loaded with antigens, CD1d chimeric antigen receptors (CD1d-CAR), or any other isomer of the five known CD1 isomers (CD1a, CD1b, CD1c, CD1e) present in humans, in any of the above forms or formulations, alone or in combination with each other or in combination with other agents.

[0520] The treatment methods described herein can be combined with other immunotherapies and therapies. For example, when used to treat cancer, the NKT cells described herein can be used in combination with cancer therapies, such as, for example, surgery, radiotherapy, chemotherapy, or a combination thereof, depending on the type of tumor, patient condition, other health problems, and various factors. In some aspects, other therapeutic agents that can be used in combination with the inhibitors described herein for cancer therapy include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor β, interferon-α, soluble KDR and FLT-1 receptors, placental proliferator-associated protein, and those listed by Carmeliet and Jain (2000). In some embodiments, the inhibitors described herein can be used in combination with a VEGF antagonist or VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a VEGFR tyrosine kinase inhibitor, and any combination thereof (e.g., the anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0521] Non-limiting examples of chemotherapeutic compounds that can be used in combination therapy include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, bacillus Calmette-Guérin (bcg), bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, , chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine ), genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine,medroxyprogesterone acetate, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate ate), pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0522] These chemotherapeutic compounds can be divided into several categories according to their mechanism of action, for example, the following: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine));Antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones, and navelbine bine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide amide (cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as actinomycin D ( D), daunorubicin, adriamycin, idarubicin, anthracyclines, mitoxantrone, bleomycins, mithramycin, and mitomycin;enzymes (L-asparaginase, which metabolizes L-asparagine systemically and removes cells that do not have the ability to synthesize their own asparagine); antiplatelet drugs; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazene-dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes drugs (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents (such as thrombin inhibitors, streptokinase and urokinase); agent); antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin);mTOR inhibitors, topoisomerase inhibitors (adriamycin, amsacrine, camptothecin, daunorubicin, actinomycin D, teniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors. ;

[0523] For the treatment of viral infections, the combination therapies described herein may include administering the compositions and methods described herein in conjunction with antiviral drugs. Non-limiting examples of useful antiviral drugs 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 salt or variant thereof. See also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson PDR, Montvale NJ; 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.

[0524] Reagent test kit

[0525] The present disclosure also includes kits that can contain any of the various compositions of the present disclosure, including a peptide, a peptide-based molecule (such as a complex comprising the peptide (e.g., a peptide-MHC (pMHC) complex)), a fusion protein or a conjugate), a nucleic acid molecule, a vector, a cell or a binding moiety of the present disclosure.

[0526] In one aspect, the disclosure may include a kit comprising, for example: (a) a container comprising a pharmaceutical composition disclosed herein, e.g., a solution or a lyophilized form of the pharmaceutical composition; (b) optionally, a second container containing a diluent or a reconstitution solution for the lyophilized formulation; and / or (c) optionally, instructions for use of (i) the solution or (ii) the reconstitution and / or use of the lyophilized formulation.

[0527] In some embodiments, the kit may further comprise, for example, but not limited to, one or more of: (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, a vial, a syringe, or a test tube. In some embodiments, the container may be a multi-purpose container. In some embodiments, the pharmaceutical composition may be lyophilized.

[0528] The kit of the present disclosure may include instructions for use of the lyophilized formulation of the present disclosure in a suitable container and its compounding and / or use. Suitable containers include, for example, bottles, vials (e.g., double-chamber bottles), syringes (e.g., double-chamber syringes) and test tubes. The container can be made of a variety of materials, such as glass or plastic. The kit and / or container may include instructions for use on or associated with the container, which instructions for use indicate compounding of the lyophilized formulation and / or instructions for use of the kit. For example, a label may indicate that the lyophilized formulation will be compounded to an appropriate peptide concentration. The label may indicate that the formulation is useful for any route of administration disclosed herein or is intended for any route of administration disclosed herein, for example, a parenteral route of administration disclosed herein.

[0529] The container holding the formulation may be a multi-use vial, which allows repeated administration (eg, 2-6 administrations) of the reconstituted formulation. The kit may also comprise: a second container comprising a suitable diluent (eg, sodium bicarbonate solution).

[0530] After the diluent and the lyophilized formulation are mixed, the final peptide concentration in the reconstituted formulation is reached. The kit may also contain other materials desirable from a commercial and / or user perspective, including, but not limited to, for example, other buffers, diluents, filters, needles, syringes and / or package inserts, which may include, for example, instructions for use.

[0531] The kits of the present disclosure may have a single container containing a formulation of a pharmaceutical composition according to the present disclosure, with or without other components (e.g., other compounds or pharmaceutical compositions of these other compounds), or may have distinct containers for each component.

[0532] In some embodiments, the kits of the present disclosure may include a formulation of the present disclosure packaged for co-administration in combination with a second compound, such as an adjuvant (e.g., GM-CSF, a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer or a chelator), or a pharmaceutical composition thereof. The components of the kit may be pre-compounded, or each component may be placed in a separate, distinct container prior to administration to a patient. The components of the kit may be provided in the form of one or more liquid solutions. The liquid solutions described herein may be aqueous solutions, such as sterile aqueous solutions. The components of the kit may also be provided in solid form, which solids may be converted to liquids, such as by adding a suitable solvent, which solvents may be provided in another, distinct container.

[0533] The container of the treatment kit can be a vial, test tube, flask, bottle, syringe or any other device for encapsulating solid or liquid. When there are multiple components, the kit can include a second vial or other container, which can allow separate administration. The kit can also include another container for pharmaceutically acceptable liquid. In some embodiments, the kit can include a device (e.g., one or more needles, syringes, eye droppers, pipettes, etc.) that can allow the use of an agent of the present disclosure that is a component of the kit.

[0534] Methods and systems for identifying immunogenic virus-derived peptides

[0535] The present disclosure also includes a method and system for identifying immunogenic virus-derived peptides. Some or all of the steps of the method can be performed by a computing device. In certain aspects, some or all of the steps of the method can be stored in a memory as computer-readable instructions, such that the instructions can be executed by one or more processors to perform functions associated with the method. As a non-limiting example, the method and system for identifying immunogenic virus-derived peptides can include Figure 1A Various aspects of the HBV genome reconstruction pipeline are shown.

[0536] In one aspect, the present disclosure may include a method for identifying immunogenic virus-derived peptides, the method comprising the following steps: (a) obtaining multiple RNA contig sequences derived from an infected subject infected with a virus, wherein the multiple RNA contig sequences include multiple virus-derived RNA contig sequences and multiple infected subject endogenous RNA contig sequences; (b) identifying multiple virus-derived RNA contig sequences from the multiple RNA contig sequences; (c) assembling viral RNA sequences based on the multiple virus-derived RNA contig sequences; (d) identifying protein sequences based on the viral RNA sequences; and (e) identifying immunogenic virus-derived peptides based at least in part on the identified protein sequences.

[0537] In one aspect, a system configured to perform functions associated with 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 thereon, which, 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, such that the plurality of RNA contig sequences include a plurality of virus-derived RNA contig sequences and a plurality of infected subject endogenous RNA contig sequences, and wherein the infected subject is infected with a virus; (b) identify the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences; (c) assemble a 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 protein sequence; and (f) provide the immunogenic virus-derived peptide as output.

[0538] In some embodiments, the plurality of RNA contig sequences can be derived from an infected subject.

[0539] In some embodiments, the infected subject can be a human.

[0540] In some embodiments, the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

[0541] In some embodiments, the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

[0542] In some embodiments, identifying the multiple virus-derived RNA contig sequences from the multiple RNA contig sequences (step b) may further include: comparing at least a portion of the contig sequences of the multiple RNA contig sequences with a reference viral sequence; and identifying the multiple virus-derived RNA contig sequences such that each contig sequence in the multiple virus-derived RNA contig sequences contains at least a portion of the sequence corresponding to the reference viral sequence.

[0543] In some embodiments, each contig sequence in the plurality of virus-derived RNA contigs can be different from the plurality of infected subject endogenous RNA contig sequences.

[0544] In some embodiments, each contig sequence in the plurality of virus-derived RNA contig sequences can lack an RNA contig sequence endogenous to the infected subject.

[0545] In some embodiments, the reference viral sequence may include a reference genome.

[0546] In some embodiments, the reference genome may include a hepatitis B virus genome.

[0547] In some embodiments, assembling the viral RNA sequence based on the multiple virus-derived RNA contiguous group sequences (step c) may include: overlapping the common sequence portion at the end of at least a portion of the multiple virus-derived RNA contiguous group sequences so that at least a portion of the multiple virus-derived RNA contiguous group sequences are linearly overlapped to assemble the viral RNA sequence.

[0548] In some embodiments, identifying a protein sequence based on the viral RNA sequence such that the protein sequence includes a translation of the viral RNA sequence (step d) may include identifying the protein sequence without comparison to a viral protein database.

[0549] In some embodiments, identifying a protein sequence based on the viral RNA sequence so that the protein sequence includes a translation of the viral RNA sequence (step d) may include: identifying multiple protein sequences based on the viral RNA sequence, each of the multiple protein sequences includes a translation of the viral RNA sequence; and identifying the protein sequence as a frequently occurring protein sequence among the multiple protein sequences.

[0550] In some embodiments, the protein sequence can be identified based on viral RNA sequences associated with a single infected subject.

[0551] In some embodiments, identifying the immunogenic virus-derived peptide based at least in part on the protein sequence (step 3) can include: identifying an MHC molecule associated with a single infected subject; identifying one or more peptides based at least in part on the protein sequence such that each of the one or more peptides forms a corresponding MHC-peptide complex with the MHC molecule; and identifying the immunogenic virus-derived peptide based on the one or more peptides.

[0552] Methods and systems for identifying integration sites of viral genes within genes of a subject

[0553] The present disclosure further includes a method and system for identifying the integration site of a viral gene within a subject's gene. Some or all of the steps of the method may be performed by a computing device. In some aspects, some or all of the steps of the method may be stored in a memory as computer-readable instructions so that the instructions may be executed by one or more processors to perform functions associated with the method. As a non-limiting example, a method and system for identifying the integration site of a viral gene within a subject's gene may include Figure 1A Various aspects of the HBV integration process are shown in the .

[0554] In one aspect, the present disclosure may include a method for identifying an integration site of a viral gene within a subject's gene, the method comprising the following steps: (a) obtaining 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 viral-derived RNA contig sequences, a plurality of infected subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences comprising viral and infected subject endogenous portions; (b) identifying the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences; (c) for at least a portion of the plurality of hybrid RNA contig sequences, comparing the infected subject endogenous portion with a subject reference genome; and (d) identifying an integration site comprising the subject's gene based at least in part on the comparison of the infected subject endogenous portion with the subject reference genome.

[0555] In one aspect, a system configured to perform functions associated with 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 thereon, which, when executed by the one or more processors, cause the computing device to: (a) receive a plurality of RNA contig sequences derived from an infected subject infected with a virus as input, such that the plurality of RNA contig sequences include a plurality of viral-derived RNA contig sequences, a plurality of infected subject endogenous RNA contig sequences, and a plurality of hybrid RNA contig sequences comprising viral and infected subject endogenous portions; (b) identify the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences; (c) for at least a portion of the plurality of hybrid RNA contig sequences, compare the infected subject endogenous portion with a subject reference genome; (d) identify an integration site comprising a subject gene based at least in part on a comparison of the infected subject endogenous portion with the subject reference genome; and (e) provide the integration site as output.

[0556] In some embodiments, the plurality of RNA contig sequences can be derived from an infected subject.

[0557] In some embodiments, the infected subject can be a human.

[0558] In some embodiments, the plurality of virus-derived RNA contig sequences can be derived from a virus that infects the infected subject.

[0559] In some embodiments, the virus may include hepatitis B virus.

[0560] In some embodiments, the plurality of infected subject endogenous RNA contig sequences can be derived from RNA endogenous to the infected subject.

[0561] In some embodiments, the subject reference genome may include a human genome.

[0562] Certain embodiments and implementations of the disclosed technology are described above with reference to systems and methods and / or computer program products according to exemplary embodiments or implementations of the disclosed technology. It will be appreciated that the method steps may be implemented by computer executable program instructions. Likewise, according to some embodiments or implementations of the disclosed technology, some method steps do not need to be performed in the order presented, may be repeated, or may not need to be performed at all.

[0563] These computer executable program instructions can be loaded onto a general purpose computer, a special purpose computer, a processor or other programmable data processing device to produce a specific machine, so that the instructions executed on the computer, processor or other programmable data processing device create a means for implementing one or more functions associated with the method steps. These computer program instructions can also be stored in a computer readable memory, which can instruct a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer readable memory produce an article of manufacture including an instruction means for implementing one or more functions associated with the method steps.

[0564] Non-transitory computer-readable media may include, but are not limited to, random access memory (RAM), read-only memory (ROM), electronically erasable programmable ROM (EEPROM), flash memory or other storage technology, compact disk ROM (CD-ROM), digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other tangible, physical medium that can be used to store computer-readable information.

[0565] Embodiments or implementations of the disclosed technology may provide a computer program product, including a computer-usable medium having a computer-readable program code or program instructions contained therein, the computer-readable program code being adapted to be executed to implement one or more functions associated with the example methods presented herein. Likewise, the computer program instructions may be loaded onto a computer or other programmable data processing device to cause a series of operating elements or steps to be executed on the computer or other programmable device to produce a computer-implemented process, such that the instructions executed on the computer or other programmable device provide elements or steps for implementing the functions associated with the example methods presented herein.

[0566] Therefore, the illustration and description of the method support 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 is also understood that at least some of the method steps and combinations of method steps can be implemented by a computer system based on special-purpose hardware or a combination of special-purpose hardware and computer instructions that performs the specified functions, elements or steps.

[0567] Certain embodiments of the disclosed technology can be used with custom devices that may include mobile computing devices. Those skilled in the art will recognize that there are several categories of mobile devices, generally referred to as portable computing devices, which can operate using battery power but are not generally classified as laptops. For example, mobile devices include, but are not limited to, portable computers, tablet computers, Internet tablets, PDAs, ultra mobile computers (UMPCs), wearable devices, and smartphones.

[0568] Certain implementations of the disclosed technology may be used with medical devices, medical apparatuses, and / or related peripherals.

[0569] Example

[0570] The present disclosure is also described and illustrated by the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the present disclosure or any illustrative terms. Likewise, the present disclosure is not limited to any particular preferred embodiment described herein. In fact, many modifications and variations of the present disclosure may be apparent to those skilled in the art upon reading this specification, and these variations may be made without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is limited only by the terms of the appended claims and the full range of equivalents to which these claims are entitled.

[0571] Example 1. Generation of RNAseq database from HBV-infected patient samples.

[0572] Comparative genomic analyses are typically guided by a small number of "reference" genomes. As of January 2022, the National Center for Biotechnology and Information (NCBI) database contains a repository of more than 13,000 HBV genome sequences that are grouped into at least 10 distinct phylogenetic lineages (A to J). While each lineage is represented by a single HBV genome, these reference sequences do not capture the full genomic diversity that exists in nature. This lack of coverage can negatively impact the identification of novel viral peptides, as sequences from patient isolates can differ substantially from the reference genome sequences used for comparative analyses.

[0573] To circumvent this problem, an RNAseq-based approach was developed to generate a database of patient-specific HBV genome sequences reconstructed directly from RNA reads amplified from liver samples of patients in an Asian population ( Figure 1A ). A de novo sequencing strategy was used to reconstruct patient-specific HBV genomes and identify HBV integration sites within human chromosomes. Human total RNA reads were converted into large contigs, and contigs with loose homology to the HBV reference genome were labeled as HBV-specific sequences. The HBV contigs were then divided into two categories based on the percentage of contig sequence coverage of the HBV reference. The fully mapped contigs to the HBV reference were realigned in a reference-free manner to assemble patient-specific HBV genome sequences. Specific coding sequences from each reconstructed genome were deposited into a large patient-specific HBV database. Hybrid contigs matching the HBV reference were aligned to the human genome, and hybrid contigs matching both HBV and human sequences were selected. Specifically, hybrid contigs matching both human and HBV references were filtered using a homologous sequence alignment method to identify integration sites within the human genome. All HBV coding sequences were then extracted and translated using patient-specific viral sequences. The coverage of the HBV reference genome by total RNA reads from bulk RNA sequencing of all human liver samples is shown in Figure 1B Using this approach, a total of 80 unique coding sequences (large S = 14, medium S = 14, small S = 16, precapsid = 6, capsid = 12, X = 16, and polymerase = 2) were retrieved from virus isolates representing four phylogenetic lineages (A, B, C, D) ( Figure 1C The phylogenetic relationships between the reconstructed HBV genome and the reference HBV sequences representing eight HBV lineages (A, B, C, D, E, F, G, H) are shown in Figure 1C The tree was generated using a neighbor-joining phylogenetic method rooted at the midpoint. Read mapping analysis showed 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 coverage of the rest of the genome ( Figure 1B ). These gaps in the genome suggest that these reads may be from partially integrated HBV DNA sequences. The N-terminal region of the polymerase gene was poorly covered genome-wide, while the C-terminal portion of the polymerase gene was well detected. For most HBV+ samples, hybrid contigs combining HBV and human sequences were also identified. These sequences are likely generated by integration of HBV DNA into the coding sequences of human genes. The genomic locations of HBV sequences in these junctions are called “breakpoints.” The HBV breakpoints found in RNAseq reads with HBV / human junctions were mapped and it was found that most HBV breakpoints occurred between nucleotide positions 1601-1900 in the HBV genome, as shown in Figure 2. Figure 1DThe frequencies of the genomic positions of the breakpoints in all studied samples are shown in 100-nucleotide intervals; 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. The reconstruction of the HBV genome and HBV sequences from patient liver samples were used to discover HBV peptides presented on the surface of HCC tumors.

[0574]

[0575]

[0576]

[0577] aggrecan6; GRB10, growth factor receptor binding protein 10; GTF2IP20, general transcription factor IIi pseudogene 20; HEXD, hexosaminidase D; IGF1R, insulin-like growth factor 1 receptor; IKZF2, IKAROS family zinc finger 2; IL1RAP, interleukin 1 receptor accessory protein; ITPR2, inositol 1,4,5-trisphosphate receptor type 2; KCNMB2, potassium-calcium-activated channel subfamily M regulatory beta subunit 2; KIF1A, kinesin family member 1A; KLHL14, Kelch-like family member 14; LINC01483, long intergenic nonprotein-coding RNA 1483; LINC01572, long intergenic nonprotein-coding RNA 1572; LINC02315, long intergenic nonprotein-coding RNA 2315; MAN2A1, mannosidase alpha 2A class member 1; MASP1, MBL-associated serine protease 1; METTL15, methyltransferase-like 15; MIR548AI, microRNA 548ai; MIR924HG, MIR924 host gene; NAA25, N-alpha-acetyltransferase 25, NatB auxiliary subunit; NCOA2, nuclear receptor coactivator 2; NFX1, nuclear transcription factor, X-box binding protein 1; NME8, NME / NM23 family member 8; OPRM1, opioid receptor Mu 1; OR10J1, olfactory receptor family 10 subfamily J member 1; OR2W5, olfactory receptor family 2 subfamily W member 5 pseudogene; OSBPL5, oxysterol-binding protein-like 5; PCCA, propionyl-CoA carboxylase subunit alpha; PDZK1, PDZ domain-containing protein 1; PID1, phosphotyrosine interaction domain-containing protein 1; PLEKHH2, pleckstrin homolog, MyTH4 and FERM domain-containing protein H2; POLR2D, RNA polymerase II subunit D; PPIP5K2, inositol bisphosphate pentaphosphate kinase 2; RANBP17, RAN-binding protein 17; SCARB1, scavenger receptor class B member 1; SENP7, SUMO-specific peptidase 7; SERBP1, SERPINE1 mRNA binding protein 1; SLC25A51, solute carrier family 25 member 51; SLC35D2, solute carrier family 35 member D2; SLC5A10, solute carrier family 5 member 10; SMURF1, SMAD-specific E3 ubiquitin ligase 1; SNAP25-AS1, SNAP25 antisense RNA 1; STARD13, StAR-associated lipid transfer domain-containing protein 13; STK38, serine / threonine kinase 38; SUCO, SUN domain-containing osteoclast factor; TENT4B, terminal nucleotidyl transferase 4B; TERT, telomerase reverse transcriptase; TES, testin LIM domain protein; TK2, thymidine kinase 2;TMEM123, transmembrane protein 123; TNRC6C, trinucleotide repeat-containing adaptor protein 6C; TYW1, tRNA-YW synthesis protein 1 homolog; ZBTB5, zinc finger and BTB domain-containing protein 5; ZNF532, zinc finger protein 532.

[0578] Example 2. Immunopeptidomic characterization of HBV-infected patient samples.

[0579] HLA class I complexes were immunopurified from homogenized hepatocellular carcinoma (HCC) liver tissue samples from 48 HBV seropositive patients (Proteogenex and BioIVT, Tables 4-5) using an anti-HLA class I antibody (W6 / 32). Specifically, tissue lysates in NP-40 were incubated with W6 / 32-conjugated agarose beads in column form, followed by bead washing and HLA elution in glycine under acidic conditions. Peptides and HLA molecules were further concentrated on 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% (1 / 10 of the HLA enrichment) of the 70% acetonitrile eluate was loaded on the gel. Immunoblots were probed with anti-HLA-I W6 / 32 and goat anti-mouse HRP. Western blots of liver lysates, flow-through, and enrichment elutions showed that W6 / 32-linked agarose beads almost completely captured HLA-I ( Figure 5 After elution of the complexes, HLA-I bound peptides were identified using an Orbitrap Fusion Lumos mass spectrometer coupled to a nanoLC system. The raw mass spectrometry (MS) data files were searched against a combined database of human UniProt and HBV sequences derived from RNA HBV reads from patient samples. On average, approximately 8,500 total peptides were detected from liver samples of patients of different ages ( Figure 6 ). Figure 2A The peptide length distribution of liver tissue from all HBV-positive patients by mass spectrometry was described by serology. To generate these data, raw files were searched at a false discovery rate (FDR) of 5% at the peptide level using PEAKSX. The peptide length distribution isolated from most liver tissue samples was consistent with that of HLA class I enrichment, with peptides of 9 residues in length (9-mers) being the most abundant, followed by peptides of 10 residues in length (10-mers) and peptides of 11 residues in length (11-mers). Therefore, the vast majority of 9-mers were detected from HLA class I enrichment in a high percentage of patient liver samples. Peptides with 9 to 12 residues accounted for more than 50% of the total peptides isolated ( Figure 6 ).

[0580] Table 4 shows the patient sample ID, diagnosis (from vendor), and HBV serological test results (i.e., HBV status, from vendor) for all patient samples analyzed, as well as the number of HBV unique peptides detected by mass spectrometry (MS), HBVPOL detected 606-616 Variants, detection status of peptides STLPETTVVRR (SEQ ID NO: 43), FLLTRILTI (SEQ ID NO: 3), and SAISSTFSK (SEQ ID NO: 41), and HBV genotypes identified from RNAseq results. HCC, hepatocellular carcinoma; CCA, cholangiocarcinoma; QC, quality control.

[0581]

[0582]

[0583]

[0584]

[0585]

[0586] Tables 5A and 5B show the patient and sample information provided by the vendors. All samples were flash frozen (FF) and the sample type for all samples was liver. The HBV diagnosis corresponding to all samples was HBV positive. A single sample (sample ID: ILS24966D3-DS2) was obtained from a menopausal patient. The vendor used for all samples was BioIVT, except for the first 6 samples (sample IDs: 09945T1(3), 0000032532, 0000040312, 0000031295, 0000049186, and 0000049219), for which the vendor was Proteogenex. The Country (Ctry) column indicates the country where the sample was collected. Smoking years and drinking years refer to the duration of smoking and drinking, respectively. M, male; F, female; API, Asian Pacific ethnicity; Dx, diagnosis; Histo, histology; Grp, group; Wt, weight; Ht, height; Cigs, cigarettes; Ctry, country; VN, Vietnamese; N / A, not applicable.

[0587]

[0588]

[0589]

[0590] Example 3. A high percentage of identified peptides were predicted to bind to HLA using NetMHC pan4.0 analysis.

[0591] HLA-I polymorphisms have a profound effect on epitopes presented on tissue surfaces. Therefore, DNA sequencing was performed on liver tissue samples to determine the HLA genotype (Table 6) for prediction of binding of peptides detected by NetMHC pan 4.0.

[0592] Table 6. HLA class I alleles identified from DNA sequencing for all HBV-infected patient samples.

[0593]

[0594]

[0595] A high percentage (50–100% for most samples) of detected 9-mer peptides were predicted binders of HLA-I identified in the corresponding patient samples ( Figure 2B and Table 7). In particular, Figure 2B The figure depicted shows the percentage (%) of the allelic binding substance of HLA-A, HLA-B and HLA-C in the 9-mer peptides identified by mass spectrometry. From the DNA sequencing to each patient sample, HLA allele is identified. The overwhelming majority of nano polymer peptides detected by mass spectrometry are the predicted binding substances of the patient's HLA allele predicted by NetMHC pan 4.0. Table 7 shows the quantity of nano polymer (peptide with 9 residue lengths, 9-mer) and its predicted by NetMHC Pan4.0 with the binding percentage of the corresponding patient's allele. Strong binding substance and weak binding substance are defined by the grade predicted by NetMHC, and the grade lower than 0.5 is regarded as strong binding substance, and the grade between 0.5-2 is regarded as weak binding substance. For some samples, the combined percentage of the allelic binding substance of HLA-A, HLA-B and HLA-C exceeds 100%.

[0596] Table 7. Number of nanomers and their predicted binding percentage (%) to the corresponding patient alleles predicted by NetMHC Pan4.0.

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604] The detected peptides were then subjected to motif analysis to identify overrepresented residues at different peptide positions using Seq2Logo ( Figures 7A-7X ). Anchor residues at positions 2 and 9 (specifying HLA-I binding) were significantly overrepresented in all tissue samples, with residue identities corresponding to the HLA-I genotypes present. For example, hydrophobic anchor residues characteristic of HLA-A02 binders (i.e., Leu and Val) were enriched at positions 2 and 9 in peptides isolated from HLA-A02+ patient samples. In summary, the high percentage of predicted HLA binders and the overrepresentation of characteristic anchor residues in isolated peptides provide a high degree of confidence in HLA-I enrichment and peptide identification.

[0605] Example 4. Identification of unique HLA-associated HBV peptides using HBV genotype-specific databases.

[0606] A major challenge in developing immunotherapeutic drugs against HBV is the genomic variability between HBV strains. Using RNA sequencing, we identified the HBV genotypes present in each patient sample ( Figure 1C ). Based on the reported prevalence of HBV genotypes in Asian populations, HBV genotypes B and C were most commonly identified, found in 24 of the 48 samples (12 in each sample). HBV genotypes A and D were detected in only one patient sample each (Table 4). The RNA sequencing data for 18 samples had low HBV coverage, indicating that HBV virus was not present in these samples, possibly due to viral latency or clearance. No HBV peptides were detected in any of the 18 tissues, further validating the proteomic approach described in this example and highlighting the good correlation between the two complementary approaches. In total, HBV peptides were detected in 30 liver samples, and the RNA sequencing work was unable to determine the HBV genotype present in 4 of these samples.

[0607] The number of HBV peptides presented by HLA-I may depend on the extent of HBV infection, proteasomal processing of the antigen, and tissue sample quality. Indeed, the number of HBV-specific peptide sequences identified from infected livers varied between samples and generally showed low sequence coverage of HBV proteins (Tables 4 and Figure 1B ). A total of 49 unique HBV peptides with a length of 9-12 residues were identified using a PEAKS ion cutoff score of 20 (Table 8). Table 8 shows the HBV peptides and proteins detected by searching the patient-specific database. The peptides were initially filtered at a 5% false discovery rate (FDR) and then filtered with a -logP of 20 as the minimum cutoff score.

[0608] Table 8. HBV peptides and proteins detected by searching patient-specific databases.

[0609]

[0610]

[0611]

[0612]

[0613] * The peptide GSLPQEHIVQK (SEQ ID NO: 12), detected once at -logP of 19, was considered a true identification (see sample Liv21_499956PF). For all other peptides, a score cutoff of 20 was used.

[0614] Most of the viral peptides detected were NetMHC pan4.0 predicted binders of the HLA-I alleles present in the corresponding patient samples ( Figure 3B and Table 9). The first four HLA genotypes from HLA-A and HLA-B had the largest number of detected HBV peptides, e.g. Figure 3A As shown. Since the Asian population was the source from which the samples were obtained, a large proportion of the patient samples were HLA-02 and HLA-A11 genotypes. In particular, Figure 3B HBV peptide sequences identified from immunopeptidomics of patient liver samples are shown, along with their predicted binding to patient HLA alleles as predicted by Pan NetMHC 4.0. Strong binders have a rank below 0.5 and weak binders have a rank of 0.5–2 according to NetMHC predictions. For HBV peptides predicted to bind to more than one allele, the affinity rank of the strongest binding is shown. Target HBV peptides and detection frequencies of target HBV peptides in HBV+ samples are shown in Figure 4. Figure 3C Table 9 shows the complete list of HBV peptides and their predicted binding affinities to patient HLA alleles by Pan NetMHC 4.0.

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623] Large surface antigen proteins are the source of several identified strong HLA-A02 predicted binders (NetMHC pan4.0): 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), and the weak binder NILSPFMPLL (S 370-379 )(SEQ ID NO:37), and the frequencies of all of them were relatively low (Table 8). In contrast, some HLA-A11-associated peptides: STLPETTVVRR from capsid protein (C 141-151 )(SEQ ID NO:43) and SAISSTFSK from polymerase (SEQ ID NO:41) were found at relatively high frequencies (30-50%). The HLA-A11 restricted HBV genotype-specific polymerase peptide GSLPQEHIVQK (P 606-616 ) (SEQ ID NO: 12) and GTLPQEHIVHK (P 606-616 ) (SEQ ID NO: 13) and GTLPQEHIVQK (P 606-616 )(SEQ ID NO:14). NetMHCpan 4.0 predicted the former peptide variant to be a weak HLA-A11 binder, whereas the latter two peptides were predicted to be strong binders of HLA-A11 ( Figure 3B The combined 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 exceeded -76% (13 of 17 samples).

[0624] Subsequent analysis with synthetic peptide analogs was performed to validate the peptides identified above. NetMHC predicted HBV peptides that bind to HLA-A02 and HLA-A11, including FLLTRILTI (S 194-202 )(SEQ ID NO:3), STLPETTVVRR(C 141-151 )(SEQ IDNO:43),GSLPQEHIVQK(P 606-616)(SEQ ID NO:12) and variants were verified by retention time alignment and matching the fragmentation patterns of the peptides with their synthetic heavy forms (Figure 4 and Tables 10-13). Table 10 shows the MS2 ions produced by the fragmentation of GSLPQEHIVQK (SEQ ID NO:12) and its heavy analogs. Table 11 shows the MS2 ions produced by the fragmentation of STLPETTVVRR (SEQ ID NO:43) and its heavy analogs. Table 12 shows the MS2 ions produced by the fragmentation of GTLPQEHIVHK (SEQ ID NO:13) and its heavy analogs. Table 13 shows the MS2 ions produced by the fragmentation of FLLTRILTI (SEQ ID NO:3) and its heavy analogs. 50 fmol of synthetic heavy peptides with sequences similar to the target HBV peptide and with modified heavy leucine (Thermo) were spiked into the HLA eluted peptides and separated on a nano liquid chromatograph connected online to a mass spectrometer. MS raw data files were searched using PEAKSX against a combined database of human Uniprot and HBV using heavy leucine (+7.02) as a variable modification. Peptide validation was performed using the fragmentation patterns (b- and y-ions) of HBV peptides and their heavy analogs in the same MS1 scan, as well as their co-elution and identification. For each peptide, the ion charge was 1, except for peptides marked with an asterisk (*), which had an ion charge of 2. FR, fraction; Mod, modification.

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640] The synthetic peptide used in this analysis differs from the HBV peptide in that the heavy leucine ( 13 C(6) 15 N(1), +7.0172) and mixed with HLA eluted peptides (50 fmol) prior to LC-MS / MS analysis. In the LC-MS / MS run, all tested peptides eluted at the same retention time as their heavy analogs in the same MS1 scan and also exhibited similar fragmentation patterns ( Figures 9A-9H ) and similar fragmentation spectra. The extracted ion chromatograms were also used to estimate the expression level or copy number of some target peptides, including polymerase peptide variants (copy number ∼100–500 / cell), based on the intensity of the corresponding heavy peptide analog precursor ions. Even at relatively low surface presentation, the tripolymerase peptide P 606-616 Variants ( Figure 8 ).

[0641] The present disclosure describes the identification of unique HBV peptides. From a total of 49 HLA-I restricted BV-specific peptides in patient liver samples, 34 unique peptides were detected from envelope proteins. STLPETTVVRR (C 141-151 ) (SEQ ID NO: 43) is related to previous studies. The peptide GSLPQEHIVQK (P 606-616 )(SEQ ID NO: 12) and its variants were identified as the most common HBV epitope in the patient population analyzed. This peptide is a predicted HLA-A11 binder and is present in more than 80% of the HLA-A11+ patient population described herein.

[0642] Reconstruction of the HBV genome present in each sample disclosed herein provides insights into HBV genomic regions that may be integrated into hepatocytes during HCC development. Genomic regions containing small surface antigen proteins, X proteins, and the second half of the polymerase protein were frequently detected. Genomic regions containing the first half of the capsid protein and polymerase protein were either not detected or only partially detected.

[0643] The combined use of RNAseq data and LC / MS-MS data reported herein demonstrates a method for finding and selecting suitable peptide targets for T cell-based therapeutics. As a non-limiting example, an ideal peptide target may be highly abundant on the cell surface, commonly found in patients, conserved across multiple HBV genotypes, and located in a region of the HBV genome that is commonly integrated into HCC. Using the methods reported herein, peptides that meet all or some aspects of the above criteria can be identified. The peptide GSLPQEHIVQK (P 606-616 ) (SEQ ID NO: 12) is frequently found in HLA-A11 patient samples and is located in a region of the HBV genome where integration is prevalent. T cell-based therapeutics can, for example, surround the peptide GSLPQEHIVQK (P 606-616 ) (SEQ ID NO: 12). Using the methods described herein to examine more patient samples representing different HLA alleles, peptides that meet all or some aspects of the criteria listed above can be identified.

[0644] The following is the method used in the above examples.

[0645] HBV-positive HCC tissues were obtained. All patient tissues were purchased from BioIVT in fresh frozen form. Patient details, including age, sex, and diagnostic tests, are shown in Table 4 and Table 5A-5B. Tissues were cryopreserved until sample preparation.

[0646] Tissue lysis and HLA affinity enrichment. Tissues were pulverized in liquid nitrogen using a SPEX SamplePrep Freezer / Mill dual chamber Cryogenix grinder and lysed on ice using 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. The volume of lysis buffer was determined by the weight of the tissue (e.g., 5 ml lysis buffer for 0.5 g tissue).

[0647] Anti-HLA class I (W6 / 32) was coupled to NHS-agarose beads by overnight incubation in coupling buffer (0.2 M NaHCO3 + 0.5 M NaCl pH 8.3) at 4° C. The reaction was quenched with 0.1 M Tris-HCl pH 8.5, and the beads were washed using the same Tris-HCl solution and 0.1 M acetate buffer.

[0648] Pre-cleared tissue lysate was passed by gravity through a chromatography column packed with 1 ml of HLA class I bead bed. The column was subsequently washed with Seppro dilution buffer and 20 mM Tris-HCl pH 8, and the HLA-peptide complexes were eluted with 0.1 M glycine pH 2.7.

[0649] Sample preparation for mass spectrometry analysis. The glycine eluate was loaded onto a C18 Sep-Pak and the peptides were selectively eluted with 30% ACN / 0.1% TFA. The peptides were further purified and analyzed by nano-LC-MS / MS.

[0650] Liquid chromatography-tandem mass spectrometry (LC-MS / MS). The above HLA peptides were loaded onto a nanoViper AcclaimPepMap100 C18 capture column (75 μm id×2 cm, 3 μm, ) and a nanoViper Acclaim PepMap RSLC C18 column (75 μm id×25 cm, 2 μm, ) for separation. The gradient was provided by an EASY-nLC 1200 HPLC system at a rate of 300 nL / min. The following 120-minute elution gradient with mobile phase A (water / 0.1% formic acid) and B (80% acetonitrile / 0.1% formic acid) was used: 3% mobile phase B at 3 minutes, linearly increasing to 35% B at 100 min, and linearly increasing to 45% B at 123 min. The peptides eluted from the column were ionized by a Flex ion source at 1.9 kV and analyzed by a Thermo Fusion Lumos Tribrid mass spectrometer using an Xcalibur 4.1.31.9. Data acquisition was performed in data-dependent mode, with survey scans performed in a high-field Orbitrap analyzer (m / z range of 300-1500, resolution of 60,000), an automatic gain control target of 4.0E5, and a maximum ion fill time of 100 ms. MS / MS analysis was performed with a quadrupole using 1.2 m / z precursor ion isolation, applying a normalized HCD (high energy collision dissociation) collision energy of 32%, and analyzing fragment ions in the Orbitrap at a resolution of 15,000. The dynamic exclusion window was set to 6 seconds, monoisotopic precursor ion selection (MIPS) was set to peptide, the maximum injection time was 100 ms, and the charge state was unknown. +1-+4 charge states were included, and advanced peak determination was turned on.

[0651] For FAIMS-enabled experiments, the setup was identical except that the FAIMS device was placed between the nano-electrospray source and the mass spectrometer. FAIMS separations were performed using the following settings: internal and external electrode temperatures were set to 100 °C (unless otherwise noted), FAIMS carrier gas flow was 5.0 L / min, asymmetric waveform of DV-5000V, inlet plate voltage was 250 V, and CV settling time was 25 ms. The FAIMS carrier gas was N2 and the ion separation gap was 1.5 mm. The recorded CV was applied to the FAIMS electrodes. For external stepping or single CV experiments, the selected CV was applied to all scans during the entire analysis. For internal CV stepping experiments, each selected CV was applied to sequential measurement scans and MS / MS cycles (1 s); the MS / MS CV was always paired with the appropriate CV from the corresponding measurement scan.

[0652] HLA genotyping of patient samples. For sample preparation and sequencing, DNA sample quantity was determined by fluorescence and quality was assessed by running 25 ng of sample on a 1% precast agarose gel. Samples with high molecular weight gDNA, where most DNA fragments were larger than 20 kb and concentrations were no less than 10 ng / ul, passed the quality assessment. DNA samples were normalized to 10 ng / ul and 50 ng was used to amplify full-length HLA amplicons. Targets were amplified using LA Taq DNA polymerase in three variant-tolerant primer pools optimized for similar binding temperature and PCR conditions. The resulting amplicon pools were combined in an equimolar manner by automated capillary electrophoresis and fluorescence determination. DNA libraries were prepared using a custom NEB kit for Illumina-based sequencing. Amplicons were enzymatically fragmented to an average insert size of 250 bp, and universal adapters were ligated to the DNA fragments. Unique 10 base pair barcode sequences were added to the DNA fragments during PCR using NEBNext Ultra IIQ5 master mix to facilitate highly multiplexed sequencing. Samples were pooled and sequenced on an Illumina Nextseq500 using 150 base pair paired-end sequencing.

[0653] For data analysis, after sequencing was completed, the raw data from each Illumina Nextseq run was collected in a local buffer store and uploaded to a local high-performance computing platform for automated analysis. Reads in FASTQ format were converted from BCL files and assigned to samples identified by specific barcodes using bcl2fastq conversion software (Illumina Inc., San Diego, CA). All reads in sample-specific FASTQ files were subjected to HLA typing analysis using a reference sequence consisting of the GRCh38 genomic sequence and HLA type reference sequence in the IPD-IMGT / HLA database v3.30.0 using an updated version of the PHLAT program.

[0654] RNA sequencing. Total RNA was extracted from human liver tissue using the MagMAX kit. Strand-specific RNA-seq libraries were prepared from 1 μg of RNA using the KAPA Strand mRNA-Seq Kit (KAPA Biosystems). Twelve cycles of PCR were performed to amplify the library. The amplified library was size-selected to 400 to 600 bp using PippinHT. Sequencing was performed on an Illumina ELISA kit with 2×100 cycles of multiplexed paired-end read runs.

[0655] Patient liver RNA sequences were mapped to the HBV reference genome. The bulk RNA-seq reads were aligned to the reference genome ayw (NC_003977.2) using minimap2 (v2.17). Alignments were then sorted by coordinates and quality controlled by checking with samtoolsflagstats (v1.9) and bedtools genomeCoverageBed (v2.17.0). Duplicates were then marked and removed by the Picard toolkit (v2.18.2).

[0656] Workflow for reconstruction of HBV genome from patient liver RNAseq data. Paired-end Illumina RNA reads from each sample were de novo assembled into large contigs using megahit (options: --min-count 3 --k-min 27 --k-max 127 --prune-level 2) and mapped to the HBV reference genome ayw (NC_003977.2) using BLAST to select HBV-specific sequences. BLAST parameters used for sequence comparison included: outfmt '7std sgi stitle'; minimum E-value = 0.001; cost of opening gaps = 5; cost of extending gaps = 2; best perfect match length = 11; reward for nucleotide matches = 2; reward for nucleotide mismatches = -3. Contigs without BLAST matches and BLAST results with E-values ​​greater than 0.001, percent identities less than 79%, or alignment lengths less than 50 nucleotides were discarded. Contigs were merged using a custom script, and the final sequence covering all or part of the length of the reference sequence with the highest identity was selected. The final sequence was mapped against a reference genome to extract and translate all coding sequences. Non-repetitive protein sequences were then added to a custom database to facilitate peptide identification by mass spectrometry.

[0657] Workflow for identification of integration sites from patient liver RNAseq data. Contigs matching the HBV reference genome (ayw) were extracted and aligned to the human reference genome (GRCh38) using BLAST using the parameters described above. Contigs matching HBV and human (hybrid contigs) were collected for annotation. HBV and human integration sites of hybrid contigs were annotated based on genomic features in the HBV ayw GenBank file and the human GRCh38GTF file.

[0658] HBV peptides were identified from HLA-I immunopeptidomics of patient liver samples. Using the PEAKSDB search engine, all mass spectrometry raw files were searched in the integrated database of human UniProtKB (Homo sapiens) and HBV protein sequences obtained from RNAseq of patient liver samples as described above. PEAKSX+ (PEAKS Studio 10.5, Bioinformatics Solutions Inc.) was used to perform a de novo auxiliary database search with a precursor ion mass tolerance of 8ppm and a fragment ion tolerance of 0.02Da. Enzyme selectivity was set to none, methionine oxidation was the only variable modification, and up to two modifications were allowed for each peptide. Retrieval was performed at a false discovery rate (FDR) of 5% at the peptide level, and peptides were further filtered according to a -logP score of 20 (corresponding to 1% FDR).

[0659] Example 5. Identification of HBV antigens or epitopes.

[0660] This embodiment relates to nucleic acid assay methods and compositions for identifying HBV antigens or epitopes.

[0661] RNA or DNA is sequenced from liver tumors and / or healthy tissue from cancer patients to identify sequences in genes expressed in tumor cells that may contain HBV-associated insertions.

[0662] Tumor material is assessed for the presence of HBV-associated insertions by sequencing (such as by RNA sequencing). Sequencing data is used to investigate HBV-associated insertions expressed in genes. Peptides containing any identified HBV-associated insertions are created in silico and filtered by application of prediction algorithms or by mass spectrometry data for identification of MHC-associated epitopes. Where appropriate, suitable MHC-binding HBV epitope sets are used to identify physiologically relevant HBV epitope-specific T cell responses in either or both CD8+ and / or CD4+ T cell populations by functional assays and / or MHC multimer-based screening.

[0663] HBV epitopes are determined by sequencing the genome and / or exome of tumor tissue and / or healthy tissue of liver cancer patients using next generation sequencing (NGS) methods. Genes selected based on the presence of HBV-related insertions and the ability to act as antigens are sequenced using NGS technology. NGS is applied to, but not limited to: genome sequencing, genome resequencing, epigenome characterization, DNA-protein interactions (ChIP-sequencing) and transcriptome analysis (RNA-Seq). Similar to DNA-based assays using, for example, NGS or massively parallel sequencing (MPS) methods, RNA from tumors is analyzed by conversion to cDNA and generating libraries suitable for sequencing.

[0664] The assay method is used to identify HBV epitopes in biological samples. Suitable assay methods for identifying HBV epitopes in biological samples include, but are not limited to: proteomics, NGS, solution hybridization, array hybridization nucleic acid amplification, polymerase chain reaction (PCR), RT-PCR, quantitative PCR, branched DNA (bDNA) assay, rolling circle amplification (RCA), in situ hybridization, Northern hybridization, hybridization protection assay (HPA), single molecule hybridization assay, invasion assay and / or oligonucleotide ligation assay (OLA), hybridization and array analysis.

[0665] The sequencing data obtained by determining the presence of HBV epitopes in cancer patients is analyzed to predict personal HBV peptides that can bind to the individual's HLA molecules. The data is analyzed using a computer. In particular, the sequence data is analyzed for the presence of HBV antigens.

[0666] HBV antigens were evaluated by their affinity for MHC molecules.

[0667] A neural network-based learning approach using validated binding and non-binding peptides was used for prediction algorithms for major HLA-A and HLA-B alleles. The algorithm was used to predict missense mutations that would generate strong binding peptides to cognate MHC molecules in cancer patients. A set of peptides representing the best HBV epitopes for each patient was identified and prioritized. The HBV epitope prediction algorithm was used to predict the binding of candidate peptides to either MHC class I or MHC class II molecules.

[0668] In some cases, the peptide binding tool can be one of the following tools: Antibody Epitope Prediction, ANTIGENIC, BepiPred, CTLPred, DiscoTope, EPIPREDICT, EpitopeCluster Analysis, Epitope Conservancy Analysis, EUiPro, HLA Peptide Binding Predictions, HLA Binding, MAPPP, MHCBench, MHC-I processing predictions, Mosaic Vaccine ToolSuite, NetChop, NetCTL, NetMHC, NetMHCII, NetMHCpan, nHLAPred-I, OptiTope, PAProC, POPI, PREDEP, Prediction of Antigenic Determinants, ProPred, ProPred-1, RankPep, SMM, SVMHC, TAPPred, VaxiJen, or a combination thereof. Use other example programs such as BIMAS or SYFPEITHI, Rankpep.

[0669] In some cases, the Immune Epitope Database and Analysis Resource (IEDB) (Vita R, et al. Nucleic Acids Res. 2015; 43 (Database issue): D405-D412) is used to identify suitable tumor HBV antigens. This algorithm predicts the binding of peptides to different MHC class I variants based on artificial neural networks (ANNs), providing predicted IC50 as output. NetMHC (Lundegaard C et al. Nucleic Acids Res. 2008; 36 (Web Server issue): W509-W512.) is used. Programs such as SMMPMBEC (Kim Y et al. BMC Bio informatics. 2009; 10: 394) and / or SMM (Peters B et al. BMC Bio informatics. 2005; 6: 132) are used. These programs use position weight matrices to describe the statistical preferences of peptide-MHC I binding data. This method suppresses noise caused by, for example, a limited number of data points and / or experimental errors present in the training set.

[0670] In some cases, single nucleotide polymorphisms (SNPs) are removed from candidate HBV antigens or HBV epitopes. SNPs include a range of molecular variations: (1) SNPs, (2) multiple nucleotide polymorphisms (MNPs), (3) short deletion and insertion polymorphisms (indel / DIPs), (4) microsatellite markers or short tandem repeats (STRs), (5) heterozygous sequences, and (6) named variants.

[0671] Proteomics-based methods are used to identify tumor-specific HBV antigens, such as direct protein sequencing. Multidimensional MS techniques including tandem mass spectrometry (MS / MS) ar...

Claims

1. An isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112, wherein the isolated peptide is 8-12 amino acids in length.

2. The isolated peptide according to claim 1, wherein the isolated peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

3. The isolated peptide according to claim 1 or 2, wherein the isolated peptide consists essentially of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

4. The isolated peptide according to any one of claims 1 to 3, wherein the isolated peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-54 and 110-112.

5. An isolated peptide or a pharmaceutically acceptable salt thereof, or a fragment or derivative thereof, wherein the isolated peptide comprises two or more amino acid sequences selected from any one of SEQ ID NOs: 1-54 and 110-112.

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

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

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

9. The isolated peptide according to any one of claims 1-7, wherein the isolated peptide is produced synthetically.

10. An isolated peptide according to any one of claims 1 to 9, wherein the isolated peptide or a pharmaceutically acceptable salt thereof or a fragment or derivative thereof induces a hepatitis B virus (HBV)-specific immune response in a subject when presented on the surface of an antigen presenting cell (APC) in a complex with a major histocompatibility complex (MHC) molecule.

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

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

13. The fusion protein of claim 11, wherein the one or more heterologous molecules mediate delivery of the peptide to a specific site within a subject.

14. The fusion protein of any one of claims 11-13, wherein the one or more heterologous molecules are MHC molecules or fragments or derivatives thereof.

15. A conjugate comprising one or more isolated peptides according to any one of claims 1-10 conjugated to one or more heterologous molecules.

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

17. The conjugate of claim 15, wherein the one or more heterologous molecules mediate delivery of the peptide to a specific site within a subject.

18. The conjugate according to any one of claims 15-17, wherein the one or more heterologous molecules are MHC molecules or fragments or derivatives thereof.

19. The conjugate according to any one of claims 15-17, wherein the one or more peptides are conjugated to a particle.

20. An oligomeric complex comprising two or more isolated peptides according to any one of claims 1-10.

21. A non-covalent complex comprising: an isolated peptide according to any one of claims 1 to 10, and an MHC molecule or a fragment or derivative thereof.

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

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

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

25. The non-covalent complex of claim 24, wherein the class II MHC molecule is a class II HLA molecule.

26. A fusion protein comprising: the isolated peptide according to any one of claims 1 to 10, and an MHC molecule or a fragment or derivative thereof.

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

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

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

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

31. A conjugate comprising: an isolated peptide according to any one of claims 1 to 10, and an MHC molecule or a fragment or derivative thereof.

32. The conjugate of claim 31, wherein the MHC molecule or fragment thereof is a class I MHC molecule.

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

34. The conjugate of claim 31, wherein the MHC molecule or fragment thereof is a class II MHC molecule.

35. The conjugate of claim 34, wherein the MHC class II molecule is an HLA class II molecule.

36. A pharmaceutical composition comprising: (i) one or more isolated peptides according to any one of claims 1-10, one or more fusion proteins according to any one of claims 11-14 and 26-30, one or more conjugates according to any one of claims 15-19 and 31-35, one or more oligomeric complexes according to claim 20, or one or more non-covalent complexes according to any one of claims 21-25, or any combination thereof; and (ii) a pharmaceutically acceptable carrier or excipient.

37. The pharmaceutical composition of claim 36, further comprising an adjuvant.

38. An isolated molecule that binds to the isolated peptide of any one of claims 1-10, the fusion protein of any one of claims 11-14 and 26-30, the conjugate of any one of claims 15-19 and 31-35, the oligomeric complex of claim 20, or the non-covalent complex of any one of claims 21-25.

39. The isolated molecule of claim 38, wherein the molecule is an antibody or an antigen-binding fragment thereof.

40. The isolated molecule of claim 39, wherein the antibody is a bispecific antibody.

41. The isolated molecule of claim 38, wherein the molecule is a surrogate backbone.

42. The isolated molecule of claim 38, wherein the molecule is a chimeric antigen receptor (CAR).

43. The isolated molecule of claim 38, wherein the molecule is a T cell receptor (TCR).

44. An isolated cell comprising the CAR of claim 42.

45. The isolated cell of claim 44, wherein the isolated cell is an immune cell.

46. ​​The isolated cell of claim 45, wherein the immune cell is a T cell, a NK cell or a macrophage.

47. An isolated cell comprising the TCR of claim 43.

48. The isolated cell of claim 47, wherein the isolated cell is an immune cell.

49. The isolated cell of claim 48, wherein the immune cell is a T cell, a NK cell, or a macrophage.

50. A pharmaceutical composition comprising: (i) an isolated molecule according to any one of claims 38-43 or an isolated cell according to any one of claims 44-49; and (ii) a pharmaceutically acceptable carrier or excipient.

51. An isolated polynucleotide comprising a nucleotide sequence encoding one or more isolated peptides according to any one of claims 1-10 or a nucleotide sequence encoding a fusion protein according to any one of claims 11-14 and 26-30.

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

53. An isolated polynucleotide according to claim 51 or claim 52, wherein the isolated polynucleotide comprises DNA.

54. The isolated polynucleotide of claim 51 or claim 52, wherein the isolated polynucleotide comprises RNA.

55. The isolated polynucleotide of claim 54, wherein the RNA is mRNA.

56. The isolated polynucleotide of claim 54, wherein the RNA is a self-replicating RNA.

57. A vector comprising the isolated polynucleotide according to any one of claims 51-56.

58. The vector of claim 57, wherein the vector is an expression vector.

59. The vector of claim 57 or claim 58, wherein the vector is a viral vector.

60. A host cell comprising the isolated polynucleotide of any one of claims 51-56 or the vector of any one of claims 57-59.

61. The host cell of claim 60, wherein the host cell is a prokaryotic cell.

62. The host cell of claim 60, wherein the host cell is a eukaryotic cell.

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

64. A pharmaceutical composition comprising: (i) an isolated polynucleotide according to any one of claims 51-56 or a vector according to any one of claims 57-59; and (ii) a pharmaceutically acceptable carrier or excipient.

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

66. 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 according to any one of claims 1 to 10; b) a fusion protein according to any one of claims 11 to 14 and 26 to 30; c) a conjugate according to any one of claims 15-19 and 31-35; d) The oligomeric complex according to claim 20; e) the non-covalent complex according to any one of claims 21 to 25; f) a pharmaceutical composition according to any one of claims 36, 37, 50, 64 and 65; g) a molecule according to any one of claims 38 to 43; h) an isolated cell according to any one of claims 44-49 and 60-61; i) an isolated polynucleotide according to any one of claims 51-56; or j) A vector according to any one of claims 57 to 59.

67. 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 according to any one of claims 1-10.

68. A method of inducing an immune response against HBV infection in a subject in need thereof, the method comprising administering to the subject activated T cells generated by contacting the T cells with APCs, the APCs presenting an isolated peptide according to any one of claims 1-10 complexed with MHC molecules.

69. A method of treating an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of: a) one or more isolated peptides according to any one of claims 1 to 10; b) a fusion protein according to any one of claims 11 to 14 and 26 to 30; c) a conjugate according to any one of claims 15-19 and 31-35; d) The oligomeric complex according to claim 20; e) the non-covalent complex according to any one of claims 21 to 25; f) a pharmaceutical composition according to any one of claims 36, 37, 50, 64 and 65; g) a molecule according to any one of claims 38 to 43; h) an isolated cell according to any one of claims 44-49 and 60-63; i) an isolated polynucleotide according to any one of claims 51-56; or j) A vector according to any one of claims 57 to 59.

70. A method of preventing or reducing the likelihood of an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of: a) one or more isolated peptides according to any one of claims 1 to 10; b) a fusion protein according to any one of claims 11 to 14 and 26 to 30; c) a conjugate according to any one of claims 15-19 and 31-35; d) The oligomeric complex according to claim 20; e) the non-covalent complex according to any one of claims 21 to 25; f) a pharmaceutical composition according to any one of claims 36, 37, 50, 64 and 65; g) a molecule according to any one of claims 38 to 43; h) an isolated cell according to any one of claims 44-49 and 60-63; i) an isolated polynucleotide according to any one of claims 51-56; or j) A vector according to any one of claims 57 to 59.

71. A method of treating an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides according to any one of claims 1-10.

72. A method of preventing or reducing the likelihood of an HBV-induced disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of one or more isolated peptides according to any one of claims 1-10.

73. The method of any one of claims 70-72, wherein the HBV-induced disease or condition is liver inflammation, liver fibrosis, cirrhosis, or liver cancer.

74. The method of claim 73, wherein the liver cancer is hepatocellular carcinoma (HCC).

75. A kit comprising: (i) a) one or more isolated peptides according to any one of claims 1 to 10; b) a fusion protein according to any one of claims 11 to 14 and 26 to 30; c) a conjugate according to any one of claims 15-19 and 31-35; d) The oligomeric complex according to claim 20; e) the non-covalent complex according to any one of claims 21 to 25; f) a pharmaceutical composition according to any one of claims 36, 37, 50, 64 and 65; g) a molecule according to any one of claims 38 to 43; h) an isolated cell according to any one of claims 44-49 and 60-63; i) an isolated polynucleotide according to any one of claims 51-56; or j) a vector according to any one of claims 57 to 59; and (ii) Instructions for use of the packaging and / or kit.

76. 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, wherein the plurality of RNA contig sequences comprises a plurality of virus-derived RNA contig sequences and a plurality of RNA contig sequences endogenous to the infected subject; b) identifying a plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences; c) assembling viral RNA sequences based on the plurality of virus-derived RNA contig sequences; d) identifying protein sequences based on the viral RNA sequences; as well as e) identifying immunogenic virus-derived peptides based at least in part on the identified protein sequences.

77. The method of claim 76, wherein the plurality of RNA contig sequences are derived from an infected subject.

78. The method of claim 76, wherein the infected subject is a human.

79. The method of claim 76, wherein the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

80. The method of claim 76, wherein the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

81. The method of claim 76, wherein identifying a plurality of virus-derived RNA contig sequences from 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 viral sequence; as well as The plurality of virus-derived RNA contig sequences are identified such that each contig sequence in the plurality of virus-derived RNA contig sequences comprises at least a portion of a sequence corresponding to the reference viral sequence.

82. The method of claim 81, wherein each of the plurality of virus-derived RNA contig sequences is different from the plurality of infected subject endogenous RNA contig sequences.

83. The method of claim 81, wherein each contig sequence in the plurality of virus-derived RNA contig sequences lacks an RNA contig sequence endogenous to an infected subject.

84. The method of claim 81, wherein the reference viral sequence comprises a reference genome.

85. The method of claim 84, wherein the reference genome comprises a Hepatitis B virus genome.

86. The method of claim 76, wherein assembling a viral RNA sequence based on the plurality of viral-derived RNA contig sequences comprises: The common sequence portions overlap at the ends of at least a portion of the plurality of virus-derived RNA contig sequences so that at least a portion of the plurality of virus-derived RNA contig sequences are linearly overlapped to assemble the viral RNA sequence.

87. The method of claim 76, wherein identifying a protein sequence based on the viral RNA sequence such that the identified protein sequence comprises a translation of the viral RNA sequence comprises: Protein sequences are identified without comparison to viral protein databases.

88. The method of claim 76, wherein identifying a protein sequence based on the viral RNA sequence such that the identified protein sequence comprises a translation of the viral RNA sequence, further comprising: identifying a plurality of protein sequences, each based on the viral RNA sequence, such that each of the plurality of protein sequences comprises a translation of the viral RNA sequence, and The protein sequence is identified as a frequently occurring protein sequence among the plurality of protein sequences.

89. The method of claim 76, wherein the protein sequence identified based on the viral RNA sequence is associated with a single infected subject.

90. The method of claim 89, wherein identifying the immunogenic virus-derived peptide based at least in part on the protein sequence comprises: Identification of MHC molecules associated with individual infected subjects; identifying one or more peptides based at least in part on the protein sequence, such that each of the one or more peptides forms a corresponding MHC peptide complex with the MHC molecule; and The immunogenic virus-derived peptide is identified based on the one or more peptides.

91. A non-transitory computer readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer readable medium comprising instructions thereon that, when executed by the one or more processors, cause the computing device to: a) receiving 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 and a plurality of RNA contig sequences endogenous to the infected subject, and wherein the infected subject is infected with a virus; b) identifying the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences; c) assembling viral RNA sequences based on the plurality of virus-derived RNA contig sequences; d) identifying protein sequences based on the viral RNA sequences; e) identifying immunogenic virus-derived peptides based at least in part on the protein sequence; and f) providing said immunogenic virus-derived peptide as an output.

92. The non-transitory computer readable medium of claim 91, wherein the plurality of RNA contig sequences are derived from only one infected subject.

93. The non-transitory computer readable medium of claim 91, wherein the infected subject comprises a human.

94. The non-transitory computer readable medium of claim 91, wherein the plurality of virus-derived RNA contig sequences are derived from a virus that infects an infected subject.

95. The non-transitory computer readable medium of claim 91, wherein the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to an infected subject.

96. The non-transitory computer readable medium of claim 91, wherein the instructions that cause the computing device to identify the plurality of virus-derived RNA contig sequences from the plurality of RNA contig sequences further comprise instructions that, when executed by the one or more processors, cause the computing device to: comparing at least a portion of the contig sequences of the plurality of RNA contig sequences to a reference viral sequence; and The plurality of virus-derived RNA contig sequences are identified such that each contig sequence in the plurality of virus-derived RNA contig sequences comprises at least a portion of a sequence corresponding to the reference viral sequence.

97. The non-transitory computer readable medium of claim 96, wherein each of the plurality of virus-derived RNA contig sequences is different from the plurality of infected subject endogenous RNA contig sequences.

98. The non-transitory computer readable medium of claim 96, wherein each contig sequence in the plurality of virus-derived RNA contig sequences lacks a portion of an RNA contig sequence endogenous to an infected subject.

99. The non-transitory computer-readable medium of claim 96, wherein the reference viral sequence comprises a reference genome.

100. The non-transitory computer-readable medium of claim 99, wherein the reference genome comprises a Hepatitis B virus genome.

101. The non-transitory computer readable medium of claim 91, wherein the instructions causing the computing device to assemble the viral RNA sequence based on the plurality of viral-derived RNA contig sequences further comprise instructions that, when executed by the one or more processors, cause the computing device to: The common sequence portions overlap at the ends of at least a portion of the plurality of virus-derived RNA contig sequences so that at least a portion of the plurality of virus-derived RNA contig sequences are linearly overlapped to assemble the viral RNA sequence.

102. The non-transitory computer readable medium of claim 91, wherein the instructions that cause the computing device to identify a protein sequence based on the viral RNA sequence such that the protein sequence comprises a translation of the viral RNA sequence further comprise instructions that, when executed by the one or more processors, cause the computing device to: The protein sequence was identified without comparison to a viral protein database.

103. The non-transitory computer readable medium of claim 91, wherein the protein sequence identified based on the viral RNA sequence is a novel protein.

104. The non-transitory computer readable medium of claim 91, wherein the protein sequence identified based on the viral RNA sequence is associated with a single infected subject.

105. The non-transitory computer readable medium of claim 104, wherein the instructions that cause the computing device to identify a protein sequence based on the viral RNA sequence such that the protein sequence comprises a translation of the viral RNA sequence further comprise instructions that, when executed by the one or more processors, cause the computing device to: identifying a plurality of protein sequences, each based on the viral RNA sequence, such that each of the plurality of protein sequences, respectively, comprises a translation of the viral RNA sequence; and The protein sequence is identified as a frequently occurring protein sequence among the plurality of protein sequences.

106. The non-transitory computer readable medium of claim 104, wherein the instructions causing the computing device to identify the immunogenic virus-derived peptide based at least in part on the protein sequence further comprise instructions that, when executed by the one or more processors, cause the computing device to: identifying a major histocompatibility complex (MHC) molecule associated with the single infected subject; identifying one or more peptides based at least in part on the protein sequence, such that each of the one or more peptides is capable of forming a corresponding MHC peptide complex with the MHC molecule; and The immunogenic virus-derived peptide is identified based on the one or more peptides.

107. The non-transitory computer readable medium of claim 91, wherein the instructions, when executed by the one or more processors, further cause the computing device to: The protein sequence is stored in a database such that the protein sequence is associated with an infected subject in the database.

108. A method for identifying an integration site of a viral gene within a gene of a subject, the method comprising: a) obtaining 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 RNA contig sequences endogenous to the infected subject, and a plurality of hybrid RNA contig sequences comprising portions endogenous to the virus and to the infected subject; b) identifying the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences; c) comparing, for at least a portion of the plurality of hybrid RNA contig sequences, an endogenous portion of the infected subject to a reference genome of the subject; and d) identifying an integration site comprising a gene of the subject based at least in part on a comparison of an endogenous portion of the infected subject to a reference genome of the subject.

109. The method of claim 108, wherein the plurality of RNA contig sequences are derived from an infected subject.

110. The method of claim 108, wherein the infected subject is a human.

111. The method of claim 108, wherein the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

112. The method of claim 111, wherein the virus is hepatitis B virus.

113. The method of claim 108, wherein the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

114. The method of claim 108, wherein the subject reference genome comprises the human genome.

115. A non-transitory computer readable medium configured to communicate with one or more processors of a computing device, the non-transitory computer readable medium comprising instructions thereon that, when executed by the one or more processors, cause the computing device to: a) receiving 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 RNA contig sequences endogenous to the infected subject, and a plurality of hybrid RNA contig sequences comprising portions endogenous to the virus and to the infected subject; b) identifying the plurality of hybrid RNA contig sequences from the plurality of RNA contig sequences; c) comparing, for at least a portion of the plurality of hybrid RNA contig sequences, an endogenous portion of the infected subject to a reference genome of the subject; d) identifying an integration site comprising a gene of the subject based at least in part on a comparison of an endogenous portion of the infected subject to a reference genome of the subject; and e) providing said integration site as output.

116. The non-transitory computer readable medium of claim 115, wherein the plurality of RNA contig sequences are derived from only one infected subject.

117. The non-transitory computer readable medium of claim 115, wherein the infected subject comprises a human.

118. The non-transitory computer readable medium of claim 115, wherein the plurality of virus-derived RNA contig sequences are derived from a virus that infects the infected subject.

119. The non-transitory computer readable medium of claim 118, wherein the virus comprises a Hepatitis B virus.

120. The non-transitory computer readable medium of claim 115, wherein the plurality of infected subject endogenous RNA contig sequences are derived from RNA endogenous to the infected subject.

121. The non-transitory computer-readable medium of claim 115, wherein the subject reference genome comprises a human genome.

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