T-cell receptors produced as a result of HPV vaccine therapy and methods for treating patients using them.

Abstract

T cell receptors (TCRs) generated as a result of HPV vaccine therapy, including TCRs generated from any known HPV vaccine (e.g., HPV6 / 11 vaccine), engineered cells containing such TCRs, and a method of treating a patient using them.

Description

[Technical Field]

[0001] Description of research or development funded by the federal government. This invention was made with government support from the National Institutes of Health and the National Cancer Institute under project number ZIA BC012131. The U.S. government has certain rights to this invention. [Background technology]

[0002] Human papillomavirus (HPV) is a common sexually transmitted infection that can cause a variety of cancers, including cervical cancer, anal cancer, and head and neck cancer.

[0003] The most common HPV types are low-risk HPV-6 and HPV-11, which account for 90% of the disease known as genital warts and recurrent respiratory papillomatosis (RRP), in which tumors grow in the airways. HPV also contributes to the development of non-melanoma skin cancers (NMSC), including cutaneous squamous cell carcinoma (SCC), among patients with chronic lymphocytic leukemia (CLL) and blood and bone marrow transplants (BMT). HPV-16 and HPV-18 are the primary causes of the majority of head and neck cancers (HNSCC), as well as cancers of the cervix, anus, vagina, vulva, penis, base of the tongue, larynx, and tonsils.

[0004] Current standard treatment options for HNSCC include surgery and radiotherapy with parallel chemotherapy (e.g., cisplatin and / or cetuximab). Unfortunately, post-procedure patient burdens after such modalities are significant and persistent and may include dysphagia, dysphonia, dry mouth, scarring and facial disfigurement, as well as trismus. However, HPV-related precancerous lesions, such as those of the vulva, vagina, anus, and penis, as well as genital warts, are usually treated using cryotherapy (i.e., using extremely low temperatures to destroy tissue), chemical ablation (i.e., using chemicals to destroy tissue), and laser or surgical removal. It should be noted that in such cases, physical elimination of the precancerous lesions alone is insufficient, as more than 20-30% of cases recur, and this recurrence can occur as lesions in previously treated sites due to failure of the procedure to eliminate HPV, or as lesions in new sites due to a new infection. When this occurs, radiation therapy and chemotherapy are used and have been relatively successful, but still, about 50% of HPV-related cancer patients die from this disease.

[0005] In practice, vaccines against HPV (e.g., Gardasil, Gardasil 9, and Cervarix) have been developed and have been largely successful in preventing HPV infection and associated cancers. However, effective treatment is needed for individuals who are already infected with HPV and have developed associated cancers. Conventional cancer therapies, such as chemotherapy and radiation, often have significant side effects and are not always effective in all cases. Furthermore, cancer cells can develop resistance to these therapies, leading to disease relapse and progression. Clearly, new treatment strategies to reduce the burden of HPV-related cancers are urgently needed.

[0006] Recent advances in immunotherapy have led to the development of T cell receptor (TCR) therapies for cancer treatment. TCRs are specialized receptors on T cells that can recognize specific antigens on cancer cells and trigger an immune response against them. TCR therapy involves the steps of isolating T cells from a patient, manipulating these T cells to recognize cancer-specific antigens, and reintroducing these T cells into the patient to target and eliminate cancer cells. The use of manipulated TCR therapy offers several advantages. It allows the patient's own T cells to be given the desired specificity and to generate a sufficient number of T cells in a short period of time while avoiding T cell exhaustion. In some embodiments of the present invention, as disclosed herein, TCRs can be transduced into immune effector cells, such as central memory T cells or T cells with stem cell characteristics, thereby ensuring better persistence and function upon transfer. Preferably, TCRs can be transduced into cytotoxic T cells (CD8+ T cells; CTLs). T cells manipulated by such TCRs (TCR-T cells) can be injected into cancer patients, such as cancer patients whose lymphocytes have been reduced by chemotherapy or irradiation, resulting in efficient engraftment, but with inhibited immunosuppression. As disclosed herein, the present invention relates, at least in part, to immune cells that recombinantly express an artificial T cell receptor (TCR) that targets the HPV antigen.

[0007] In cases of HPV-associated cancer, TCRs (transcranial receptors) generated as a result of HPV vaccine therapy may be particularly effective in recognizing and targeting cancer cells. This is because the HPV vaccine stimulates the immune system to produce T cells that recognize and respond to HPV antigens. By isolating these TCRs and manipulating them to recognize cancer-specific antigens, they can be used to treat cancers associated with HPV infection.

[0008] Accordingly, the present invention relates to T cell receptors (TCRs) generated as a result of HPV vaccine therapy, and their use in the treatment of cancers associated with HPV infection. TCRs can be generated from any known HPV vaccine and can be engineered to recognize specific antigens on cancer cells associated with HPV infection, such as the E6 and E7 proteins of HPV. TCRs can be introduced into patients with cancers associated with HPV infection using adoptive T cell transfer or genetic modification. The introduction of engineered TCRs can result in a reduction in tumor size and an improvement in the patient's overall survival.

[0009] Embedding by reference All published documents, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual published document, patent, or patent application is specifically and individually indicated as being incorporated by reference. [Overview of the project]

[0010] The present invention relates in part to T cell receptors (TCRs) produced as a result of treatment with an HPV vaccine. In certain embodiments, the HPV vaccine is selected from an HPV quadrivalent recombinant vaccine; an HPV nonavalent recombinant vaccine; or an HPV bivalent recombinant vaccine.

[0011] In certain embodiments, the HPV vaccine comprises a nucleic acid sequence having at least 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with a bivalent recombinant vaccine, such as SEQ ID NO: 81. In certain embodiments, the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81 or a codon degenerate variant thereof. In certain such embodiments, the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81.

[0012] In certain embodiments, the HPV vaccine is Gardasil.

[0013] In certain embodiments, the HPV vaccine is Gardasil 9 or Cervavac.

[0014] In certain embodiments, the HPV vaccine is Cervarix, Cecolin, or Walrinvax.

[0015] In certain embodiments, the TCR comprises an amino acid sequence having at least 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 1 to 80. In certain such embodiments, the TCR comprises any one of SEQ ID NOs: 1 to 80 or a conservatively substituted variant thereof. In certain such embodiments, the TCR comprises any one of SEQ ID NOs: 1 to 80.

[0016] In certain embodiments, the TCR recognizes a specific antigen on cancer cells associated with HPV infection. In certain such embodiments, the specific antigen is the HPV E6 or E7 protein.

[0017] In certain embodiments, the TCR further includes modifications that enhance the effectiveness of the TCR against HPV infection.

[0018] In certain embodiments, the TCR further includes modifications that enhance the specificity of the TCR to the HPV epitope.

[0019] In certain embodiments, the TCR is linked to an effector molecule. In certain such embodiments, the effector molecule is selected from the group consisting of cytokines, toxins, radioisotopes, chemotherapeutic agents, antibodies, antibody fragments, and antibody-drug conjugates.

[0020] In part, the present invention also relates to a method for treating a disease or disorder associated with HPV infection in a subject requiring such treatment, the method comprising the step of introducing the TCR of the present invention into the subject.

[0021] In certain specific embodiments, the TCR is introduced into a patient using adoptive cell transfer.

[0022] In certain specific embodiments, the disease or disorder is cancer. In certain such embodiments, the cancer is cervical cancer, anal cancer, or head and neck cancer.

[0023] In certain specific embodiments, the disease or disorder is recurrent respiratory papillomatosis.

[0024] The invention further relates, in part, to the use of the TCR of the invention in the preparation of a medicament for the treatment of a disease or disorder associated with HPV infection.

[0025] In addition, the invention relates to a method of preparing TCR-T cells for use in the treatment of a disease or disorder associated with HPV infection in a subject that has received administration of an HPV vaccine, the method comprising isolating T cells from the subject, stimulating the T cells with an HPV antigen, and isolating HPV-specific TCR-T cells, wherein the HPV vaccine is selected from a quadrivalent HPV recombinant vaccine, a nonavalent HPV recombinant vaccine, and a bivalent HPV recombinant vaccine. In certain specific embodiments, the HPV vaccine comprises a nucleic acid sequence having at least 80%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 81. In certain specific embodiments, the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81 or a codon-degenerate variant thereof. In certain such embodiments, the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81.

[0026] In addition, the invention relates, in part, to engineered or isolated cells comprising the TCR of the invention. In certain specific embodiments, the cell is a mammalian cell. In certain such embodiments, the cell is a mammalian cell.

[0027] In certain specific embodiments, the engineered or isolated cell is an immune cell. In certain such embodiments, the cell is a T cell.

[0028] The present invention further relates to nucleic acid molecules encoding the TCR of the present invention.

[0029] The present invention also relates further to vectors containing the aforementioned nucleic acids. In certain embodiments, the vector is an adenovirus vector.

[0030] In addition, the present invention relates to a pharmaceutical composition comprising the above-described manipulated or isolated cells and a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is for use in the preparation of a medicament for use in the treatment of a disease or disorder associated with HPV infection.

[0031] In addition, the present invention also relates to a method for producing engineered cells expressing a TCR, comprising the step of introducing the nucleic acid of the present invention encoding the TCR into the cells. In certain embodiments, the cells are T cells.

[0032] Furthermore, the present invention relates to a kit comprising the TCR of the present invention, the manipulated cells of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, or the pharmaceutical composition of the present invention, and instructions for use in the treatment of diseases or disorders associated with HPV infection.

[0033] Furthermore, the present invention also relates to a method for treating a disease or disorder associated with HPV infection in a subject requiring such treatment, comprising the step of administering to the subject, in combination with a therapy selected from chemotherapy, radiotherapy, or checkpoint inhibitor therapy, the TCR of the present invention, the engineered cells of the present invention, the nucleic acid molecules of the present invention, the vectors of the present invention, or the pharmaceutical compositions of the present invention.

[0034] A further aspect of the present invention is a method for detecting HPV infection in a subject, the method comprising the steps of contacting a sample from the subject with the TCR of the present invention, and detecting the binding of the TCR to the sample, wherein the binding of the TCR to the sample indicates the presence of HPV infection in the subject.

[0035] Further aspects of the present invention are methods for producing manipulated cells according to any one of claims 43 to 47, comprising the steps of culturing cells and introducing nucleic acids encoding the TCR of the present invention into cells under conditions sufficient for the expression of the TCR in the cells. [Brief explanation of the drawing]

[0036] [Figure 1-1]Figure 1A is a dot plot illustrating the logarithmically transformed change in HPV-specific T cell response from peripheral blood 6 weeks after completion of HPV 6 / 11 vaccine treatment compared to previous levels. Each dot represents the logarithmically transformed change in IFNγ concentration after peptide stimulation by individual pools of HPV peptides encoded in the vaccine. Pools in which no IFNγ response was detected in pre- or post-treatment samples are not shown. Patient N=14; sufficient pre-treatment PBMCs were not available from patient 5. Respondents are shown in blue, and non-respondents in gold. Figure 1B is a dot plot illustrating the changes in HPV-specific peripheral blood response. Significance was determined by the Mann-Whitney two-tailed test. Figure 1C is a bar graph illustrating the fraction of the post-treatment TCRβ repertoire represented by the top 10 CDR3 frequencies determined to be HPV-specific in the HPV 6 / 11 peptide-stimulated FEST assay. The Simpson clonality index is shown above each bar graph. The top horizontal bar indicates the response to the treatment. R: Respondent; NR: Non-respondent. Figure 1D is a dot plot showing the log-transformed change in the frequencies (determined by FEST assay) of the top 10 HPV-specific CDR3 sequences from peripheral blood 6 weeks after completion of HPV6 / 11 vaccine treatment compared to pre-treatment without peptide stimulation. Figure 1E is a dot plot showing a summary of the changes in the top 10 HPV-specific CDR3 sequences. Significance was determined by an unpaired two-sided t-test. Figure 1F is a bar graph summarizing the fractions (determined by FEST assay) of the top 10 peripheral blood HPV-specific CDR3 sequences detected in post-treatment peripheral blood, either not detected (appeared), detected at a lower frequency (enlarged), or detected at a higher frequency (contracted), compared to pre-treatment peripheral blood. The top horizontal bar indicates the response to the treatment. Figure 1G is a dot plot illustrating the log-2 converted change in HPV-specific papilloma-infiltrating lymphocytes (PILs) after completion of HPV6 / 11 vaccine treatment compared to before. Each dot represents the log-converted change in the number of IFNγ spots after co-culture of antigen-presenting cells loaded with individual pools of HPV peptides encoded in PRGN-2012 and PILs.Pools in which no IFNγ response was detected in pre- or post-treatment samples are not shown. Patient N=9; post-treatment biopsy material was unavailable for patients 7, 10, 11, and 13, and pre-treatment PIL culture establishment failed for patients 2 and 3. Figure 1H is a dot plot summarizing the changes in HPV-specific PIL response. Significance was determined by the Mann-Whitney two-tailed test. Figure 1I is a clinical endoscopic image showing the appearance of the pharynx of patient 5 before treatment and at 6 weeks. Red arrows indicate the pre-treatment and 6-week biopsy sites that produced PIL for the experiments shown in Figures 1J and 1K. Figure 1J is a representative IFNγELISpot well (with IFNγ spot numbers inserted) showing IFNγ spots after stimulation with pool 2 peptides and negative (DMSO alone) and positive (PMA / ionomycin) controls before treatment and at 6 weeks. Figure 1K is a bar graph showing the IFNγ concentration after co-culturing antigen-presenting cells loaded with individual peptides contained in Pool 2 with PIL samples for 6 weeks. [Figure 1-2] Same as Figure 1-1. [Figure 2]Figure 2A shows representative micrographs of T cell immunofluorescence in baseline papilloma biopsy material collected from responders (top row) and non-responders (bottom row) after treatment with the HPV6 / 11 vaccine. Figure 2B shows dot plots indicating the density of CD8 T cells in the papilloma and stroma, or in responders and non-responders, after treatment with the HPV6 / 11 vaccine. Significance was determined by the Mann-Whitney two-tailed test. Figure 2C shows box plots indicating the number of normalized HPV gene transcripts in papilloma cells in responders and non-responders after treatment with the HPV6 / 11 vaccine, determined from single-cell RNA-seq. Significance was determined by two-way INOVA. Figure 2D is a box plot showing cell-normalized reactome IFNγ signaling scores in different cell types (x axis) in responders and non-responders, determined from single-cell RNA-seq. Significance was determined by two-way INOVA. Figure 2E is a heatmap showing the number of bio-normalized chemokine transcripts in different cell types (y axis). The bar graph on the right shows mean expression. The top horizontal bar shows the response to treatment with HPV 6 / 11 vaccine. The significance of the difference between responders and non-responders was determined by two-way INOVA. Figure 2F is a violin plot showing the number of CXCR3 transcripts for CD8 and CD4 papilloma T cells, determined from single-cell RNA-seq. Significance was determined by the Mann-Whitney two-tailed test. Figure 2G is a violin plot showing the percentage of (total) cells positive for CXCL9 or CXCL10. Significance was determined by a two-tailed Mann-Whitney test. Representative micrographs of RNAscope immunofluorescence are shown. Figure 2H depicts dot plots showing the expression of selected T cell-related genes across T lymphocyte clusters, identified by single-cell RNA-seq and sorted by the ratio of change in the number of cells detected in responders and non-responders (responders / non-responders; bar graph below) after treatment with the HPV6 / 11 vaccine.The left column shows T cells that were frequently observed in non-responders, and the right column shows cells that were frequently observed in responders. The color of the circles corresponds to the scaled average expression, and the size of the circles indicates the percentage of cells with non-zero gene expression for the corresponding gene. The bar graph at the top represents the total number of cells. [Modes for carrying out the invention]

[0037] Detailed description of the invention It should be understood that this disclosure is not limited to the specific embodiments described herein and is subject to modification. While various features of this disclosure may be described in the context of a single embodiment, features may also be provided separately or in any preferred combination. Those skilled in the art will recognize that variations and modifications of this disclosure exist that fall within their scope.

[0038] All terms are intended to be understood in the same way that they are understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are generally understood by those skilled in the art in which this disclosure relates.

[0039] Section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described.

[0040] definition The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0041] In this application, the use of the singular form includes the plural form unless otherwise specifically indicated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise.

[0042] In this application, the use of “or” means “and / or” unless otherwise specified. The terms “and / or” and “any combination thereof,” as well as their grammatical equivalents, can be used interchangeably as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjugate or disjunctively unless the context specifically refers to a disjunctive use.

[0043] The use of the term "including," as well as other forms such as "include," "includes," and "included," is not restrictive; that is, "including" does not mean "limited to."

[0044] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in conjunction with embodiments are included in at least some embodiments of this disclosure, but not necessarily in all embodiments of this disclosure.

[0045] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional possible components, elements, or steps of the method. Any embodiment discussed herein is intended to be performed with respect to any method or composition of the Disclosure, and vice versa. Furthermore, compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0046] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on the method of measuring or determining the value, i.e., the limits of the measuring system. For example, “about” may mean within 1 or a standard deviation greater than 1, according to practice in the art. Alternatively, “about” may mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the quantity “about 10” includes 10 and any quantities from 9 to 11. In yet another example, the term “about” in relation to a reference number may also include a range of values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or plus or minus 1% from that value. Alternatively, particularly in relation to biological systems or processes, the term “about” may mean within one order of magnitude of the value, preferably within five times, more preferably within two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” means within an acceptable margin of error for the particular value to be assumed.

[0047] The term “isolated” and its grammatical equivalent, as used herein, refers to the removal of nucleic acids, proteins, polypeptides, cells, or other materials from their natural environment. The term “purified” and its grammatical equivalent, as used herein, refers to molecules or compositions whose purity has been increased, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA), and / or amplified under laboratory conditions, where “purity” is a relative term, not “absolute purity.” However, it should be understood that nucleic acids and proteins may be formulated with diluents or adjuvants and still be isolated for practical purposes. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalent, as used herein, refers to nucleic acid sequences, polypeptides, proteins, or other compounds that essentially do not contain, i.e., no more than about 50%, no more than about 70%, or no more than about 90% of the polynucleotides, proteins, polypeptides, and other molecules to which the nucleic acid, polypeptide, protein, or other compound relates in nature.

[0048] "Polynucleotide," "oligonucleotide," "polynucleotide construct," "gene," "gene construct," "heterogene," and their grammatical equivalents, as used herein, refer to polymeric forms of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of molecules. Therefore, this term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. This also includes polynucleotides modified, for example, by methylation and / or capping, as well as polynucleotides in their unmodified forms. This term also means that molecules include nucleotides that do not exist naturally or are synthetic, and nucleotide analogs. Nucleic acid sequences and vectors disclosed or contemplated herein may be introduced into cells, for example, by transfection, transformation, or transduction.

[0049] The terms and phrases “polynucleotide encoding a polypeptide” or similar terms, as used herein, include any polynucleotide that can be used to express (i.e., encode) a polypeptide. In practice, methods for constructing any number of different polynucleotide sequences that can encode the same polypeptide (i.e., a polypeptide having the same amino acid sequence but encoded by different codon sequences (nucleotide triplets)) are well known and understood to those skilled in the art. For example, various polynucleotide sequences encoding a particular polypeptide can be generated by using various codons in a standard gene code. Furthermore, various polynucleotide sequences encoding a particular polypeptide can also be generated by using codon selection, such as by using “codon usage frequencies” found in a given organism, for example, the Human (Homo sapiens) Codon Usage Frequency Table, for example. See, for example, https: / / www.genscript.com / tools / codon-frequency-table.

[0050] As used herein, the term “codon degenerate variant” refers to a modified nucleic acid sequence that codes for the same amino acid sequence as the original sequence, but with different specific nucleotides containing codons. The genetic code is degenerate, meaning that multiple codons can code for the same amino acid. For example, the amino acid leucine can be coded by six different codons: CTG, CTT, CTC, CTA, TTG, and TTA. A codon degenerate table, also known as a genetic code table or codon table, is a chart that provides information about the relationships between codons (sequences of three nucleotides) and the corresponding amino acids they code for. The table lists 64 possible codons and indicates which amino acid each codon represents. Table 1 is an example of a codon degenerate table.

[0051] [Table 1]

[0052] Furthermore, publicly available software resources are readily available for computer-generated "reverse transcription" (also known as "reverse translation" of polypeptide sequences, i.e., the conversion of polypeptide sequences to the nucleotide sequences that encode them). See, for example, Madeira, F., et al., Nucleic Acids Res, 47(Wl), W636-W641 (2019); Madeira, F., et al., Curr Protoc in Bioinformatics, 66(1):e74 (2019); Chojnacki, S, et al., Nucleic Acids Res. 2017 Jul 3;45(Wl):W550-W553 (2017); Athey, J., et al., BMC Bioinformatics 18:391 (2017).

[0053] Therefore, a "codon degenerate variant" refers to a nucleic acid sequence that has been modified to contain different codons, but still maintains the same amino acid sequence when translated. Codon degenerate variants can be used to optimize gene expression or enhance protein production. By modifying codons within a nucleic acid sequence, it is possible to utilize codons that are more frequently used or preferred by the translation mechanism of the host organism. This can result in increased efficiency of protein expression or improved compatibility with specific host organisms.

[0054] "Polypeptide," "peptide," "polypeptide construct," and "peptide construct," as well as their grammatical equivalents, refer to polymers of amino acid residues as used herein. "Mature protein" is a full-length protein, optionally including glycosylation or other modifications typical of a protein in a given cellular environment. Embodiments of the present invention, as disclosed herein, include the HPV antigen / antigenic polypeptides, peptides, and mature proteins described herein, and also include polynucleotides (DNA or RNA) encoding them. Polypeptides and proteins disclosed herein (including their functional portions and functional variants) may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 Examples include 2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0055] In the context of two nucleic acid or amino acid sequences of a polypeptide, the terms “identical” and their grammatical equivalents, or “sequence identity,” as used herein, refer to residues in two sequences that are identical when aligned for maximum match across a defined comparison window. “Comparison window,” as used herein, refers to a segment of at least about 20, typically about 50 to about 200, and more commonly about 100 to about 150, consecutive positions over which the sequences can be compared to the same number of consecutive positions of a reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.The optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Nat. Acad Sci US.A., 85:2444 (1988); or by computer execution of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program from Intelligentics, Mountain View Calif, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA, GAP, BESTFIT, BLAST, FASTA, and TFASTA); the CLUSTAL program is from Higgins and Sharp, Gene, This is well described in 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by inspection and manual alignment. In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or a fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example.Similarly, nucleic acids can also be described by reference to a starting nucleic acid, for example, a nucleic acid may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, 99%, or 100% identical to a reference nucleic acid or a fragment thereof, as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters, for example. When a molecule is said to have a certain percentage of sequence identity with a larger molecule, this means that, when the two molecules are optimally aligned, the said percentage of residues in the smaller molecule will find matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0056] The term “substantially identical” and its grammatical equivalent, applied to nucleic acids or amino acid sequences, means that the nucleic acid or amino acid sequence contains a sequence that, using standard parameters and a program described above, e.g., BLAST, has sequence identity of at least 90%, at least 95%, at least 98%, and at least 99%, compared to a reference sequence. For example, the BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequences in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In embodiments, substantial identity exists over a region of sequence that is at least about 50 residues long, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding region.

[0057] The term “functional fragment” or its grammatical equivalent is used herein to mean a portion, fragment, or segment of a biomolecule that retains essential functional characteristics or activity of the original biomolecule. The term “functional variant” or its grammatical equivalent is used herein to mean a modified form of a biomolecule that retains essential functional characteristics or activity of the original molecule, while exhibiting some degree of variation. This includes biomolecules that have been modified, for example, by genetic engineering or mutagenesis techniques, to introduce specific changes while preserving the overall functionality of the biomolecule. A functional variant may have one or more amino acid substitutions, insertions, or deletions compared to the original molecule, while still maintaining the desired biological activity or function.

[0058] When used herein, "T cells" or "T lymphocytes" refer to a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface.

[0059] Unless otherwise specified, the term "TCR" should be understood to include the entire TCR as well as its antigen-binding moiety or antigen-binding fragment. In some embodiments, the TCR is a intact or full-length TCR, e.g., a TCR containing α and β chains. In some embodiments, the TCR is an antigen-binding moiety that is shorter than the full-length TCR but binds to a specific peptide bound within an MHC molecule, e.g., an MHC-peptide complex. In some examples, the antigen-binding moiety or fragment of the TCR may contain only a portion of the structural domain of the full-length or intact TCR, but can still bind to the peptide epitope to which the entire TCR binds, e.g., an MHC-peptide complex. In some examples, the antigen-binding moiety contains the variable domain of the TCR, e.g., the variable α (Vα) and variable β (Vβ) chains of the TCR, or a sufficient antigen-binding fragment to form a binding site for binding to a specific MHC-peptide complex.

[0060] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH9, TH17, TH22, or TFH (follicular helper T cells), which secrete different cytokines that promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[0061] Cytotoxic T cells (TC cells, or CTLs) or cytotoxic T lymphocytes destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8+ cells can be inactivated into an anerogenic state that prevents autoimmune diseases by IL-10, adenosine, and other molecules secreted by regulatory T cells.

[0062] "Memory T cells" refer to a subset of antigen-specific T cells that persist for a long period after an infection has resolved. They rapidly expand into a large number of effector T cells upon re-exposure to their congener antigens, thus providing an immune system with memory of past infections. Memory T cells include three subtypes: central memory T cells (TcM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA, and / or CCR7.

[0063] Regulatory T cells (Treg cells), previously known as suppressor T cells, play a role in maintaining immune tolerance. Their primary roles are to shut down T cell-mediated immunity toward termination of the immune response and to suppress autoreactive T cells that have evaded the negative selection process in the thymus.

[0064] Natural killer T cells (NKT cells – not to be confused with natural killer cells of the innate immune system) bridge the gap between the adaptive immune system and the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CDs. When activated, these cells can perform functions attributed to both helper T cells (THs) and cytotoxic T cells (TCs) (i.e., cytokine production and release of cytotoxic / death-causing molecules). They can also recognize and eliminate some tumor cells and cells infected with herpesviruses.

[0065] The terms "conservative amino acid substitution" or "conservative mutation" refer to the substitution of one amino acid with another amino acid that shares common properties. A functional method for defining the common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, a group of amino acids can be defined as one in which the amino acids within the group preferentially exchange with each other, and therefore their effects on the overall protein structure are most similar to each other (Schulz, GE and Schirmer, RH, above). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., lysine and arginine, and vice versa, where the positive charge can be maintained; glutamic acid and aspartic acid, and vice versa, where the negative charge can be maintained; serine and threonine, where the free -OH can be maintained; and glutamine and asparagine, where the free -NH2 can be maintained. Examples of conservative amino acid substitutions are shown in the chart below.

[0066] [Table 2] An amino acid sequence that differs from a reference amino acid sequence solely through conservative amino acid substitutions is referred to herein as a "conservatively substituted variant" of the reference sequence.

[0067] In some embodiments, a functional variant may include the amino acid sequence of a reference protein having at least one non-conserved amino acid substitution. The term “non-conserved amino acid substitution” includes amino acid substitutions between different groups, such as the substitution of tryptophan by lysine or serine by phenylalanine. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conserved amino acid substitutions can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the homologous parent protein. The substitutability of amino acids is discussed in more detail, for example, LY Yampolsky and A. Stoltzfus, “The Exchangeability of Amino acids in Proteins,” Genetics 2005 Aug.; 170(4):1459-1472.

[0068] As used herein, “antibody” refers to either a monoclonal antibody or a polyclonal antibody. The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, “polyclonal antibody” refers to a group of antibodies produced by different B cells and bound to different epitopes of the same antigen. A total antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, and each region contains four framework regions whose sequences are relatively conserved. The framework region is connected by three complementarity-determining regions (CDRs). These three CDRs, known as CDR1, CDR2, and CDR3, form the antibody's "hypervariable region," which is responsible for antigen binding.

[0069] An "antibody-like molecule" can be, for example, a protein that is a member of the Ig superfamily and can selectively bind to a partner. MHC molecules and T cell receptors are such molecules. In one embodiment, the antibody-like molecule is a TCR. In one embodiment, the TCR is modified to increase its MHC binding affinity.

[0070] The terms “functional antibody fragment” and “functional fragment of an antibody” or their grammatical equivalents are used interchangeably herein to mean a portion, fragment, or segment of an antibody that retains essential functional characteristics or activity of the original antibody. In one embodiment, the activity is the ability to specifically bind to an antigen (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) in general). A functional antibody fragment may include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination thereof. Non-limiting examples of functional antibody fragments include: (i) an antigen-binding fragment (Fab), which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in the stem region; (iii) a variable fragment ("Fv") consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to synthesize a single polypeptide chain (e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778). Examples include dimers of polypeptide chains, (see 1998), and (v) dimers of polypeptide chains, where each polypeptide chain contains a VH connected to the VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule having two functional antigen-binding sites. Functional antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950.

[0071] The "antigen recognition portion" or "antigen recognition domain" refers to a molecule or a part of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition portion is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0072] Where used herein, the term “proliferative disorders” refers to a unified concept, including cancer, in which excessive cell proliferation and / or turnover of the intracellular matrix significantly contribute to the pathogenesis of the disease.

[0073] Where used herein, “patient” or “subject” refers to a mammalian subject that has been diagnosed with, has, or is suspected of having, a proliferative disorder such as cancer. In some embodiments, the term “patient” refers to a mammalian subject that has a higher-than-average likelihood of developing a proliferative disorder such as cancer. Exemplary patients may be humans, apes, dogs, pigs, cattle, cattle, horses, goats, sheep, rodents, and other mammals that may benefit from the therapies disclosed herein. Exemplary human patients may be male and / or female. “Patient in need of it” or “subject in need of it” is used herein to refer to a patient who has been diagnosed with, or is suspected of having, a disease or disorder, but is not limited to, for example, human papillomavirus (HPV) infection.

[0074] "Administering" is used herein to refer to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of a composition, e.g., by injection, may be carried out by intravenous, subcutaneous, intradermal, intraperitoneal, or intramuscular injection. One or more such routes may be used. Parenteral administration may be, for example, by bolus injection or by progressive perfusion over time. Alternatively, or in combination, administration may be by an oral route. In addition, administration may also be by surgical deposition or placement of a medical device. Pharmaceutical compositions may include the compositions of the present invention described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0075] As used herein, the terms “treatment,” “to treat,” or their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms or pathological signs that may contribute to the disease. For this purpose, the method of the present invention comprises the step of administering a therapeutically effective amount of a composition of the present invention expressing the nucleic acid sequence of the present invention, or a vector comprising the nucleic acid sequence of the present invention.

[0076] As used herein, “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that may be repeated on a regular schedule. In some embodiments, the dosage regimen may have one or more periods during the treatment interval in which no therapeutic agent is administered.

[0077] The terms “administered in combination,” “simultaneously administered,” “to administer simultaneously,” or “to provide simultaneously,” as used herein, mean that two (or more) different treatments are delivered to a subject during the course of the subject’s suffering due to a disease or disorder, for example, that two or more treatments are delivered after the subject has been diagnosed with a disease or disorder and before the disease or disorder is cured or eliminated, or before the treatments are discontinued for any other reason. In some embodiments, the delivery of one treatment is still taking place when the delivery of a second treatment is initiated, resulting in an overlap in the duration of administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment is initiated. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation is observed with the first treatment. In some embodiments, the delivery is such that the reduction of symptoms or other parameters relating to the impairment is greater than that observed with a treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more than additive. The delivery may be such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0078] As used herein, the term "decomposition" refers to a significant reduction in the volume (i.e., bulk) of a tumor, without the aim of complete eradication. Decomposition is usually achieved by surgical removal.

[0079] In some embodiments of the present invention, the first and second treatments may be administered simultaneously (e.g., at the same time) or sequentially, using the same or separate compositions. Sequential administration refers to the administration of one treatment prior to the administration of an additional (e.g., secondary) treatment (e.g., immediately before; less than 5, 10, 15, 30, 45, or 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 hours or longer before; 4, 5, 6, 7, 8, 9 days or longer before; or 1, 2, 3, 4, 5, 6, 7, 8 weeks or longer before). The order of administration of the first and secondary treatments may also be reversed.

[0080] The terms “therapeutic effective dose,” “therapeutic dose,” “immunological effective dose,” “antiotumor effective dose,” and “tumor inhibitory effective dose,” or their grammatical equivalents, refer to an effective dose, which is the amount and duration of medication required to achieve the desired therapeutic outcome. The therapeutic effective dose may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compositions described herein to induce a desired response in one or more subjects.

[0081] Alternatively, the pharmacological and / or physiological effects of administering one or more compositions described herein to a patient or subject may be “preventive,” meaning the effect completely or partially prevents the disease or its symptoms. “Preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive outcome (e.g., prevention of disease or prevention of signs of a target condition).

[0082] With respect to the numerical range limitations specified herein, each intervening number between them is explicitly intended to be of the same precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0083] T cell receptor (TCR) T cell receptors (TCRs) are protein molecules found on the surface of T cells, a type of leukocyte involved in adaptive immune responses. TCRs are involved in recognizing and binding to specific antigens presented on the surface of antigen-presenting cells (APCs) or any nucleated cells (e.g., all human cells in the body except red blood cells), such as peptides derived from pathogens or abnormal cells. The variable domain of the TCR contains a highly pleomorphic loop called the complementarity-determining region (CDR), which is responsible for binding to the peptide-presenting MHC. There are two main forms of TCR: αβ TCR and γδ TCR. Both forms consist of two protein chains, known as alpha (α) and beta (β) chains for αβ TCR and gamma (γ) and delta (δ) chains for γδ TCR. These chains combine to form a heterodimer structure. 1. αβ TCR: The majority of T cells in the human immune system express αβ TCRs. The α and β chains of the αβ TCR are encoded by separate gene segments, which undergo recombination during T cell development to produce diverse TCR specificities. The α and β chains contain variable (V), diverse (D), and conjugating (J) gene segments, respectively, as in the antibody gene rearrangement process. The combination of the V, D, and J gene segments contributes to the unique antigen-binding specificity of the αβ TCR. The αβ TCR recognizes antigen peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. 2. γδ TCRs: In contrast to αβ TCRs, γδ TCRs play a less dominant but still important role in the immune system. The γ and δ chains of γδ TCRs are also encoded by separate gene segments and undergo recombination during T cell development. The γδ TCR gene rearrangement process differs from that of αβ TCRs. γδ T cells often exhibit tissue-specific distribution and are found in epithelial tissues, such as the skin and intestines. γδ TCRs can recognize a variety of antigens, including certain peptide and non-peptide molecules, independently of MHC presentation.

[0084] Both αβ TCRs and γδ TCRs participate in and respond to immune surveillance, but they have different functions and specificities. αβ TCRs are primarily involved in the recognition of peptides presented by major histocompatibility complex (MHC) molecules, while γδ TCRs may possess a wider range of antigen recognition capabilities.

[0085] TCRs and constructs encoding TCRs that recognize MHC-antigen complexes can be created and introduced into T cells (known as TCR T cells), and the following TCR-peptide-MHC interactions can be utilized to induce an immune response. Greenbaum et al., Cancer Immunol Res 1 November 2021; 9 (11): 1252-1261. There is interest in the use of TCRs with higher affinity than the normal range for peptide-MHC antigens (type I), referred to as high-affinity TCRs, to generate soluble TCRs that can be used directly against target cells, either 1) to drive the activity of CD4 helper T cells (which lack CD8 coreceptors), or 2) by attaching to "effector" molecules (e.g., antibody Fc region, toxic drugs, or antibody scFv for forming bispecific proteins, e.g., anti-CD3 antibodies) (Ashfield and Jakobsen, IDrugs, 9, 554-9 (2006); Foote and Eisen Proc Natl Acad Sci USA, 97, 10679-81 (2000); Holler et al., Proc Natl Acad Sci USA, 97, 5387-92 (2000); Molloy et al., Curr Opin Pharmacol, 5,438-43 (2005); Richman and Kranz, Biomol Eng, 24,361-73 (2007)). This approach can also overcome the problem faced by some cancer patients because these T cells do not express TCRs with sufficient specificity and binding affinity to basic tumor antigens. For example, more than 300 MHC-restricted T cells that define tumor antigens have been identified (Cheever et al., Clin Cancer Res. 2009;15(17):5323-5337). These tumor antigens include mutated peptides, differentiation antigens, and overexpressed antigens, all of which function as targets for therapy.Since most cancer antigens described to date originate from intracellular proteins that can only be targeted on the cell surface within the context of MHC molecules, TCRs are ideal candidates for therapy, as they have evolved to recognize this class of antigens.

[0086] Similarly, TCRs can detect peptides derived from viral proteins that are naturally processed in infected cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have optimized TCRs that bind to and destroy infected cells. Finally, in a highly specific manner, TCRs can be used as receptor antagonists for autoimmune targets or as delivery agents to immunosuppress local immune cell responses, thereby avoiding general immunosuppression.

[0087] The present invention provides a TCR generated as a result of HPV vaccine therapy, which can be expressed in immune effector cells and enhance activity against a specific target (e.g., antitumor activity). In certain embodiments, T cells expressing such an engineered TCR (e.g., cytotoxic T cells; CTLs) are useful for the prevention and / or treatment of HPV infection and / or cancer (e.g., cancer expressing an HPV epitope) and / or precancerous lesions. In some embodiments, the TCR sequence (and the HPV epitope sequence to which it specifically binds) comprises at least one polypeptide or a functional variant thereof from the group consisting of SEQ ID NOs: 1 to 80 (e.g., a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 80, or a conservatively substituted variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 80).

[0088] The TCRs disclosed herein may be specific to HPV antigens, including HPV peptides E6 and E7. In certain embodiments, the TCR is specific to an antigen comprising at least one epitope or functional variant having an amino acid sequence selected from the amino acid sequences disclosed in International Publication No. 2022 / 115470 (e.g., an epitope having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences disclosed in International Publication No. 2022 / 115470, or a conservatively substituted variant of any one of the amino acid sequences disclosed in International Publication No. 2022 / 115470).

[0089] The TCRs of the present invention can be generated from any known HPV, HPV6 / 11 vaccine, or HPV vaccine disclosed in International Publication No. 2022 / 115470. The TCRs can be isolated from T cells of patients who have received HPV vaccine therapy. The TCRs can be engineered to recognize specific antigens on HPV-associated cancer cells. Currently, three HPV vaccines are approved for use in the United States by the U.S. Food and Drug Administration ("FDA"), as shown in the table below.

[0090] [Table 3] See Petrosky et al., Use of 9-valent human papillomavirus (HPV) vaccine: updated HPV vaccination recommendations of the advisory committee on immunization practices. MMWR Morb Mortal Wkly Rep. 2015 Mar 27;64(11):300-4; also see FDA, Human Papillomavirus Vaccine https: / / www.fda.gov / vaccines-blood-biologics / human-papillomavirus-vaccine (last updated Jan. 31, 2018).

[0091] Cervarix, Gardasil, and Gardasil 9 are the HPV vaccines currently approved for use in the United States. However, a great many other HPV vaccines, including at least Cecolin, Wallinvax, and Cervavac, are known in the art and are approved for use in countries outside the United States. Mo, Bi, Sheng et al., Hum Vaccin Immunother. 2022;18(6):2092363; World Health Organization, Wkly Epidemiol Rec. 50 (97) (2022): 645-672; Chu, Bi, Huang et al., Lancet Reg. Health 2023; 100731.

[0092] Other HPV vaccines that may be used to generate the TCR of the present invention include at least those disclosed in the following publications: Japanese Patent No. 7098330; U.S. Patent No. 10772947; Chinese Patent No. 104039833; U.S. Patent No. 7871816; Cyprus Patent No. 1107457 (translation of European patent); U.S. Patent Application Publication No. 2022152189; Dominican Republic Patent Application Publication No. 2022000168; International Publication No. 2019151760; European Patent No. 2386645; International Publication No. Patent No. 2017192418; US Patent Application Publication No. 2009232842; US Patent No. 10125175; US Patent Application Publication No. 2009317415; Patent No. 7229151; Korean Patent Application Publication No. 20170032813; Canadian Patent Application Publication No. 2965498; Chinese Patent Application Publication No. 114681603; Spanish Patent No. 2519490 (translation of European patent); Russian Patent No. 2356943; Chinese Patent No. 101765607; US Patent No. 7371390 ; US Patent No. 8187606; European Patent No. 3095798; European Patent No. 1984388; Canadian Patent Application Publication No. 2649555; US Patent No. 8263560; Brazilian Pipeline Patent Application Publication No. 0414845; Korean Patent Application Publication No. 20090005011; Brazilian Pipeline Patent Application Publication No. 0909547; Brazilian Patent No. 112013000031; Brazilian Pipeline Patent No. 0810959; Brazilian Pipeline Patent Application Publication No. Specification No. 0915076; Specification No. 7885, Iceland; Specification No. 101487009, China; Specification No. 107080833, China; Japanese Patent Publication No. 2017507117; Specification No. 178140, Israel; Specification No. 102154325, China; Specification No. 0508722, Brazil; Specification No. 112013030150, Brazil; Specification No. 340633, Norway; Specification No. 101487010, China;European Patent Application Publication No. 2940133; Brazilian Patent Application Publication No. 112017004181; Chinese Patent No. 104338126; Hungarian Patent Additional Certificate of Guarantee Application No. 1500061; Brazilian Pipeline Patent No. 0810951; Chinese Patent Application Publication No. 109706144; European Patent Application Publication No. 1758609; Brazilian Patent No. 112012004928; Chinese Patent Application Publication No. 112891526; Chinese Patent Application Publication No. 111529699; Republic of South Africa National Patent No. 200504907; Brazilian Patent Application Publication No. 112013000996; Chinese Patent Application Publication No. 102008721; Chinese Patent Application Publication No. 110639013; Chinese Patent Application Publication No. 105597092; Brazilian Patent Application Publication No. 112022009429; International Publication No. 2011068934; US Patent Application Publication No. 2010285058; Argentine Patent Application Publication No. 054259; US Patent No. 7758866; Chinese Patent Application Publication No. 103992395 Specification; Chinese Patent Application Publication No. 102343103; Chinese Patent Application Publication No. 104998260; International Publication No. 2008145745; Chinese Patent No. 105363029; Chinese Patent Application Publication No. 108424926; Russian Patent No. 2509570; Chinese Patent Application Publication No. 1900118; Chinese Patent No. 109200270; Chinese Patent No. 103667319; Argentine Patent Application Publication No. 053715; Chinese Utility Model Patent No. 213677887; JP 2023516904 Publication No. 202313658; Thai Patent No. 75524; Chinese Patent Application Publication No. 109381699; Taiwan Patent Application Publication No. 202313658; Thai Patent Application Publication No. 118552; British Patent Application No. 0920319; Chinese Patent Application Publication No. 114594068; Chinese Patent Application Publication No. 113984947; Brazilian Pipeline Patent Application Publication No. 0610396; Philippine Patent Application Publication No. 20061434; International Publication No. 2022171681; US ​​Patent Application Publication No. 2023123584;Thai Patent No. 118552; International Publication No. 2017179232; International Publication No. 2023014853; Chinese Patent Application Publication No. 111228477; Chinese Patent Application Publication No. 114594067; European Patent No. 1007551; New Zealand Patent Application Publication No. 505108; New Zealand Patent Application Publication No. 535085; Chinese Patent Application Publication Patent Application Publication No. 115569189; Chinese Patent Application Publication No. 1656116; New Zealand Patent Application Publication No. 516725; Taiwan Patent Application Publication No. 201729837; Austrian Patent Application No. 354662 (translation of European patent); Korean Patent Application Publication No. 20040030599; Slovenian Patent Application No. 1210112 (translation of claims from European patent) ;Korean Patent No. 100785397;Specification of Chinese Patent No. 114134165;Specification of Chinese Patent No. 110680918;International Publication No. 2022244815;Taiwan Patent Application Publication No. 202307211;Specification of South African Patent No. 200808893;Specification of Chinese Patent No. 1308036;Specification of Chinese Patent Application Publication No. 114958741;Czech Republic Patent Application Publication No. 20 Specification No. 001244; Chinese Patent Application Publication No. 111440811; Australian Patent Application Publication No. 2002310802; European Patent Application Publication No. 3037103; Cyprus Patent No. 1115704 (translation of the European patent); Brazilian Pipeline Patent No. 0017420-B1; and Brazilian Pipeline Patent Application Publication No. 0610032.

[0093] In one embodiment, the TCR is generated from a quadrivalent (types 6, 11, 16, 18) recombinant HPV vaccine (e.g., Gardasil). The TCR can be isolated from the T cells of a patient who has received the vaccine. The TCR can be engineered to recognize specific antigens on cancer cells associated with HPV infection, such as E6 and E7 proteins. The engineered TCR can then be introduced into the patient to treat the cancer.

[0094] In one embodiment, TCRs are generated from a recombinant HPV 9-valent vaccine (e.g., Gardasil 9). TCRs can be isolated from T cells of a patient who has received the vaccine. TCRs can be engineered to recognize specific antigens on cancer cells associated with HPV infection, such as E6 and E7 proteins. The engineered TCRs can then be introduced into the patient to treat the cancer.

[0095] In another embodiment, the TCR is generated from a bivalent (types 16 and 18) recombinant HPV vaccine (e.g., Cervarix). The TCR can be isolated from the T cells of a patient who has received the vaccine. The TCR can be engineered to recognize specific antigens on cancer cells associated with HPV infection, such as E6 and E7 proteins. The engineered TCR can then be introduced into the patient to treat the cancer.

[0096] In yet another embodiment, the TCR is generated from the HPV vaccine disclosed in International Publication No. 2022 / 115470. The TCR can be isolated from T cells of a patient who has received the HPV vaccine disclosed in that patent application. The TCR can be engineered to recognize specific antigens on cancer cells associated with HPV infection, such as E6 and E7 proteins. The engineered TCR can then be introduced into a patient to treat the cancer.

[0097] In another embodiment, the TCR generated from the HPV vaccine disclosed in International Publication No. 2022 / 115470 has one of the amino acid sequences from the group of SEQ ID NOs: 1 to 80. In a particular embodiment, the TCR includes an amino acid sequence or a functional variant thereof that has at least 90% sequence identity with one of the amino acid sequences of SEQ ID NOs: 1 to 80 (e.g., a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with one of the amino acid sequences of SEQ ID NOs: 1 to 80, or a conservatively substituted variant of one of the amino acid sequences of SEQ ID NOs: 1 to 80).

[0098] TCRs can be introduced into a patient using any known method of T-cell therapy, such as adoptive cell transfer or genetic modification of T cells.

[0099] Provided herein are methods for producing cells of any of the embodiments provided, comprising the step of introducing one of the vectors provided into the cells in vitro or ex vivo. In certain embodiments, the vector is a viral vector, and the introduction step is carried out by transduction.

[0100] A composition comprising any of the manipulated cells described herein is provided herein. In any part of any such embodiment, the manipulated cells comprise CD4+ and / or CD8+ T cells. In certain embodiments, the manipulated cells comprise CD4+ and CD8+ T cells.

[0101] Compositions comprising engineered CD8+ cells and engineered CD4+ cells as described herein are also provided herein. In certain embodiments, the TCR binds to or recognizes a peptide epitope of HPV 6 / 11 in association with an MHC molecule that is at least partially CD8-independent. In some of the such embodiments, the CD8+ cells and CD4+ cells are engineered using the same TCR and / or each using a TCR that binds to or recognizes the same peptide epitope of HPV 6 / 11 in association with an MHC molecule. In certain embodiments, the composition further comprises pharmaceutically acceptable excipients.

[0102] A treatment method comprising the step of administering one of the provided manipulated cells to a subject having an HPV-related disease or disorder is also provided herein. A treatment method comprising the step of administering one of the provided compositions to a subject having an HPV-related disease or disorder is also provided herein. In certain embodiments, the disease or disorder is related to HPV 6 / 11. In certain embodiments, the disease or disorder is cancer.

[0103] A treatment method comprising the step of administering manipulated cells described herein to a subject having an HPV-related disease or disorder is provided herein. A treatment method comprising the step of administering a composition described herein to a subject having cancer is also provided herein. Any one of the compositions described herein for use in the treatment of an HPV-related disease or disorder in a subject is provided herein.

[0104] The use of compositions for the manufacture of pharmaceuticals for treating HPV-related diseases or disorders in subjects is provided herein. In some of the such embodiments, the disease or disorder is related to HPV6 / 11. In certain embodiments, the disease or disorder is cancer. Compositions described herein for use in treating cancer in subjects are also provided herein. The use of any of the compositions described herein for the manufacture of pharmaceuticals for treating cancer in subjects is also provided herein. In further such embodiments, the cancer is related to HPV6 / 11 infection. In certain embodiments, the subject is human.

[0105] Nucleic acids and vectors that deliver and / or express TCRs Also disclosed are polynucleotides and polynucleotide vectors encoding the disclosed HPV antigen-specific TCR, enabling the expression of the disclosed HPV antigen-specific TCR in the disclosed immune effector cells (e.g., T cells). In certain aspects of the present invention, isolated nucleic acids are provided herein that comprise a peptide comprising an amino acid sequence from the group consisting of SEQ ID NOs: 1 to 80 or a functional variant thereof (e.g., a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 1 to 80, or a conservatively substituted variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 80).

[0106] The term "suitable for host cell transformation" means that the recombinant expression vector is a regulatory sequence selected based on the nucleic acid molecule of the present invention and the host cell used for expression, and is operably ligated to the nucleic acid molecule. The terms "operably ligated" or "operably ligated" are used interchangeably and are intended to mean that the nucleic acid is ligated to the regulatory sequence in a manner that enables the expression of the nucleic acid.

[0107] Accordingly, the present invention provides a recombinant expression vector comprising a nucleic acid encoding an HPV-specific TCR and a regulatory sequence essential for the transcription and translation of the inserted protein sequence. Suitable regulatory sequences may be derived from various sources, including bacterial, fungal, or viral genes. Generally, the vector encoding the TCR used to transfer target T cells should not replicate in the target T cells. Numerous virus-based vectors are known in which the number of viral copies retained in the cell is low enough to maintain cell viability. Exemplary vectors include the AdenoVerse® vector and the Gorilla Adeno Vector, as well as vectors based on HIV, SV40, EBV, HSV, and BPV.

[0108] The selection of appropriate regulatory sequences is generally dependent on the selected host cell and can be easily performed by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase-binding sequences, and ribosome-binding sequences containing transcription initiation sequences. In addition, other sequences, such as origins of replication, additional DNA restriction sites, enhancers, and sequences that confer transcriptional induction ability, may also be included in the expression vector, depending on the selected host cell and the vector used. It will also be understood that essential regulatory sequences may be provided by native proteins and / or contiguous regions thereof.

[0109] The disclosed nucleic acids can be cloned into a wide variety of vectors. For example, nucleic acids can be cloned into vectors including, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Of particular interest are expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0110] Alternatively, TCR constructs can be introduced into T cells using viral vectors (e.g., retroviral vectors, adenovirus vectors, adeno-associated virus vectors, or lentiviral vectors). Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a suitable restriction endonuclease site, and one or more selectable markers. In some embodiments, the polynucleotide vector is a lentiviral or retroviral vector.

[0111] Numerous virus-based systems have been developed for gene delivery into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Preferably, gene delivery is into mammalian cells (e.g., PBMCs).

[0112] A suitable example of a promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence that can drive high levels of expression of any polynucleotide operably ligated to it.

[0113] Another example of a suitable promoter is elongation growth factor-1a (EF-1a; EF1a). However, other constitutive promoter sequences may be used, including, but are not limited to, the monkey virus 40 (SV40) early promoter, the MND (myeloprenovatory sarcoma virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus pre-early promoter, the Roussarcoma virus promoter, and human gene promoters, such as, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Alternatively, the promoter may be an inductive promoter. Examples of inductive promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progestosterone promoter, and the tetracycline promoter.

[0114] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to also contain functional elements downstream of the initiation site. Spacing between promoter elements is often flexible, and as a result, promoter function is preserved when elements are inverted or moved relative to each other.

[0115] To evaluate the expression of a TCR polypeptide or a portion thereof, the expression vector introduced into cells may contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells being sought for transfection or infection with the viral vector. In other embodiments, the selectable marker may be supported on a separate DNA fragment and used in the cotransfection procedure. Both the selectable marker and the reporter gene may be flanked with appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes. The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequence. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is manifested by some readily detectable characteristic, such as enzymatic activity. The expression of the reporter gene is assayed at an appropriate time after the DNA has been introduced into the recipient cells.

[0116] Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct having a minimal 5' flanking region that exhibits the highest level of expression of the reporter gene is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used for drug evaluation of its ability to modulate promoter-driven transcription.

[0117] Methods for introducing and expressing genes in cells are well known in the art. Regarding expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art to produce transformed or transfected host cells. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0118] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0119] Biological methods for introducing a target polynucleotide into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.

[0120] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, e.g., macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). In examples where non-viral delivery systems are utilized, the exemplary delivery vehicle is liposomes. In another embodiment, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and oligonucleotides, captured within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in "disintegrated" structures. They can also simply be scattered in solution and, if necessary, form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources.For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0121] Immune effector cells including TCRs Most preferably, host cells engineered to express all or part of the TCR disclosed in the present invention include immune cells (e.g., immune effector cells). Such cells may be obtained from the subject being treated (i.e., a donor) (i.e., autologous cells). However, in some embodiments, immune cell lines or donor cells other than the subject's own cells (i.e., allogeneic cells) may be used.

[0122] Immune effector cells can be obtained from numerous sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. Immune effector cells can also be obtained from blood collected from subjects and / or donors using any number of techniques known to those skilled in the art, such as Ficoll® isolation. For example, cells from an individual's circulating blood can be obtained by apheresis.

[0123] In some embodiments, immunoeffector cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by a PERCOLL® gradient or countercurrent centrifugation. Furthermore, specific subpopulations of immunoeffector cells can be isolated by positive or negative selection techniques. For example, immunoeffector cells can be isolated using a combination of antibodies against surface markers specific to positively selected cells, for example, by incubation with antibody conjugate beads for a sufficient time to positively select the desired immunoeffector cells. Alternatively, enrichment of the immunoeffector cell population can be achieved by negative selection using a combination of antibodies against surface markers specific to negatively selected cells.

[0124] In some embodiments, immune effector cells include any leukocytes involved in the body's defense against infectious diseases and foreign substances. For example, immune effector cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells may include any subset of T cells (e.g., helper T cells, cytotoxic T cells, regulatory T cells, memory T cells, natural killer T cells).

[0125] TCR T cells Methods for the prevention and treatment of HPV-related diseases and cancers by adoptive transfer of autologous or allogeneic HPV-specific, TCR-expressing cells (e.g., TCR T cells as described herein) are provided herein. Such methods involve T cells containing the TCR of the present invention (e.g., CTLs, CD8 + T cells and / or CD4 + This may include the generation and / or use of T cells.

[0126] The generation of peptide-specific T cells is known in the art and may include, for example, inducing sensitization (e.g., activation and proliferation) of peptide-specific T cells by incubating a sample containing T cells (e.g., a PBMC sample, a concentrated sample, or a sample of isolated T cells) with an antigen peptide (i.e., a peptide containing a T cell epitope) or an antigen-presenting cell (APC) that displays one or more such T cell epitopes (e.g., an APC that displays a peptide containing a CTL HPV epitope on a class I MHC complex). In the present invention, such antigen peptides and epitopes are HPV-specific.

[0127] In certain embodiments, a sample containing CTLs (i.e., a PBMC sample) is incubated under culture conditions with an antigenic HPV peptide (e.g., an antigenic HPV6 / 11 peptide, e.g., disclosed in International Publication No. 2022 / 115470) or with HPV antigen-presenting cells (APCs) provided herein (e.g., "peptide pulse" cells that present the peptide containing the HPV epitopes described herein on a class I MHC complex).

[0128] In some embodiments, the APC is self-identified to the subject from which the T cells are obtained. In some embodiments, a sample containing T cells is incubated with the APC provided herein two or more times. In some embodiments, T cells are incubated with the APC in the presence of at least one cytokine. In some embodiments, the cytokines are IL-4, IL-7, and / or IL-15. An exemplary method for inducing T cell proliferation using the APC is provided, for example, in U.S. Patent Application Publication No. 2015 / 0017723, which is thus incorporated herein by reference. In some embodiments, the antigen is an HPV antigen (e.g., E6 and E7).

[0129] In certain embodiments, allele HLA restriction of such TCR-T cells (i.e., restriction to specific HLA-A, HLA-B, or HLA-C alleles) is known. In some embodiments, the T cells used to generate the TCR-T cells of the present invention are peptide-specific (i.e., sensitized to antigenic peptides such as viral peptides). In some embodiments, the T cells used to generate the TCR-T cells of the present invention are pluripotent T cells, i.e., T cells capable of inducing multiple immune effector functions that result in a more effective immune response against a pathogen than cells that produce only a single immune effector (e.g., a single biomarker, e.g., cytokine or CD107a). Less pluripotent, monofunctional, or even “exhausted” cells may be dominant during chronic infection and thus may negatively impact defense against virus-associated complications. In a more preferred embodiment, the TCR-T cells of the present invention are pluripotent. In certain embodiments, at least 50% of the T cells used to generate the TCR-T cells of the present invention are CD4 + These are T cells. In some such embodiments, the T cells are less than 50% CD4 + These are T cells. In further embodiments, these T cells are mainly CD4 + These are T cells. In some embodiments, at least 50% of the T cells used to generate the TCR-T cells of the present invention are CD8 + These are T cells. In some such embodiments, the T cells are less than 50% CD8 + These are T cells. In further embodiments, the T cells are mainly CD8 + These are T cells. In some embodiments, the T cells (e.g., donor samples, sensitized T cells, and / or TCR-T cells as described herein) are stored in a cell library or bank before being administered to the subject.

[0130] In some embodiments, engineered TCR-T cells expressing the disclosed TCR further express dominant-negative mutations that perform immune checkpoint blockade (e.g., expressing a dominant-negative form of an immune checkpoint molecule, e.g., PD-1). While not intended to be an exhaustive list, immune checkpoint molecules are selected from programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA), T cell immunoglobulin mucin-3 (HM-3), lymphocyte activating protein 3 (LAG-3), T cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4), and CD 160. Immune checkpoint molecules may also be transforming growth factor β (TGF-β) receptors. In certain preferred embodiments, the immune checkpoint molecule is CTLA-4. In certain embodiments, the immune checkpoint molecule is PD-1.

[0131] In some embodiments, one or more T cell populations contain a high concentration or deficiency of cells that are positive for or negative for specific markers, such as surface markers. In some cases, these markers are absent or expressed at relatively low levels in certain T cell populations (e.g., non-memory cells), but present or expressed at relatively high levels in other specific T cell populations (e.g., memory cells).

[0132] The T cells of the present invention can be isolated by any isolation method or combination thereof known in the art. The isolation method may vary depending on the desired purity, cell yield, and downstream application. Researchers and clinicians can select the most suitable technique based on the specific requirements of their research or clinical procedure.

[0133] In certain specific embodiments, T cells can be isolated from a cell sample of a patient or subject by density gradient centrifugation. Methods Mol Biol. 2019;1930:11-17. In density gradient centrifugation, the cell sample from the patient or subject is loaded onto a density gradient medium, typically a solution such as Ficoll or Percoll. Accordingly, upon centrifugation, different cell types are separated based on their density. T cells, which are relatively dense, will form distinct layers within the gradient, allowing their isolation.

[0134] In certain specific embodiments, T cells can be isolated from a cell sample of a patient or subject by magnetic cell separation. Nanoscale, 2017;9:13592-13599. In magnetic cell separation, T cells are targeted using specific antibodies attached to magnetic beads. The cell sample is incubated with antibody-coated beads, which allows the beads to bind to T cells. The cell suspension is then passed through a magnetic field, whereby T cells (bound to the beads) are retained, while other cells are washed away. The T cells can then be released from the beads for future use.

[0135] In certain specific embodiments, T cells can be isolated from a cell sample of a patient or subject by flow cytometry sorting. Cytometry A., 2019;95(6):647-654. Fluorescently labeled antibodies specific for T cell surface markers (e.g., CD3, CD4, CD8) are used to identify and sort T cells. The cell sample is stained with the antibodies, and the labeled cells are then passed through a flow cytometer. The flow cytometer detects the labeled cells and separates them based on their fluorescent properties, enabling the isolation of T cells.

[0136] In some embodiments, T cells are isolated from peripheral blood mononuclear cell (PBMC) samples by negative selection for markers expressed on non-T cells, such as CD14 expressed on B cells, monocyte cells or other leukocyte cells. In some embodiments, CD4 + or CD8+ The selection step is CD4 + Helper and CD8 + These CD4 cells are used to isolate cytotoxic T cells. + and CD8 + The group can be further classified into subgroups by positive or negative selection of one or more unsensitized memory and / or effector T cell subgroups or markers that are relatively highly expressed.

[0137] In some embodiments, the T cells are autologous T cells. In one method, tumor cells are obtained from a patient to obtain a single-cell suspension. The single-cell suspension can be achieved by any suitable method, for example, mechanically or enzymatically. The single-cell suspension of tumor enzyme lysate can be cultured in interleukin-2 (IL-2). The cells are then compacted (e.g., about 2 × 10⁻⁶). 6 Cells can be cultured for, for example, about 5 to 21 days, preferably about 10 to 14 days, until they reach a certain number of lymphocytes. For example, cells can be cultured for 5, 5.5, or 5.8 days to 21 days, 21.5, or 21.8 days, for example, 10, 10.5, or 10.8 days to 14 days, 14.5, or 14.8 days.

[0138] Cultured T cells can be collected and rapidly proliferated. Rapid proliferation increases the number of antigen-specific T cells by at least about 50 times (e.g., 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times or more) over a period of about 10 to about 14 days. More preferably, rapid proliferation increases the number by at least about 200 times (e.g., 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, or 900 times or more) over a period of about 10 to about 14 days.

[0139] The T cells of the present invention may be proliferated by any of the various proliferation methods known in the art. For example, T cells can be rapidly increased by nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15) (IL-2 is preferred). T cells can also be rapidly proliferated by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more cancer cell antigens that can be expressed (including their antigenic moieties, e.g., epitopes or cells). In vitro-induced T cells can be rapidly proliferated by resttimulation with the same cancer antigen(s) pulsed onto antigen-presenting cells expressing HLA-A2. Alternatively, T cells can be proliferated, for example, by irradiated autologous lymphocytes or irradiated HLA-A2 cells. + It can be restimulated with allogeneic atypical lymphocytes and IL-2.

[0140] In other embodiments, autologous T cells can be modified to express T cell growth factors that promote the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukins IL-2, IL-7, IL-15, and IL-12. Suitable modification methods are known in the art. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rded., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, modified autologous T cells express high levels of T cell growth factors. Coding sequences for T cell growth factors, such as IL-12, are readily available in the art, as are promoters, and the operable binding of promoters to T cell growth factor coding sequences promotes high levels of expression.

[0141] In certain embodiments, T cell growth factors that promote the growth and activation of autologous T cells are administered to the subject simultaneously with or after the autologous T cells. The T cell growth factors can be any suitable growth factors that promote the growth and activation of autologous T cells. Examples of suitable T cell growth factors are interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a preferred T cell growth factor.

[0142] The T cells of the present invention can be manipulated to express novel T cell receptors (TCRs) using various genetic engineering techniques known in the art. Careful design and optimization techniques, quality control measures, and extensive characterization of the manipulated T cells are crucial to ensure proper expression and function of TCR cells, as well as their safety and efficacy in clinical applications.

[0143] In certain embodiments, retroviral or lentiviral transduction is used to manipulate T cells to express a desired TCR. Viruses, 2021;13(8):1528; Hum Gene Ther., 2009;20(6):630-40. Thus, in such embodiments, a retroviral or lentiviral vector is used to deliver the TCR gene to T cells, and the vector comprises a nucleic acid sequence encoding the desired TCR, along with nucleic acid sequences encoding other necessary elements such as a promoter and a selection marker. In such embodiments, T cells are isolated from a patient and activated to allow them to accept gene transfer. The vector containing the TCR gene is then introduced into the activated T cells by transduction. The viral vector integrates the TCR gene into the T cell genome, allowing the T cells to express the desired TCR on their surface.

[0144] In certain embodiments, genome editing techniques such as TALEN (transcription activator-like effector nuclease) or CRISPR / Cas9 (clustered, regularly arranged, short palindromic repeat sequences / CRISPR-related protein 9) are used to manipulate T cells to express a desired TCR. Gene Therapy, 2014;21:539-548; Blood, 2018;131(3):311-322. TALEN and CRISPR involve targeted modifications of the T cell genome for insertion into a desired TCR gene or modification of an endogenous TCR locus. TALEN or CRISPR / Cas9 are designed to recognize and cleave specific DNA sequences at TCR loci. This allows for the introduction of a desired TCR gene or the disruption of an endogenous TCR gene, resulting in the expression of the manipulated TCR.

[0145] In certain embodiments, engineered T cells expressing the TCR of the present invention (i.e., TCR-T cells) are expanded under culture conditions to generate a sufficient number of cells for therapeutic purposes or for research applications disclosed herein.

[0146] Treatment method Immune effector cells expressing the disclosed TCR can induce an immune response beneficial to the treatment of HPV antigen-expressing cancer cells (e.g., HPV-associated cancers). For example, the antitumor immune response induced by the disclosed TCR-modified immune effector cells may be an active or passive immune response. In addition, TCR-mediated immune responses may be part of adoptive immunotherapy approaches in which TCR-modified immune effector cells induce an immune response specific to HPV antigens.

[0147] Adoptive transfer of immunoeffector cells expressing a modified TCR is a promising anticancer therapy. Accordingly, in certain aspects of the present invention, a method for treating HPV-related cancer in a subject is provided, comprising the step of administering an effective amount of an adoptive immunotherapy composition containing TCR-expressing cells as intended herein. After collecting immunoeffector cells from a patient, the cells can be genetically engineered to express the disclosed HPV antigen-specific TCR, thereby modifying the specific antigenicity of the immunoeffector cells (e.g., T cells), and they can be injected back into the patient. Furthermore, immunoeffector cells obtained from a non-patient donor (i.e., allogeneic to the patient) can be genetically engineered to express the disclosed HPV antigen-specific TCR, and the TCR-containing cells are then injected into the patient. In certain specific embodiments, the immunoeffector cells containing the anti-HPV antigen TCR polypeptide are allogeneic HPV-specific cytotoxic T cells.

[0148] For example, a particular embodiment of the present invention is a therapeutic dose composition comprising TCR-T cells provided herein. In some embodiments, such a composition is used to treat cancer and / or HPV infection in a subject by administering an effective dose of the composition to the subject. In some embodiments, the engineered TCR-T cells are not of the subject's own. In some embodiments, the TCR-T cells are of the subject's own. In some embodiments, the TCR-T cells are stored in a cell bank before being administered to the subject. Thus, in some embodiments, the disclosed immune effector cells comprising one or more of the engineered TCR polypeptides of the present invention are allogeneic or autoimmune effector cells.

[0149] The disclosed TCR-modified immunoeffector cells (e.g., TCR-T cells) may be administered either alone or as a pharmaceutical composition in combination with diluents and / or other components, e.g., IL-2, IL-15, or other cytokines or cell populations. Briefly, the pharmaceutical composition may comprise the targeted cell population described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers, e.g., neutral buffered saline, phosphate-buffered saline; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions for use in the disclosed methods are formulated for intravenous administration in some embodiments. The pharmaceutical composition may be administered in any form suitable for treating MM. The dosage and frequency of administration will be determined by factors such as the patient's condition and the severity of their illness, but the appropriate dosage can be determined through clinical trials.

[0150] Where an "immunologically effective dose," "antitumor effective dose," "tumor inhibitory effective dose," or "therapeutic dose" is indicated, the exact amount of the composition of the present invention administered may be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.

[0151] In certain embodiments, it may be desirable to administer activated T cells to a target, then re-collect blood (or have the target undergo apheresis), activate T cells from there according to the disclosed method, and re-inject these activated, expanded T cells into the patient. This process can be performed multiple times at intervals of several weeks. In certain embodiments, T cells can be activated from 10cc to 400cc of blood. In certain embodiments, T cells are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc of blood. The use of this multiple blood collection / multiple re-injection protocol may be useful for selecting a particular population of T cells.

[0152] The disclosed compositions may be administered in any convenient manner, including by injection, infusion, or indwelling. The compositions described herein may be administered to a patient by direct administration to an organ, subcutaneous administration, intradermal administration, intratumoral administration, intranodal administration, intramedullary administration, intramuscular administration, intrapleural administration, intracranial administration, intravenous (IV) injection, or intraperitoneal administration. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by IV injection. The compositions may also be administered directly to a tumor, lymph node, or site of infection.

[0153] The appropriate dosage of TCR therapy may be determined based on the type of disease being treated, the severity and course of the disease, the patient's clinical condition, their medical history and response to treatment, and the discretion of the attending physician.

[0154] In some embodiments, naked DNA encoding the TCR of the present invention, or a suitable vector containing such a TCR, can be introduced into target T cells (e.g., T cells obtained from a human patient with cancer caused by HPV infection). Methods for stably transfecting T cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319.

[0155] If the manipulated immune cells (e.g., TCR-T cells) can express the TCR of the present invention at a desired level, such cells can be reintroduced or administered to a target to activate an antitumor response in the target.

[0156] To facilitate administration, transduced cells (e.g., TCR T cells) may be prepared in a pharmaceutical composition or indwelling suitable for in vivo administration using a suitable pharmaceutically acceptable carrier or diluent. Methods for preparing such compositions or indwellings are well known in the art. See, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mack, ed. (1980). Where appropriate, engineered cells expressing the TCR of the present invention (e.g., TCR T cells) may be formulated in semi-solid or liquid preparations, such as capsules, solutions, injections, inhalations, or aerosols, in a manner conventional for their respective routes of administration.

[0157] Using methods known in the art, the release and absorption of a composition can be prevented or minimized until it reaches a target tissue or organ, or the composition can be sustained. Generally, it is desirable to use a pharmaceutically acceptable form that enables cells expressing TCRs. Therefore, preferably, T cells can be prepared as a pharmaceutical composition containing an equilibrium salt solution such as Hanks equilibrium salt solution or conventional physiological saline.

[0158] Diagnostic methods In addition to the therapeutic applications of TCR described herein, TCR can also be used for diagnostic purposes, such as the detection of HPV infection in subjects. HPV infection is a major cause of several types of cancer, including cervical, anal, and head and neck cancers. Early detection of HPV infection can facilitate timely intervention and treatment, and thus potentially prevent the development of these cancers.

[0159] One method for detecting HPV infection using TCR according to the present invention comprises the steps of contacting a sample from a subject with TCR and detecting the binding of TCR to the sample. The sample may be any biopsy material that may contain HPV-infected cells, such as a tissue biopsy, a swab from a potentially infected site, or a blood sample. Binding of TCR to the sample indicates the presence of HPV antigens and therefore HPV infection in the subject. Detection of TCR binding can be performed using various assays known in the art, such as ELISA, flow cytometry, or immunohistochemical tests.

[0160] In one embodiment, the TCR used to detect HPV infection is linked to a detectable label, such as a fluorescent dye, enzyme, or radioisotope. The labeled TCR is then brought into contact with a sample, and the presence of the label is detected by an appropriate method, such as fluorescence microscopy, colorimetric assay, or autoradiography. In another embodiment, the TCR is unlabeled, and the binding of the TCR to the sample is detected using a secondary reagent, such as a labeled antibody specific to the TCR. Diagnostic methods using the TCR of the present invention may be performed in vitro or in vivo, depending on the specific requirements and constraints of the clinical setting.

[0161] Pharmaceutical preparation In carefully selected embodiments, cells expressing the TCR of the present invention, or polynucleotides encoding the TCR of the present invention, may be included in a vaccine composition administered to a subject. The administration is expected to induce a therapeutic immune response against cancer caused by HPV infection in the subject. The therapeutic composition for pharmaceutically use in the subject may include a TCR composition, e.g., a soluble TCR (optionally attached to a contrast agent), and a pharmaceutically acceptable carrier.

[0162] As used herein, "protective immune response" refers to the response of the mammalian host immune system to cancer. Protective immune responses may demonstrate therapeutic effects on cancer treatment, such as reducing tumor size or increasing survival rates.

[0163] The appropriate formulation of a pharmaceutical composition depends on the chosen route of administration. An overview of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). The formulation and dosage of the composition also depend on factors such as the patient's weight, the severity of the disease, the type of disease to be treated, existing or current therapeutic interventions, and the specificity of idiopathic disease.

[0164] The pharmaceutical compositions disclosed herein may be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intrabladderally, intramucosally, pericardially, intranasally, intraocularly, orally, topically, as well as by inhalation, injection, infusion, continuous infusion, lavage, and topical perfusion. The therapeutic compositions may be administered to a target by catheter, as a cream, as a lipid composition, by ballistic microparticle delivery, or by other methods or combinations thereof, as is known to those skilled in the art (e.g., Remington, The Science and Practice of Pharmacy, 21 st See Ed. Lippincott Williams and Wilkins, 2005.

[0165] Any suitable carrier known to those skilled in the art may be used in the pharmaceutical composition of the present invention, but the type of carrier will vary depending on the method of administration. Carriers for parenteral administration, such as subcutaneous injection, preferably contain water, saline solution, alcohol, fat, wax, or buffer. For oral administration, any of the above carriers, or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharate, talc, cellulose, glucose, sucrose, and magnesium carbonate may be used. Biodegradable microspheres (e.g., polylactic acid galactide) may also be used as carriers for the pharmaceutical composition of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent No. 4,897,268 and U.S. Patent No. 5,075,109.

[0166] Suitable pharmaceutical compositions include aqueous and non-aqueous isotonic sterile solutions that may contain antioxidants, buffers, and bacteriostatic agents, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Pharmaceutical compositions may be presented in sealed containers of unit doses or multiple doses, such as ampoules and vials, and can be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier, such as water, immediately before use. Immediate solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the carrier is a buffered saline solution.

[0167] In some embodiments, the vaccine composition may be administered by microstructural subcutaneous delivery or ballistic particle delivery. Microstructures as carriers for vaccine formulations are well known in the art as preferred configurations for vaccine use (Gerstel and Place 1976 (U.S. Patent No. 3,964,482), Ganderton and McAinsh 1974 (U.S. Patent No. 3,814,097); U.S. Patents No. 5,797,898, No. 5,770,219 and No. 5,783,208, and U.S. Patent Application Publication No. 2005 / 0065463). Such vaccine compositions formulated for ballistic particle delivery may include isolated TCR peptides disclosed herein immobilized on the surface of a carrier support. In such embodiments, the carrier support may include, but is not limited to, microcapsules, microparticles, microspheres, nanocapsules, nanoparticles, nanospheres, or combinations thereof.

[0168] Microstructures or ballistic microparticles that function as carrier supports for TCRs disclosed herein, such as soluble TCRs, may be composed of biodegradable and non-biodegradable materials, such carrier supports being synthetic polymers, silica, lipids, carbohydrates, proteins, lectins, ionic agents, crosslinkers, and other microstructural components that may be used in the art. Protocols and reagents for immobilizing the peptides of the present invention on carrier supports composed of these materials are widely available and can be obtained in the art.

[0169] In other embodiments, the vaccine composition comprises an immobilized or encapsulated TCR or a soluble TCR as disclosed herein, and a carrier support. In this embodiment, the carrier support is, but is not limited to, lipid microspheres, lipid nanoparticles, etosomes, liposomes, niosomes, phospholipids, sphingosomes, surfactants, transferosomes, emulsions, or combinations thereof. The production and use of liposomes and other lipid nanocarriers and microcarrier formulations are generally known to those skilled in the art, and liposomes, microparticles, nanocapsules, etc., are widely used for the delivery of therapeutic agents (see, for example, U.S. Patent No. 5,741,516). Numerous methods, including peptide encapsulation, are being re-examined for liposomes and liposome-like drugs as potential drug carriers (e.g., U.S. Patents Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587).

[0170] In addition to the delivery methods described herein, a great many alternative techniques for administering the disclosed vaccine compositions are also envisioned. In non-limiting examples, a vaccine composition may include sonophoresis (i.e., ultrasound), as described in U.S. Patent No. 5,656,016, which is used to improve drug penetration through (and into) the circulatory system and the efficacy of drug penetration, and may be administered by intradermal injection (e.g., U.S. Patent No. 5,779,708) or by feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0171] A variety of optional adjuvants can be used in the vaccines of the present invention to nonspecifically improve the immune response. Most adjuvants are substances designed to protect antigens from rapid catabolism; for example, aluminum hydroxide or mineral oil, and lipid A, Bortella pertussis, or Mycobacterium tuberculosis. Nonspecific immune response stimulants. Suitable adjuvants include, for example, Freund's incomplete adjuvant and complete Freund's adjuvant ([manufactured by Difco Laboratories, Detroit, MI]) and Merck Adjuvant 65 [Raway, New Jersey, USA] (commercially available from Merck and Company, Inc.). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A, and quill A.

[0172] Soluble TCRs can be formulated by incorporating them into neutral or salt-formulated compositions. Examples of pharmaceutically acceptable salts include acid addition salts (formed as free amino acid groups of proteins) formed from inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Furthermore, salts formed with free carboxyl groups include inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, and procaine.

[0173] In some embodiments, the composition may contain various antioxidants to slow the oxidation of one or more components. In addition, preservatives such as various antimicrobial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof, may be used to control microbial activity.

[0174] If necessary, the TCR of the present invention can be incorporated in a suitable amount in a suitable solvent together with various other components listed above, then filtered and sterilized to prepare a sterile injectable solution. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and / or other components. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, preferred preparation methods are vacuum drying and lyophilization, from which the powder of the active ingredient and additional desired optional components is obtained from a pre-sterilized filtered liquid medium. The liquid medium should be appropriately buffered if necessary, and the liquid diluent should be isotonic before injection with sufficient saline or glucose. Preparation of high-concentration compositions for direct injection is also intended, in this example, the use of DMSO as a solvent is considered to induce very rapid penetration and delivery of high concentrations of the active agent into a small area.

[0175] In certain embodiments, an absorption retarder, such as aluminum monostearate, gelatin, or a combination thereof, may be used in the composition to delay the absorption of the injectable composition.

[0176] In certain embodiments, the pharmaceutical composition may contain one or more pH adjusters or buffers, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the pharmaceutical composition within an acceptable range.

[0177] In certain embodiments, the pharmaceutical composition may contain one or more salts in amounts necessary to bring the osmotic pressure of the composition into an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citric acid, ascorbic acid, boric acid, phosphoric acid, bicarbonate, sulfuric acid, thiosulfate, or bisulfite anions; preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0178] The pharmaceutical composition may be formulated in any suitable dosage form, including, but is not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, rapid-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, delayed-release formulations, sustained-release formulations, pulsed-release formulations, multi-particle formulations, and mixed formulations of immediate-release and controlled-release, for oral administration by the individual being treated. In some embodiments, the pharmaceutical composition is formulated in capsule form. In some embodiments, the pharmaceutical composition is formulated in solution (e.g., for IV administration). In some examples, the pharmaceutical composition is formulated as an intravenous infusion. In some examples, the pharmaceutical composition is formulated as an injectable.

[0179] In certain embodiments, the pharmaceutical composition is a liquid. In some embodiments, the composition may be freeze-dried and then restored before use.

[0180] In certain embodiments, the pharmaceutical composition may contain one or more preservatives, for example, to inhibit microbial activity. Suitable preservatives include mercury-containing substances, such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0181] In certain embodiments, the pharmaceutical composition may contain one or more defoaming agents. These defoaming agents can reduce foaming during processing, which can result in aggregation of aqueous dispersions, bubbles in the finished film, or generally, impaired processing. Exemplary defoaming agents include silicone emulsions or sorbitan sesquioleates.

[0182] In certain embodiments, the pharmaceutical composition may contain one or more antioxidants. Exemplary antioxidants include butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite, and tocopherol. In certain embodiments, one or more antioxidants enhance the chemical stability of the composition.

[0183] In certain embodiments, the pharmaceutical composition may contain one or more stabilizers. Examples of stabilizers include, for example: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations, such as magnesium and zinc; or (n) combinations thereof.

[0184] In certain embodiments, the pharmaceutical composition may contain one or more binders. These binders can impart cohesiveness. Exemplary binders include alginic acid and its salts; cellulose derivatives, e.g., carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®); microcrystalline dextrose; amylose; aluminum magnesium silicate; acidic polysaccharides; bentonite; gelatin; polyvinylpyrrolidone / vinyl acetate copolymer; crospovidone; povidone; starch; and aluminum. Examples include fermented starch; tragacanth, dextrin, sugars, e.g., sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), and lactose; natural or synthetic gums, e.g., acacia, tragacanth, gatchum, isapole bark mucus, polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone® XL-10), larch alabogalactan, Veegum®, polyethylene glycol, wax, sodium alginate, etc.

[0185] In certain embodiments, the pharmaceutical composition may include a carrier or a pharmaceutically compatible carrier material. These may include any excipient commonly used in pharmaceuticals, which should be selected based on their compatibility with the pharmaceutical compounds described herein. Exemplary carrier materials include binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. Exemplary pharmaceutically compatible carrier materials include acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglycerides, diglycerides, and pregelatinized starch. For example, see Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).

[0186] In certain embodiments, the pharmaceutical composition may include one or more diffusion promoters, dispersants, and / or viscosity modifiers to control the diffusion and homogeneity of the composition, for example, by a liquid medium or granulation or blending method. In some embodiments, these agents also promote the effectiveness of the coating or erosive matrix. Exemplary diffusion promoters and dispersants include hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and carbohydrate-based dispersants, such as hydroxypropyl cellulose (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC 4-(1,1,3,3-tetramethylbutyl)-phenol polymer (also known as tyloxapol) with ethylene oxide and formaldehyde, such as K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinylpyrrolidone / vinyl acetate copolymer (S630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer (also known as tyloxapol) with ethylene oxide and formaldehyde, poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, which are tetrafunctional block copolymers derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF) Corporation, Parsippany, NJ)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone / vinyl acetate copolymer (S-630), polyethylene glycol, for example polyethylene glycol having a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gum, for example, Examples of dispersants include sugars and cellulose-derived materials such as lagacant gum and acacia gum, guar gum, xanthan gum, and xanthan gum, as well as polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomer, polyvinyl alcohol (PVA), alginates, chitosan, and combinations thereof. Plasticizers, such as cellulose or triethylcellulose, can also be used as dispersants. Particularly useful dispersants in liposome dispersions and self-emulsifying dispersions are dimyristoyl phosphatidylcholine, egg-derived natural phosphatidylcholine, egg-derived natural phosphatidylglycerol, cholesterol, and isopropyl myristate.

[0187] In a particular embodiment, the pharmaceutical composition comprises a combination of one or more erosion accelerators and one or more diffusion accelerators.

[0188] In certain embodiments, the pharmaceutical composition may contain one or more diluents. Diluents are chemical compounds used to dilute the substance of interest before delivery. Diluents can also be used to stabilize the substance, as they can provide a more stable environment. Salts dissolved in buffer solutions (which may also provide pH control or maintenance) are used as diluents in the art, but are not limited to phosphate-buffered saline solutions. In certain embodiments, diluents increase the volume of the composition to facilitate compression or to create a sufficient volume for a homogeneous blend for capsule filling. Examples of such compounds include lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, e.g., Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugars, e.g., Di-Pac® (Amstar); mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, powdered sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrose; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[0189] In certain embodiments, the pharmaceutical composition may contain one or more bulking agents. Examples of bulking agents include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrate, dextran, starch, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, and polyethylene glycol.

[0190] In certain embodiments, the pharmaceutical composition may contain one or more lubricants or flow promoters, which are compounds that prevent, reduce, or inhibit adhesion or friction of materials. Examples of lubricants include stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, hydrocarbons such as mineral oil or hydrogenated vegetable oils such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum salts, calcium salts, magnesium salts, zinc salts, stearic acid, sodium stearate, glycerol, talc, wax, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax®, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid®, Cab-O-Sil®, starch such as corn starch, silicone oil, and surfactants.

[0191] In certain embodiments, the pharmaceutical composition may contain one or more plasticizers. These are compounds used to soften microencapsulated materials or film coatings to prevent them from becoming more brittle. Examples of plasticizers include polyethylene glycol, e.g., PEG-300, PEG-400, PEG-600, PEG-1450, PEG-3350, and PEG-800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. In some embodiments, the plasticizer may also function as a dispersant or wetting agent.

[0192] In certain embodiments, the pharmaceutical composition may contain one or more solubilizers. Examples of solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrin, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycoflor, transktol, propylene glycol, and dimethyl isosorbide.

[0193] In certain embodiments, the pharmaceutical composition may contain one or more stabilizers. Exemplary stabilizers include any antioxidant, buffer, acid, preservative, and the like.

[0194] In certain embodiments, the pharmaceutical composition may contain one or more suspending agents. Exemplary suspending agents include polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30), vinylpyrrolidone / vinyl acetate copolymer (S630), polyethylene glycol (e.g., polyethylene glycol can have molecular weights of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400), sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, and hydroxymethylcellulose acetate stearate. Examples of compounds include polysorbate-80, hydroxyethylcellulose, sodium alginate, gums such as tragacanth gum and acacia gum, guar gum, xanthan gum, sugars, cellulose-derived compounds (e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose), polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, and povidone.

[0195] In certain embodiments, the pharmaceutical composition may contain one or more surfactants. Exemplary surfactants include sodium lauryl sulfate, sodium doxate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbate, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, such as Pluronic® (BASF). Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, such as octoxynol 10 and octoxynol 40. In some embodiments, surfactants may be included in the pharmaceutical composition to enhance physical stability or for other purposes.

[0196] In certain embodiments, the pharmaceutical composition may contain one or more thickeners. Examples of thickeners include methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof.

[0197] In certain embodiments, the pharmaceutical composition may contain one or more wetting agents. Exemplary wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium doxert, sodium oleate, sodium lauryl sulfate, sodium doxert, triacetin, Tween 80, vitamin E TPGS, and ammonium salts.

[0198] In certain embodiments, the pharmaceutical composition may be manufactured in a conventional manner, for example, by conventional mixing, dissolving, granulation, sugar coating, grinding, emulsification, encapsulation, sealing, or compression processes.

[0199] In certain embodiments, the pharmaceutical compositions for the administration of TCRs described herein (including cells containing such TCRs and / or polynucleotide sequences encoding such TCRs) can be conveniently provided in unit dosage forms and may be prepared by any method well known in the art of compounding. Generally, the pharmaceutical compositions may be prepared by associating the active ingredient with a carrier and then, if necessary, shaping the product into a desired formulation. The pharmaceutical compositions contain the TCRs (or polynucleotides encoding them) described herein in an amount sufficient to produce the desired effect on the process, condition, or disease to be treated.

[0200] In some embodiments, the pharmaceutical composition may be stored by freezing at temperatures of approximately 0°C to approximately -120°C, approximately -10°C to approximately -110°C, approximately -20°C to approximately -100°C, approximately -30°C to approximately -90°C, approximately -40°C to approximately -90°C, approximately -50°C to approximately -90°C, approximately -60°C to approximately -90°C, approximately -65°C to approximately -85°C, or approximately -70°C to approximately -80°C. In some embodiments, the pharmaceutical composition may be stored by freezing at a temperature of about -60°C, about -61°C, about -62°C, about -63°C, about -64°C, about -65°C, about -66°C, about -66°C, about -67°C, about -68°C, about -69°C, about -70°C, about -71°C, about -72°C, about -73°C, about -74°C, about -75°C, about -76°C, about -77°C, about -78°C, about -79°C, about -80°C, about -81°C, about -82°C, about -83°C, about -84°C, about -85°C, about -86°C, about -87°C, about -88°C, about -89°C, or about -90°C. The pharmaceutical composition may be thawed in a water bath, for example, before use, and prolonged exposure of the thawed composition to the water bath can be avoided. The temperature of the water bath used to thaw the pharmaceutical composition may be, for example, between approximately 30°C and 44°C, 31°C and 43°C, 32°C and 42°C, 33°C and 41°C, 34°C and 40°C, or 35°C and 39°C. In some embodiments, the temperature of the water bath used to thaw the pharmaceutical composition may be approximately 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. The thawed composition may be stored at ambient temperature for up to approximately 15 minutes, 30 minutes, 45 minutes, 1 hour, 75 minutes, 90 minutes, 105 minutes, or 2 hours before administration. In some embodiments, the thawed composition appears as a clear to slightly milky colorless liquid and is substantially free of visible particles.

[0201] Kit / Product Another aspect of the present invention is kits and products for use using one or more methods described herein. A preferred kit may include packaging or containers comprising a TCR as described herein, or a polynucleotide encoding such a TCR, or a composition comprising the same. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the containers are formed from various materials, such as glass or plastic. A preferred product may include packaging materials. Examples of pharmaceutical packaging materials include blister packaging, bottles, tubes, bags, containers, bottles, and any packaging materials suitable for the selected formulation and the intended manner of administration and treatment.

[0202] A kit typically includes a label listing the contents and / or instructions for use, as well as a package insert containing instructions for use. A set of instructions may also be included. In some embodiments, the label is on the container or associated with the container. In some embodiments, the label is on the container if the letters, numbers, or other characters forming the label are attached, molded, or engraved on the container itself; the label is associated with the container if it is located in a container or carrier that also holds the container, for example, as a package insert. In some embodiments, the label is used to indicate that the contents should be used for a specific therapeutic application. The label also indicates instructions for use of the contents, for example, in the methods described herein.

[0203] In some embodiments, the kit contains vials containing the compositions of the present invention. In some such embodiments, the vials contain, for example, about 0.1 to about 20 ml of pharmaceutical composition, about 0.1 to about 15 ml of pharmaceutical composition, about 0.1 to about 10 ml of pharmaceutical composition, about 0.1 to about 9 ml of pharmaceutical composition, about 0.1 to about 8 ml of pharmaceutical composition, about 0.1 to about 7 ml of pharmaceutical composition, about 0.1 to about 6 ml of pharmaceutical composition, about 0.1 to about 5 ml of pharmaceutical composition, about 0.2 to about 5 ml of pharmaceutical composition, and about It contains approximately 0.2 to 4 ml of pharmaceutical composition, approximately 0.2 to 3 ml of pharmaceutical composition, approximately 0.2 to 2 ml of pharmaceutical composition, approximately 0.2 to 1 ml of pharmaceutical composition, approximately 0.5 to 2 ml of pharmaceutical composition, approximately 0.5 to 1.75 ml of pharmaceutical composition, approximately 0.5 to 1.5 ml of pharmaceutical composition, approximately 0.5 to 1.25 ml of pharmaceutical composition, approximately 0.5 to 1 ml of pharmaceutical composition, and approximately 0.75 to 1.25 ml of pharmaceutical composition. In other such embodiments, the vial contains, for example, about 0.5 ml of pharmaceutical composition, about 0.55 ml of pharmaceutical composition, about 0.6 ml of pharmaceutical composition, about 0.65 ml of pharmaceutical composition, about 0.7 ml of pharmaceutical composition, about 0.75 ml of pharmaceutical composition, about 0.8 ml of pharmaceutical composition, about 0.85 ml of pharmaceutical composition, about 0.9 ml of pharmaceutical composition, about 0.95 ml of pharmaceutical composition, about 1 ml of pharmaceutical composition, about 1.05 ml of pharmaceutical composition, about 1.1 ml of pharmaceutical composition, about 1.15 ml of pharmaceutical composition, or about 1.2 ml of pharmaceutical composition.

[0204] Pharmaceutical manufacturing The present invention also relates in part to the use of TCRs as described herein, polynucleotides encoding TCRs as described herein, or compositions comprising them, in the manufacture of pharmaceuticals for use in treating diseases or disorders in subjects requiring such treatment. In certain embodiments, the disease or disorder may be a proliferative disorder or disorder, such as cancer. In some embodiments, the disease or condition is caused by HPV infection.

[0205] In one embodiment, the present invention provides a method for producing engineered cells expressing the TCR of the present invention. The method comprises the steps of culturing cells and introducing nucleic acids encoding the TCR into the cells under conditions sufficient for TCR expression in the cells. The cells may be any cells suitable for TCR expression, such as mammalian cells, human cells, immune cells, or T cells. The nucleic acids can be introduced into the cells using various methods known in the art, such as viral transduction, electroporation, or lipofection.

[0206] In one embodiment, the method includes the steps of isolating T cells from a subject, such as a patient with an HPV-related disease or disorder, and manipulating the T cells to express the TCR of the present invention. The manipulated T cells can then be expanded in vitro and reintroduced into the subject to elicit an immune response against HPV-infected cells. Known as adoptive cell therapy, this approach has shown promising results in the treatment of various types of cancer.

[0207] Manipulated cells expressing the TCR of the present invention can be produced in large quantities using standard cell culture techniques. For example, cells can be cultured in a bioreactor or large-scale cell culture system, such as a wave bioreactor or perfusion bioreactor. Culture conditions, such as temperature, pH, and nutrient composition, can be optimized to maximize cell growth and TCR expression. Manipulated cells can be harvested, purified, and formulated into pharmaceutical compositions for administration to targets requiring them.

[0208] In another embodiment, the method includes the step of introducing a nucleic acid encoding a TCR into cells in vivo, for example, by direct injection into a tumor or by systemic administration using a targeted delivery system. In vivo expression of the TCR in the target cells may lead to the generation of TCR-expressing T cells that can initiate an immune response against HPV-infected cells.

[0209] The manipulated cells expressing the TCR of the present invention can also be used in in vitro applications, such as studying the mechanisms of HPV infection and the immune response to HPV, or for screening compounds that can modulate TCR activity or enhance the efficacy of TCR-based therapies.

[0210] Combination therapy In certain embodiments, the compositions and methods of the present invention can be combined with at least one additional therapy. Such additional therapies include radiotherapy, surgery (e.g., weight loss), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the aforementioned therapies. The additional therapy may be in the form of an adjuvant or neoadjuvant therapy.

[0211] Any suitable agent that can be combined with the TCR of the present invention or the polynucleotide encoding it may be used. For example, the agent may be a therapeutic agent, such as a chemotherapeutic agent, an anti-inflammatory agent, an analgesic, a bio-response modifier, a vector containing such an agent, or a cell containing a therapeutic agent or nucleic acid encoding it.

[0212] In certain embodiments, the additional agent is administered at or near the same site as the TCR of the present invention, or a composition comprising the polynucleotide encoding it. In certain other embodiments, the additional agent is administered at a different site, for example, to the opposite or opposite limb.

[0213] The administration of an additional agent may occur simultaneously with the administration of a composition comprising the TCR (or the corresponding polynucleotide encoding it) of the present invention. In certain embodiments, the additional agent may be contained in the same formulation as the formulation containing the vector and administered together with the vector in a single unit dose. In certain other embodiments, the additional agent may not be contained in the same formulation but may be administered simultaneously with the administration of the vector or within a limited time frame (e.g., one day, one hour, or fraction of an hour) after the administration of the vector.

[0214] Alternatively, the administration of additional agents may be sequential in relation to the administration of the composition comprising the TCR of the present invention. Such administration may be preferred in cases where it is desirable to minimize adverse reactions. In such embodiments, the additional agents may be administered according to a schedule based on the approved dosing regimen for those agents. Alternatively, the agents may be administered according to a schedule that helps to better maximize the therapeutic effect of the combination therapy while minimizing adverse reactions.

[0215] In certain embodiments, blood tests, Pap tests, or clinical biopsies may be used to detect HPV-infected cells before administering additional medication.

[0216] In certain embodiments, the TCR of the present invention (including cells containing it) or a polynucleotide encoding the TCR may first be administered to a subject for the treatment of a disease or disorder, and then additional agents may be administered at a later date. In one embodiment, the additional agents are administered when, after administration of the TCR of the present invention, the disease or condition is thought to have relapsed, progressed, or become unresponsive to the TCR.

[0217] Anti-inflammatory agents for use in such combination therapies include steroids and glucocorticoids, including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone; non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mimicophenolates; and sphingosine 1-phosphate receptor modulators, including fingolimod (Gilenya®), ozanimod (Zeposia®), and amicelimod. In some embodiments, the NSAID is selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialates.

[0218] Examples of analgesics for use in combination therapy include acetaminophen, oxycodone, tramadol, or propoxifen hydrochloride.

[0219] Examples of bio-response modifiers suitable for use in combination therapy according to the present invention include, for example, molecules with directional properties towards cell surface markers (e.g., CD4, CD5); cytokine inhibitors, for example, TNF inhibitors (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)); chemokine inhibitors; cell signaling inhibitors, for example, EGFR inhibitors (e.g., gefitinib (IRESSA®) and erlotinib (TARCEVA®), nucleotide analogs (e.g., cidofovir); angiogenesis inhibitors, for example, bevacizumab (AVASTIN®); nonsteroidal anti-inflammatory compounds (NSAIDs), for example, COX-2 selective agents (e.g., celecoxib (CELEBREX®)); and immunosuppressants. Examples of inhibitors include PD-1 inhibitors (e.g., pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®, and semiprimab (LIBTAYO®))) and PD-L1 inhibitors (e.g., atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®, and durvalumab (IMFINZI®))), adhesion molecule inhibitors, and other adjuvant therapies. Bio-response modifiers include not only monoclonal antibodies but also recombinant molecules. Exemplary disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0220] In certain embodiments, the TCRs provided herein are co-delivered and / or co-expressed together with other cytokines. In certain embodiments, the TCRs provided herein are polynucleotides encoding gene switch polypeptides and cytokines, or variants or derivatives thereof, as well as methods and systems incorporating them. Cytokines are a category of small molecules between approximately 5 and 20 kDa that are involved in cell signaling. In some examples, cytokines include chemokines, interferons, interleukins, colony-stimulating factors, or tumor necrosis factors. In some embodiments, chemokines act as chemotaxis that guide cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include the CC subfamily: CCLI, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCLI0), CCLI 1, CCL12, CCL13, CCL14, CCL15, CCL16, CCLI 7, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC subfamily: CCXCLI, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CCXCLI0, CXCLII, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CCXCLI 7. Examples include chemokines from the XC subfamily: XCLI and XCL2, and the CX3C subfamily: CX3CL1.

[0221] In some embodiments, the cytokine is a membrane-bound cytokine co-expressed with a chimeric antigen receptor as described herein. In some embodiments, one or more methods described herein further include the administration of a cytokine. In some examples, the cytokine includes chemokines, interferons, interleukins, colony-stimulating factors, or tumor necrosis factors.

[0222] In some examples, one or more methods described herein further include the administration of cytokines selected from chemokines, interferons, interleukins, colony-stimulating factors, or tumor necrosis factors. In some examples, one or more methods described herein further include the administration of cytokines selected from IL2, IL7, IL12, IL15, a fusion of IL-15 and IL-15Rα, IL21, IFNγ, or TNF-α.

[0223] In certain embodiments, the TCRs provided herein are co-delivered and / or co-expressed with interferons. Interferons (IFNs) include interferon type I (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), interferon type II (e.g., IFN-γ), and interferon type 111. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNAI7, and IFNA21. In certain embodiments, the TCRs provided herein are co-delivered and / or co-expressed with other interleukins. Interleukins are expressed by leukocytes or white blood cells and promote the development and differentiation of T and B lymphocytes as well as hematopoietic cells. Exemplary interleukins include IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36. In some embodiments, the interleukin is IL-2, IL-12, IL-15, IL-21, or a fusion of IL-15 and IL-15α.

[0224] In some embodiments, interleukins may include IL-12. Interleukin-12 (IL-12) is spontaneously produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two distinct genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (called p70) and homodimer of p40 are formed after protein synthesis. IL-12 is a major regulator of the immune system. IL-12 promotes the immune response by activating NK cells and T cells.

[0225] In some embodiments, IL-12 is single-stranded IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or intercalated IL-12. In some examples, IL-12 variants are as described in International Publication Nos. 2015 / 095249, 2016 / 048903, and 2017 / 062953.

[0226] In some embodiments, the interleukin comprises mbIL-15. In some embodiments, mbIL-15 is a membrane-bound chimeric IL-15 that can be co-expressed with the modified effector cells described herein. In some embodiments, mbIL-15 comprises full-length IL-15 (e.g., native IL-15 polypeptide) or a functional fragment or variant thereof, fused in frame with full-length IL-15Rα, its functional fragment or variant. In some examples, IL-15 is indirectly linked to IL-15Rα by a linker. In some examples, mbIL-15 is as described in Hurton et al., “Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells,” PNAS 2016.

[0227] In certain embodiments, the TCRs provided herein are co-delivered and / or co-expressed together with tumor necrosis factor. Tumor necrosis factor (TNF) is a group of cytokines that modulate apoptosis. In some examples, the TNF family includes about 19 members, but is not limited to, TNFα, lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-associated apoptosis-inducing ligand (TRAIL).

[0228] In certain embodiments, the TCRs provided herein are co-delivered and / or co-expressed together with colony-stimulating factors. Colony-stimulating factors (CSFs) are secreted glycoproteins that interact with receptor proteins on the surface of hematopoietic stem cells, thereby modulating cell proliferation and differentiation into specific types of blood cells. In some examples, CSFs include macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), or promegaprotein.

[0229] In certain embodiments, the TCR provided herein is delivered to and / or expressed in a subject in conjunction with the delivery and / or expression of interleukin-12 cytokines.

[0230] In certain embodiments, in conjunction with the delivery or expression of the TCR described herein, IL-12 expression in the subject is controlled by constitutive or inductive regulation of expression. In preferred embodiments, in conjunction with the delivery or expression of the HPV vaccine antigen, IL-12 expression in the subject is controlled by inductive regulation of expression (also known as inductively regulated expression of IL-12).

[0231] In certain embodiments, the TCRs provided herein may be co-delivered and / or co-expressed together with surfactants such as immunostimulatory complexes (ISCOMS). Freund's incomplete adjuvants, LPS analogs including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs and vesicles, such as squalene and hyaluronic acid, may also be administered in conjunction with the TCR.

[0232] In some embodiments, additional therapy is the administration of small molecule enzyme inhibitors or anti-transfer agents. In some embodiments, additional therapy is the administration of side effect limiting agents (e.g., drugs aimed at reducing the incidence and / or severity of side effects of the procedure, such as nausea). In some embodiments, additional therapy is radiotherapy. In some embodiments, additional therapy is surgery. In further embodiments, the surgery is debulking surgery. In some embodiments, additional therapy is a combination of radiotherapy and surgery. In some embodiments, additional therapy is gamma irradiation.

[0233] In certain embodiments, T-cell therapy may be administered before, during, or after additional cancer therapies, such as immune checkpoint therapy, or in various combinations with additional cancer therapies. Dosage may occur at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments where T-cell therapy is provided to the patient separately from additional therapeutic agents, the operator may generally ensure that no significant time elapses between each delivery time so that the two compounds continue to exert a beneficial combination effect on the patient. Antibody therapy and anticancer therapy may be provided to the patient within approximately 12–24 hours or 72 hours of each other, and more specifically, within approximately 6–12 hours of each other. In some examples, the treatment period is significant, ranging from days (2, 3, 4, 5, 6, or 7 days) to weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each dose. Extending the period may also be desirable.

[0234] Various combinations can be used. In the following example, the TCR is "A" and the additional therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0235] The administration of any compound or therapy of this embodiment to a patient will follow general protocols for compound administration, taking into account any drug toxicity present. Therefore, some embodiments include a step to monitor toxicity resulting from the combination therapy.

[0236] chemotherapy In some embodiments, the additional therapy is chemotherapy, such as dacarbazine or temozolomide. The additional therapy may be one or more chemotherapeutic agents known in the art.

[0237] The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapy agents" refer to compounds or compositions administered during cancer treatment. These drugs are classified according to their mode of activity within cells, for example, whether and at what stage of the cell cycle they affect it. Alternatively, drugs can be characterized based on their ability to directly crosslink DNA, insert into DNA, or affect nucleic acid synthesis to induce chromosomal and mitotic mutations.

[0238] Exemplary chemotherapeutic agents that can be administered in combination with the composition of the present invention include alemtuzumab (Campath®), alitretinoin (Panretin®), anastrozole (Arimidex®), bevacizumab (Avastin®), bexarotene (Targretin®), bortezomib (Velcade®), bosutinib (Bosulif®), brentuximab vedotin (Adcetris®), cabozantinib (Cometriq®), and cabozantinib. Rufilzomib (Kyprolis trademark), cetuximab (Erbitux®), crizotinib (Xalkori®), dasatinib (Sprycel®), deniroikin difutitox (Ontak®), erlotinib hydrochloride (Tarceva®), everolimus (Afinitor®), exemestane (Aromasin®), fulvestrant (Faslodex®), gefitinib (Iressa®), ibritumomab tiuxetan (Zeva) lin(registered trademark), imatinib mesylate (Gleevec(registered trademark)), ipilimumab (Yervoy(trademark)), lapatinib ditosylate (Tykerb(registered trademark)), letrozole (Femara(registered trademark)), nilotinib (Tasigna(registered trademark)), ofatumumab (Arzerra(registered trademark)), panitumumab (Vectibix(registered trademark)), pazopanib hydrochloride (Votrient(registered trademark)), pertuzumab (Perjeta(trademark)), pralatrexate (Folotyn(registered trademark)), regorafenib ( Stivarga®, rituximab (Rituxan®), romidepsin (Istodax®), sorafenib tosylate (Nexavar®), sunitinib malate (Sutent®), tamoxifen, temsirolimus (Torisel®), toremifene (Fareston®), tositumomab and 1311-tositumomab (Bexxar®), trastuzumab (Herceptin®), tretinoin (Vesanoid®),Examples include, but are not limited to, vandetanib (Caprelsa®), vemurafenib (Zelboraf®), vorinostat (Zolinza®), and Ziv-aflibercept (Zaltrap®). Further examples of chemotherapeutic agents, including but not limited to such examples, include: alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and piposulfan; aziridines, e.g., benzodopa, carbocon, metsuredopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including the synthetic analog topotecan); briostatin; callistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (synthetic analogs, KW-2189 and Including CB1-TM1); eryuterobin; pancratistatin; sarcodicin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlornafadin, cyclophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobembitin, fenesterine, prednimustine, trophosphamide, uracil mustard; nitrosourea, e.g., carmustine, Chlorozotosin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engineeric antibiotics (e.g., calicheamicin, in particular calicheamicin gamma II and calicheamicin omega II; dynemycin, including dynemycin A; bisphosphonates, e.g., clodronate; esperamicin; and neocardinostatin chromophore and related pigment protein engineeric antibiotic chromophore, acrasinomycin, actinomycin,Austramycin, azacerin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-F-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin Esolubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folic acid analogs, e.g., denopterin, methotrexate, pterop Therin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal drugs, e.g., aminoglutethimide, mitotane , trilostane; folic acid supplements, e.g., folinic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bistolene; edatraxate; dehofamine; demecolsin; diazicon; eflornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; lonidainine; meitansinoids, e.g.,Maytansine and anthamitosine; Mitoguazone; Mitoxantrone; Mopidammole; Nitracrine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex); Lazoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triadicone; 2,2',2”-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Verracurin A) A) Loridine A and Anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Paclitaxel and Docetaxel; Chlorambucil; Gemcitabine; 6-Thiogunine; Mercaptopurine; Methotrexate Sart; platinum-coordinated complexes, e.g., cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, e.g., retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0239] Radiation therapy In certain embodiments, radiotherapy may be used in combination with any of the TCR treatment methods described herein. "Radiotherapy" refers to treatment for a disease or disorder (typically cancer) in which radioactive energy is used to destroy cells and terminate their division.

[0240] Modern radiotherapy systems utilize relatively high-energy beams of radiation from radioisotopes or electron beam X-rays, or from generators in the case of gamma rays. Radiotherapy includes external beam radiation, intensity-modulated radiation therapy (IMRT), focused radiation, and any form of surgical irradiation, including gamma knife, cyberknife, linear accelerator, and interstitial irradiation (e.g., implanted radiation seeds, GliaSite balloons), as well as / or surgery. Other forms of DNA damage that can be performed in radiotherapy include microwaves, proton beam irradiation (US Patent Nos. 5,760,395 and 4,870,287), and ultraviolet irradiation. The dose range for X-rays varies from 50 to 200 rotgens per day over a long period (3 to 4 weeks) to 2,000 to 6,000 lentgens per day in a single dose. The range of radioisotope irradiation can vary widely depending on the half-life of the isotope, the intensity and type of radiation emitted, and the absorption rate of neoplastic cells.

[0241] In certain embodiments, radiotherapy is provided, which may include irradiation of a patient or the administration of an associated radiopharmaceutical. The radiation source may be either external or internal to the patient to be treated (the radiotherapy may take the form of, for example, external beam radiotherapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be used to carry out such a method include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

[0242] immunotherapy In some embodiments, the subject is also administered immunotherapy agents. Immunotherapy refers to treatments that use the subject's immune system to treat cancer, such as the use of cancer vaccines, cytokines, cancer-specific antibodies, T-cell therapy, and dendritic cell therapy. Immunotherapy in relation to cancer therapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. In some embodiments, the subject is also administered immunomodulatory proteins.

[0243] Examples of immunomodulatory proteins include, but are not limited to, B lymphocyte chemoattractants ("BLC"), CC motif chemokine 11 ("eotaxin-1"), eosinophil chemotactic protein 2 ("eotaxin-2"), granulocyte colony-stimulating factor ("G-CSF"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), 1-309, intercellular adhesion molecule 1 ("ICAM-1"), interferon-gamma ("IFN-gamma"), interleukin-1 alpha ("IL-1 alpha"), interleukin-1 beta ("IL-1 beta"), and interleukin-1 receptor antagonists ("IL-1") Interleukin-2 ("IL-2"), Interleukin-4 ("IL-4"), Interleukin-5 ("IL-5"), Interleukin-6 ("IL-6"), Interleukin-6 soluble receptor ("IL-6 sR"), Interleukin-7 ("IL-7"), Interleukin-8 ("IL-8"), Interleukin-10 ("IL-10"), Interleukin-11 ("IL-11"), Interleukin-12 subunit beta ("IL-12 p40" or IL-12p70), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 ("IL-17"), chemokine (CC motif) ligand 2 ("MCP-1"), macrophage colony-stimulating factor ("M-CSF"), gamma interferon-induced monokine ("MIG"), chemokine (CC motif) ligand 2 ("MIP-1 alpha"), chemokine (CC motif) ligand 4 ("MIP-1 beta"), macrophage inflammatory protein-1-delta ("MIP-1 delta"), platelet-derived growth factor subunit B ("PDGF-BB"), chemokine (CC motif) ligand 5, regulated upon activation and expressed and secreted by normal T cells ("RANTES"), TIMP metallopeptidase inhibitor 1 ("TIMP-1"), TIMP metallopeptidase inhibitor 2 ("HMR-2"), tumor necrosis factor, lymphotoxin-alpha ("TNF-alpha"), tumor necrosis factor, lymphotoxin-beta ("TNF-beta"), soluble TNF receptor type 1 ("sTNFRI"), sTNFRIIAR, brain-derived neurotrophic factor ("BDNF"), basic fibroblast growth factor ("bFGF"), osteomorphogenetic protein 4 ("BMP-4"), osteomorphogenetic protein 5 ("BMP 5") ("BMPS'1)), Bone morphogenetic protein 7 ("BMP-7"), nerve growth factor ("b-NGF"), epidermal growth factor ("EGF"), epidermal growth factor receptor ("EGFR"), endocrine gland-derived vascular endothelial growth factor ("EG-VEGF"), fibroblast growth factor 4 ("FGF-4"), keratinocyte growth factor ("FGF-7"), growth and differentiation factor 15 ("GDF-15"), glial cell-derived neurotrophic factor ("GDNF"), growth hormone, heparin-bound EGF-like growth factor ("HB-EGF"), Hepatocyte growth factor ("HGF"), insulin-like growth factor-binding protein 1 ("IGFBP-1"), insulin-like growth factor-binding protein 2 ("IGFBP-2"), insulin-like growth factor-binding protein 3 ("IGFBP-3"), insulin-like growth factor-binding protein 4 ("IGFBP-4"), insulin-like growth factor-binding protein 6 ("IGFBP-6"), insulin-like growth factor 1 ("IGF-1"), insulin, macrophage colony-stimulating factor ("M-CSF") Nerve growth factor receptor ("NGF R"), neurotrophin-3 ("NT-3"), neurotrophin-4 ("NT-4"), osteoclast inhibitor ("osteoprotegerin"), platelet-derived growth factor receptor ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp-karin-F-box complex ("SCF"), stem cell factor receptor ("SCF R"), transforming growth factor alpha ("TGF alpha"), transforming growth factor beta-1 ("TGF beta-1"), transforming growth factor 3 ("TGF beta-3"), vascular endothelial growth factor ("VEGF"), vascular endothelial growth factor receptor 2 ("VEGFR2"), vascular endothelial growth factor receptor 3 ("VEGFR3"), VEGF-D6Ckine, tyrosine-protein kinase receptor UFO ("Axl"), beta-cellulin ("BTC"), mucosa-associated epithelial chemokine ("CCL28"), chemokine (Chemokme) (CC motif) ligand 27 ("CTACK"), chemokine (CXC motif) ligand 16 ("CXCL16"), CXC motif chemokine 5 ("ENA-78"), chemokine (CC motif) ligand 26 ("Eotaxin-3"), granulocyte chemotactic protein 2 ("GCP-2"), GRO, chemokine (CC motif) - Ligand 14 ("HCC-1"), Chemokine (CC motif) ligand 16 ("HCC-4"), Interleukin-9 ("IL-9"), Interleukin-17F ("IL-17F"), Interleukin-18 binding protein ("IL-18BPa"), Interleukin-28A ("IL-28A"), Interleukin-29 ("IL-29"), Interleukin-31 ("IL-31"), CXC motif chemokine 10 ("IP-10"), Chemokine receptor CXCR3 ("I-TAC"), Leukemia Disease suppressor ("LIF"), light, chemokine (C motif) ligand ("lymphotactin"), monocyte chemoattractant protein 2 ("MCP-2"), monocyte chemoattractant protein 3 ("MCP-3"), monocyte chemoattractant protein 4 ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibitor ("MIF"), chemokine (CC motif) ligand 20 ("MIP-3 alpha"), CC motif chemokine 19 ("MIP-3 beta"), chemokine (CC motif) ligand 23 ("M PIF-1), macrophage-stimulating protein alpha chain (MSP-alpha), nucleosome assembly protein 1-like 4 (NAP-2), secreted phosphorylated protein 1 (osteopontin), lung and activation regulatory cytokine (PARC), platelet factor 4 (PF4), stromal cell-derived factor-1-alpha (SDF-1-alpha), chemokine (CC motif) ligand 17 (TARC), thymic-expressed chemokine (TECK), thymic-stromal lymphocyte neoplastic factor (TSLP4-IBB), CD166 antigens ("ALCAM"), differentiation antigen group 80 ("B7-1"), tumor necrosis factor receptor superfamily member 17 ("BCMA"), differentiation antigen group 14 ("CD14"), differentiation antigen group 30 ("CD30"), differentiation antigen group 40 ("CD40 ligand"), carcinoembryonic antigen-associated cell adhesion molecule 1 (bile glycoprotein) ("CEACAM-1"), cell death receptor 6 ("DR6"), deoxythymidine kinase ("Dtk"), type 1 membrane glycoprotein Protein ("endoglin"), receptor tyrosine-protein kinase erbB-3 ("ErbB3"), endothelial leukocyte adhesion molecule 1 ("E-selectin"), apoptosis antigen 1 ("Fas"), Fms-like tyrosine kinase 3 ("Flt-3L"), tumor necrosis factor receptor superfamily member 1 ("GITR"), tumor necrosis factor receptor superfamily member 14 ("HVEM"), intercellular adhesion molecule 3 ("ICAM-3"), IL-1 R4, IL-1 RI, IL-10 R-beta, IL-17R, IL-2 R-gamma, IL-21R, Lysosomal membrane protein 2 ("LIMPII"), Neutrophil gelatinase-associated lipocalin ("Lipocalin-2"), CD62L ("L-selectin"), Lymphatic endothelium ("LYVE-1"), MHC class I polypeptide-associated sequence A ("MICA"), MHC class I polypeptide-associated sequence B ("MICB"), NRG I-beta I, Beta-type platelet-derived growth factor receptor ("PDGF R-beta"), Platelet endothelial cell adhesion molecule ("PECAM-1"), RAGE, Hepatitis A virus cell receptor 1 ("TIM-1"), Tumor necrosis factor receptor superfamily member IOC ("TRAIL") R3), Trappin protein transglutaminase binding domain ("Trappin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDAR, activin A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, cathepsin S, CD40, Cryptic family protein IB ("Cripto-1"), DAN, Dickkopf-related protein 1 ("DKK-1"), E-cadherin, epithelial cell adhesion molecule ("EpCAM"), Fas ligand (FasL or CD95L), FcgRIIB / C, follistatin (FoUistatin), galectin-7, intercellular adhesion molecule 2 ("ICAM-2"), IL-13 R1, IL-13 R2, IL-17 B, IL-2 Ra, IL-2 Rb, IL-23, LAP, neuronal adhesion molecule ("NrCAM"), plasminogen activator inhibitor-1 ("PAI-1"), platelet-derived growth factor receptor ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1 beta"), sgp130, secreted Frizzled-related protein 2 ("ShhN"), sialic acid-binding immunoglobulin lectin ("Sigrec-5"), ST2, transforming growth factor-beta 2 ("TGF-beta 2"), Tie-2, thrombopoietin ("TPO"), tumor necrosis factor receptor superfamily member 10D ("TRAIL") R4), induced receptor 1 expressed in bone marrow cells ("TREM-1"), vascular endothelial growth factor C ("VEGF-C"), VEGFR1, adiponectin, adipsin ("AND"), alpha-fetoprotein ("AFP"), angiopoietin-like 4 ("ANGPTL4"), beta-2-microglobin ("B2M"), basal cell adhesion molecule ("BCAM"), glycosylated antigen 125 ("CA125"), cancer antigen 15-3 ("CA15-3"), carcinoembryonic antigen ("CEA"), cAMP receptor protein ("CRP"), human epidermal growth factor receptor 2 ("ErbB2"), follistatin, follicle-stimulating hormone ("FSH"), chemokine (CXC motif) ligand 1 ("GRO-alpha"), human chorionic gonadotropin ("beta-HCG"), insulin-like growth factor 1 receptor ("IGF-1") sR”), IL-1 sRII, IL-3, IL-18Rb, IL-21, leptin, matrix metalloproteinase-1 ("MMP-1"), matrix metalloproteinase-2 ("MMP-2"), matrix metalloproteinase-3 ("MMP-3"), matrix metalloproteinase-8 ("MMP-8"), matrix metalloproteinase-9 ("MMP-9"), matrix metalloproteinase-10 ("MMP-10"), matrix metalloproteinase-13 ("MMP-13"), neuronal cell adhesion molecule ("NCAM-1"), enteractin ("Nidogen-1"), neuron-specific enolase ("NSE"), oncostatin M ("OSM"), procalcitonin, prolactin, prostate-specific antigen ("PSA"), sialic acid-binding Ig-like lectin 9 ("Sigrec-9"), ADAM 17 Endopeptidase ("TACE"), thyroglobulin, metalloproteinase inhibitor 4 ("TIMP-4"), TSH2B4, disintegrin and metalloproteinase domain-containing protein 9 ("ADAM-9"), angiopoietin 2, tumor necrosis factor ligand superfamily member 13 / acid leucine-rich nuclear phosphate protein 32 family member B ("APRIL"), bone morphogenetic protein 2 ("BMP-2"), bone morphogenetic protein 9 ("BMP-9") ), complement component 5a ("C5a"), cathepsin L, CD200, CD97, chemerin, tumor necrosis factor receptor superfamily member 6B ("DcR3"), fatty acid binding protein 2 ("FABP2"), fibroblast activating protein, alpha ("FAP"), fibroblast growth factor 19 ("FGF-19"), galectin-3, hepatocyte growth factor receptor ("HGF R"), IFN-alpha / beta R2, insulin-like growth factor 2 ("IGF-2"), insulin-like growth factor 2 receptor ("IGF-2") "R"), Interleukin-1 receptor 6 ("IL-1R6"), Interleukin 24 ("IL-24"), Interleukin 33 ("IL-33"), Kallikrein 14, Asparaginyl endopeptidase ("Regmain"), Oxidized low-density lipoprotein receptor 1 ("LOX-1"), Mannose-binding lectin ("MBL"), Neprilysin ("NEP"), Notch homolog 1, Translocation-related (Drosophila) ("Notch-1"), Overexpressed in Nephroblastoma ("NOV"), Osteoactivin, Programmed Cell Death Protein 1 ("PD") F), N-acetylmuramoyl-L-alanine amidase ("PGRP-5"), serpine A4, secreted Frizzled-related protein 3 ("sFRP-3"), thrombomodulin, Toll-like receptor 2 ("TLR2"), tumor necrosis factor superfamily member 10A ("TRAIL R1"), transferrin ("TRF"), WIF-IACE-2, albumin, AMICA, angiopoietin 4, B-cell activator ("BAFF"), glycosylation antigen 19-9 ("CA19-9"), CD 163, clusterin, CRT AM, chemokine (CXC motif) ligand 14 ("CXCL14"), cystatin C, decorin ("DCN"), Dickkopf-related protein 3 ("Dkk-3"), delta-like protein 1 ("DLL1"), fetuin A, heparin-binding growth factor 1 ("aFGF"), folate receptor alpha ("FOLR1"), furin, GPCR-related sorting protein 1 ("GASP-1"), GPCR-related sorting protein 2 ("GASP-2"), granulocyte colony-stimulating factor receptor ("GCSF")"TRAIL R"), serine protease hepsin ("HAI-2"), interleukin-17B receptor ("IL-17B R"), interleukin-27 ("IL-27"), lymphocyte activator gene 3 ("LAG-3"), apolipoprotein AV ("LDL R"), pepsinogen I, retinol-binding protein 4 ("RBP4"), SOST, heparan sulfate proteoglycan ("Syndecan-1"), tumor necrosis factor superfamily member 13B ("TACT"), tissue factor pathway inhibitor ("TFPI"), TSP-1, tumor necrosis factor receptor superfamily, member 10b ("TRAIL"). The following are also included: R2", TRANCE, troponin I, urokinase-type plasminogen activator ("uPA"), cadherin 5, type 2 or VE-cadherin (vascular endothelial) ("VE-cadherin") also known as CD144, WNT1-inducible signaling pathway protein 1 ("WISP-1"), and nuclear factor-κB activating receptor ("RANK"). In certain preferred embodiments, the subject is also administered IFN-gamma (IFNγ). In particularly preferred embodiments, the subject is pre-treated with IFNγ, for example, a low dose of IFNγ, before administration of TCR-modified immunoeffector cells disclosed herein (e.g., adaptive immunotherapy compositions disclosed herein comprising TCR-T cells disclosed herein).

[0244] In some embodiments, immunotherapy may involve immune checkpoint inhibitors. Immune checkpoints either amplify or reject signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted for blocking include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4; also known as CD152), indoleamine-2,3-deoxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitors of T cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0245] Immunotherapy checkpoint inhibitors can be small molecules or other drugs, such as recombinant ligands or receptors, or antibodies such as human antibodies (e.g., International Publication No. 2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; all of the above references are incorporated herein by reference). Known inhibitors of immunotherapy checkpoint proteins or their analogues, in particular chimeric, humanized, and human-form antibodies, can be used. As will be understood by those skilled in the art, alternative names and / or synonymous names may be used for the specific antibodies referred to in this invention. These alternative names and / or synonymous names are interchangeable in the context of this invention. For example, lambrolizumab is also known by the alternative names and / or synonymous names MK-3475 and pembrolizumab.

[0246] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain embodiments, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In other embodiments, a PDL1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In certain embodiments, the PDL1 binding partner is PD-1 and / or B7-1. In other embodiments, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain embodiments, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoconjugate, a fusion protein, or an oligopeptide. Representative antibodies are described in U.S. Patent No. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art and are described, for example, in U.S. Patent Publication Nos. 20140294898, 2014022021, and 20110008369.

[0247] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized form of the antibody, or a chimeric form of the antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist includes an extracellular or PD-1 binding moiety of PDL1 or PDL2 fused to an immunoconjugate. In some embodiments, the PD-1 conjugated antagonist is AMP-224. MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, also known as nivolumab (nibol rumap), are the PD-1 antibodies described in International Publication No. 2006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in International Publication No. 2009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in International Publication No. 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in International Publication Nos. 2010 / 027828 and International Publication Nos. 2011 / 066342.

[0248] Another immune checkpoint that may be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 is registered under Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein CD28, and both molecules bind to CD80 and CD86, also known as B7-1 and B7-2, respectively, on antigen-presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA4 is also found on regulatory T cells and may be important for the function of regulatory T cells. T cell activation by T cell receptors and CD28 increases the expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0249] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, an antibody in human form, or an antibody in chimeric form), its antigen-binding fragment, immunoconjugate, fusion protein, or oligopeptide.

[0250] Suitable anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) for use in this method can be prepared using methods well known in the art. Alternatively, anti-CTLA-4 antibodies known in the art can be used. See, for example, U.S. Patent No. 8,119,129, International Publication No. 01 / 14424, International Publication No. 98 / 42752; International Publication No. 00 / 37504 (also known as tremelimumab; formerly known as tisilimmab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206) and Mokyr et al. (1998). Anti-CTLA-4 antibodies disclosed in Cancer Res 58:5301-5304 can be used in the methods disclosed herein. Antibodies that compete with any of the antibodies known in the art for binding to CTLA-4 can also be used. For example, human-form CTLA-4 antibodies are described in International Patent Application Publication No. 2001014424, International Publication No. 2000037504, and U.S. Patent No. 8017114.

[0251] Examples of anti-CTLA-4 antibodies include ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or its antigen-binding fragments and variants (see, for example, International Publication No. 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes and / or competes for binding to the same epitope on CTLA-4 as the antibody described above, or binds to the same epitope on CTLA-4 as the antibody described above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab). Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, e.g., those described in U.S. Patent No. 5,844,905, U.S. Patent No. 5,885,796, and International Patent Applications International Publication No. 1,995,001,994 and International Publication No. 1,998,042,752, as well as immunoconjugates, e.g., those described in U.S. Patent No. 8,329,867.

[0252] Hormone therapy In some embodiments, therapeutic agents for use in combination with TCR to treat the disorders described above may be hormonal regulators (e.g., hormone therapy), such as anti-androgen and anti-estrogen therapies. Examples of such hormone regulators include tamoxifen, idoxifen, fulvestrant, droxifen, toremifene, raloxifen, diethylstilbestrol, ethinylestradiol / estinyl, antiandrogens (e.g., flutamide / eurexin), progestins (e.g., hydroxyprogesterone caproate, medroxyprogestrone / provera, megestrol acetate / megase), corticosteroids (e.g., hydrocortisone, prednisone), progestin-releasing hormone (and its analogues as well as other LHRH agonists, e.g., buserelin and goserelin), aromatase inhibitors (e.g., anastrozole / arimidex, aminoglutethimide / citraden, exemestane), or hormone inhibitors (e.g., octreotide / sandostatin).

[0253] surgery In some embodiments, the disclosed TCR (and associated constructs embodying it) is administered in conjunction with surgery.

[0254] Therapeutic surgery includes resection, treatment, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies of this embodiment, in which all or part of the cancerous tissue is physically removed, incised, and / or destroyed. Therapeutic surgery may be used in conjunction with other therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical procedures may include laser surgery, cold surgery, electrosurgery, and microsurgery. Weight loss refers to a significant reduction in the volume (i.e., bulk) of the tumor, without aiming for complete eradication. Weight loss is usually achieved by surgical removal.

[0255] When some or all cancer cells are removed, a cavity may form in the body. Treatment is achieved by perfusion, direct injection, or topical application of additional anticancer drugs to the affected area. Such treatments may be, for example, every 1, 2, 3, 4, 5, 6, or 7 days, every 1, 2, 3, 4, or 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Such treatments can also be achieved with varying dosages.

[0256] Other combination therapies It is also intended that other agents may be used in combination with certain embodiments of this model to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell proliferation inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis-inducing factors, or other biological agents. Increased intercellular signaling resulting from an increase in the number of gap junctions will enhance the anti-hyperproliferative effect on adjacent hyperproliferating cell populations.

[0257] In other embodiments, cell proliferation inhibitors or differentiation agents may be used in combination with certain aspects of this embodiment to improve the anti-overgrowth efficacy of the treatment. Cell adhesion inhibitors are thought to improve the efficacy of this embodiment. Examples of cell adhesion inhibitors include local adhesion kinase (FAK) inhibitors and lovastatin. In addition, it is intended that other agents that increase the sensitivity of overgrowth cells to apoptosis, such as the antibody c225, may be used in combination with certain aspects of the embodiment to improve therapeutic efficacy.

[0258] Exemplary embodiments of the present invention The foregoing description of specific embodiments will fully illustrate the general characteristics of the invention, which otherwise can be readily modified and / or adapted for various applications without excessive experimentation and without departing from the general concept of the invention, by applying knowledge within the skill of the art. Accordingly, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that any expressions or terms used herein are for illustrative purposes only and not limitation, and as a result, any terms or expressions used herein should be interpreted by those skilled in the art, taking into account the teachings and guidance.

[0259] Other embodiments of the present invention will be apparent to those skilled in the art from consideration of the specifications and practices of the present invention disclosed herein. Additional exemplary embodiments ("E") of some parts of the present invention include, but are not limited to, the following:

[0260] E1. T-cell receptors (TCRs) produced as a result of HPV vaccine therapy.

[0261] E2. The TCR of E1, generated from any known HPV vaccine.

[0262] E3. A TCR of E2 generated from a quadrivalent (types 6, 11, 16, 18) recombinant HPV vaccine.

[0263] E4. E3 TCR generated from Gardasil.

[0264] E5. A TCR of E2 generated from the HPV 9-valent recombinant vaccine.

[0265] E6. E5 TCR generated from Gardasil 9.

[0266] E5 TCR generated from E7.Cervavac.

[0267] E8. An E2 TCR produced from a bivalent HPV (types 16 and 18) recombinant vaccine.

[0268] E9. An E8 TCR produced from Cervarix.

[0269] E10. An E8 TCR produced from Cecolin.

[0270] E11. An E8 TCR produced from Walrinvax.

[0271] E12. An E2 TCR produced from one or more HPV vaccines disclosed in WO 2022 / 115470.

[0272] E13. An E12 TCR produced from an HPV vaccine comprising a nucleic acid sequence that is a functional variant of SEQ ID NO: 81.

[0273] E14. An E13 TCR produced from an HPV vaccine comprising a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 81.

[0274] E15. An E14 TCR produced from an HPV vaccine comprising a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 81.

[0275] E16. An E15 TCR produced from an HPV vaccine comprising a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 81.

[0276] E17. An E16 TCR produced from an HPV vaccine comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 81.

[0277] E18. An E17 TCR produced from an HPV vaccine comprising the nucleic acid sequence of SEQ ID NO: 81.

[0278] E19. An E12 TCR comprising an amino acid sequence that is a functional variant of any one of the amino acid sequences of SEQ ID NOs: 1 to 80.

[0279] E20. A TCR of E19 containing an amino acid sequence that has at least 80% sequence identity with any one of the amino acid sequences of sequence numbers 1-80.

[0280] E21. A TCR of E20 containing an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences of sequence numbers 1-80.

[0281] E22. A TCR of E21 containing an amino acid sequence that has at least 90% sequence identity with any one of the amino acid sequences of sequence numbers 1-80.

[0282] E23. A TCR of E22 containing an amino acid sequence that has at least 95% sequence identity with any one of the amino acid sequences of sequence numbers 1-80.

[0283] E24. A TCR of E23 containing an amino acid sequence that is one of the amino acid sequences of sequence numbers 1-80.

[0284] E25. A method for treating cancer associated with HPV infection in a patient, comprising the step of introducing a TCR generated as a result of HPV vaccine therapy to the patient.

[0285] E26. The method of E25, in which a TCR is generated from any known HPV vaccine.

[0286] E27. The HPV vaccine is a recombinant HPV quadrivalent (types 6, 11, 16, 18) vaccine, as described in E26.

[0287] E28. The HPV vaccine is Gardasil, according to method E27.

[0288] E29. The HPV vaccine is an HPV 9-valent recombinant vaccine, according to method E25.

[0289] E30. The HPV vaccine is Gardasil 9, according to method E29.

[0290] The method of E29, wherein the HPV vaccine is Cervavac.

[0291] The method of E26, wherein the HPV vaccine is a bivalent HPV (types 16 and 18) recombinant vaccine.

[0292] The method of E32, wherein the HPV vaccine is Cervarix.

[0293] The method of E32, wherein the HPV vaccine is Cecolin.

[0294] The method of E32, wherein the HPV vaccine is Walrinvax.

[0295] The method of E26, wherein the HPV vaccine is one or more of those disclosed in International Publication No. WO 2022 / 115470.

[0296] The method of E36, wherein the HPV vaccine comprises a nucleic acid sequence that is a functional variant of SEQ ID NO: 81.

[0297] The method of E37, wherein the HPV vaccine comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 81.

[0298] The method of E38, wherein the HPV vaccine comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 81.

[0299] The method of E39, wherein the HPV vaccine comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 81.

[0300] The method of E40, wherein the HPV vaccine comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 81.

[0301] The method of E41, wherein the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81.

[0302] E43. The method of E36, wherein the HPV vaccine contains an amino acid sequence that is a functional variant of any one of the amino acid sequences of SEQ ID NOs: 1-80.

[0303] The method of E43, wherein E44.TCR contains an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0304] The method of E44, wherein E45.TCR contains an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0305] The method of E45, wherein E46.TCR contains an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0306] The method of E46, wherein E47.TCR contains an amino acid sequence having at least 95% sequence identity with any one of the amino acid sequences of sequence numbers 1 to 80.

[0307] The method of E47, wherein E48.TCR contains an amino acid sequence having one of the amino acid sequences of sequence numbers 1 to 80.

[0308] E49. A method of E25 in which a TCR is engineered to enhance or increase the recognition of specific antigens on cancer cells associated with HPV infection.

[0309] E50. Method E49, in which the specific antigen is the HPV E6 or E7 protein.

[0310] E51. The method of E49 in which a TCR is introduced into a patient using adoptive cell transfer.

[0311] E52. The E49 method, in which a TCR is introduced into a patient using genetic modification of T cells.

[0312] E53. The method of E49, where the cancer associated with HPV infection is cervical cancer, anal cancer, or head and neck cancer.

[0313] E54. A method for preparing TCR-T cells for use in the treatment of cancer associated with HPV infection in patients, comprising the steps of isolating T cells from a patient who has received an HPV vaccine, stimulating T cells with an HPV antigen, and isolating HPV-specific TCR-T cells.

[0314] E55. The method of E54, wherein the HPV vaccine is any known HPV vaccine.

[0315] E56. HPV is a recombinant HPV quadrivalent (types 6, 11, 16, 18) vaccine, as described in E55.

[0316] E57. The HPV vaccine is Gardasil, according to method E56.

[0317] E58. The HPV vaccine is a 9-valent recombinant HPV vaccine, according to method E55.

[0318] E59. The HPV vaccine is Gardasil 9, according to method E58.

[0319] E60. The HPV vaccine is Cervavac, according to method E58.

[0320] E61. The HPV vaccine is a bivalent (types 16 and 18) recombinant HPV vaccine, according to the method of E55.

[0321] E62. The HPV vaccine is Cervarix, according to E61.

[0322] E63. The HPV vaccine is Cecolin, as described in E61.

[0323] E64. The HPV vaccine is Wallinvax, according to method E61.

[0324] E65. The method of E55, wherein the HPV vaccine is one or more of those disclosed in International Publication No. 2022 / 115470.

[0325] E66. The method of E65, wherein the HPV vaccine contains a nucleic acid sequence that is a functional variant of SEQ ID NO: 81.

[0326] E67. The HPV vaccine contains a nucleic acid sequence that is at least 80% identical to sequence number 81, according to the method of E66.

[0327] E68. The HPV vaccine contains a nucleic acid sequence that is at least 85% identical to sequence number 81, according to the method of E67.

[0328] E69. The HPV vaccine contains a nucleic acid sequence that is at least 90% identical to sequence number 81, according to method E68.

[0329] E70. The HPV vaccine contains a nucleic acid sequence that is at least 95% identical to sequence number 81, according to the method of E69.

[0330] E71. The HPV vaccine contains the nucleic acid sequence of sequence number 81, according to method E70.

[0331] Method E65, wherein E72.TCR contains an amino acid sequence that is a functional variant of any one of the amino acid sequences of SEQ ID NOs. 1-80.

[0332] The method of E72, wherein E73.TCR contains an amino acid sequence having at least 80% sequence identity with any one of the amino acid sequences of sequence numbers 1 to 80.

[0333] The method of E73, wherein E74.TCR contains an amino acid sequence having at least 85% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0334] The method of E74, wherein E75.TCR contains an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0335] The method of E75, wherein E76.TCR contains an amino acid sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs. 1 to 80.

[0336] The method of E76, wherein E77.TCR contains an amino acid sequence in which one of the amino acid sequences of sequence numbers 1 to 80 is selected.

[0337] E78. The method of E54, further comprising the step of manipulating the isolated TCR to enhance or increase the recognition of specific antigens on cancer cells associated with HPV infection.

[0338] E79. Method E78, in which the specific antigen is the HPV E6 or E7 protein.

[0339] E80. The method of E78, further comprising the step of introducing an engineered TCR into a patient with cancer associated with HPV infection.

[0340] E81. The method of E78, where the cancer associated with HPV infection is cervical cancer, anal cancer, or head and neck cancer.

[0341] E82. The E78 method, in which a TCR is introduced into a patient using adoptive cell transfer.

[0342] E83. The E78 method, in which a TCR is introduced into a patient using genetic modification of T cells.

[0343] E84. The method described in E78, in which the patient did not respond to standard cancer therapy.

[0344] E85. The method of E78, in which the patient received the HPV vaccine before the introduction of the manipulated TCR.

[0345] E86. The method described in E78, in which the introduction of an manipulated TCR results in a reduction in tumor size and an improvement in overall patient survival.

[0346] E87. A TCR of E1 that recognizes specific antigens on cancer cells associated with HPV infection.

[0347] E88. TCR of E87, where the specific antigen is the HPV E6 or E7 protein.

[0348] E89. A TCR of E1, further comprising at least one modification for enhancing the efficacy or specificity of the TCR.

[0349] E90. The TCR of E89, in which at least one modification enhances the specificity of the TCR to at least one HPV epitope.

[0350] E91. The TCR of E89, with at least one modification that enhances the effectiveness of the TCR against HPV infection.

[0351] E92. A TCR of E89 in which the modification is selected from the group consisting of amino acid substitutions, deletions, insertions, and domain swaps.

[0352] E93. TCR of E1 linked to the effector molecule.

[0353] E94. The TCR of E93, wherein the effector molecule is selected from the group consisting of cytokines, toxins, radioisotopes, chemotherapeutic agents, antibodies, antibody fragments, and antibody-drug conjugates.

[0354] E95. Manipulated or isolated cells containing any one TCR according to claims 1-24 and 87-94.

[0355] E96. Mammalian cells, which are manipulated or isolated cells of E95.

[0356] E97. Human cells, which are manipulated or isolated cells of E96.

[0357] E98. Immune cells, which are manipulated or isolated cells of E95.

[0358] E99. T cells, which are manipulated or isolated E98 cells.

[0359] E100. A nucleic acid molecule encoding any one of the TCRs described in claims 1 to 24 and claims 87 to 94.

[0360] A vector containing nucleic acid molecules E101 and E100.

[0361] E102 is an adenovirus vector, the vector for E101.

[0362] A composition comprising E103.E95 cells and pharmaceutically acceptable excipients.

[0363] A composition comprising E104.E100 nucleic acid and a pharmaceutically acceptable excipient.

[0364] A composition comprising the E105.E101 vector and a pharmaceutically acceptable excipient.

[0365] E106. A treatment method comprising the step of administering a composition according to any one of claims 103 to 105 to a subject having a disease or disorder related to HPV.

[0366] E107. Treatment method for E106 in patients with cervical, anal, or head and neck cancer.

[0367] E108. Treatment method for E106, where the disease to be treated is recurrent respiratory papillomatosis (RRP).

[0368] E109. The treatment method of E106, wherein the treatment includes the step of treating a patient with one or more additional therapies.

[0369] E110. An additional treatment is weight-loss surgery, the procedure described in E109.

[0370] E111. The treatment method of E109, wherein the additional therapy comprises chemotherapeutic agents, anti-inflammatory agents, analgesics, bio-response modifiers, vectors containing such agents, or cells containing agents or nucleic acids encoding such agents.

[0371] E112. A method for producing engineered cells expressing any one TCR according to claims 1 to 24 and claims 87 to 94, comprising the step of introducing a vector according to any one of claims 101 to 102 into cells, wherein the vector causes the expression of the TCR in the cells.

[0372] E113. The method of E112, in which manipulated cells expressing TCRs are T cells.

[0373] While the foregoing disclosures have been described in some detail by description and examples for the purpose of clarity of understanding, it will be readily apparent to those skilled in the art, in consideration of the teachings of this disclosure, that certain changes and modifications may be made to them without departing from the spirit or scope of the appended claims. [Examples]

[0374] The following embodiments are included to illustrate preferred embodiments of the invention, in addition to the embodiments previously disclosed herein. Those skilled in the art will understand that the techniques disclosed in the subsequent embodiments are representative of those techniques that the inventors have found to function well in carrying out the invention, and therefore may constitute a preferred mode for its implementation. However, those skilled in the art will also understand that, in view of this disclosure, modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or comparable results can still be obtained.

[0375] [Example 1] TCR generation from HPV quadrivalent vaccine T cells are isolated from patients treated with a quadrivalent (types 6, 11, 16, 18) recombinant HPV vaccine (e.g., Gardasil). The T cells are then stimulated with HPV antigens, and T cells expressing HPV-specific TCRs are isolated by density gradient centrifugation, magnetic cell separation, or flow cytometry sorting. TCR-T cells and / or polynucleotides encoding HPV-specific TCRs are then manipulated to enhance or increase recognition of HPV E6 and E7 proteins using retroviral / lentiviral transduction or TALEN / CRISPR genome editing techniques. The manipulated TCR-T cells or manipulated polynucleotides are cultured and then introduced into patients with cervical, anal, or head and neck cancer associated with HPV infection. The patients show a significant reduction in tumor size and improved overall survival.

[0376] [Example 2] TCR generation from HPV 9-valent vaccine T cells are isolated from patients treated with a recombinant HPV 9-valent vaccine (e.g., Gardasil 9). The T cells are then stimulated with HPV antigens, and T cells expressing HPV-specific TCRs are isolated by density gradient centrifugation, magnetic cell separation, or flow cytometry sorting. TCR-T cells and / or polynucleotides encoding HPV-specific TCRs are then manipulated to enhance or increase recognition of HPV E6 and E7 proteins using retroviral / lentiviral transduction or TALEN / CRISPR genome editing techniques. The manipulated TCR-T cells or manipulated polynucleotides are cultured and then introduced into patients with cervical, anal, or head and neck cancer associated with HPV infection. The patients show a significant reduction in tumor size and improved overall survival.

[0377] [Example 3] TCR generation from HPV bivalent vaccine T cells are isolated from patients treated with a bivalent (types 16 and 18) recombinant HPV vaccine (e.g., Cervarix). The T cells are then stimulated with HPV antigens, and T cells expressing HPV-specific TCRs are isolated by density gradient centrifugation, magnetic cell separation, or flow cytometry sorting. TCR-T cells and / or polynucleotides encoding HPV-specific TCRs are then manipulated to enhance or increase recognition of HPV E6 and E7 proteins using retroviral / lentiviral transduction or TALEN / CRISPR genome editing techniques. The manipulated TCR-T cells or manipulated polynucleotides are cultured and then introduced into patients with cervical, anal, or head and neck cancer associated with HPV infection. The patients show a significant reduction in tumor size and improved overall survival.

[0378] [Example 4] TCR generation from HPV6 / 11 vaccine T cells are isolated from patients administered one of the HPV6 / 11 vaccines disclosed in International Publication No. 2022 / 115470. For example, T cells can be isolated from patients administered an HPV vaccine containing the nucleic acid sequence of Sequence ID No. 81 or a functional variant thereof. The T cells are then stimulated with an HPV antigen, and T cells expressing an HPV-specific TCR are isolated by density gradient centrifugation, magnetic cell separation, or flow cytometry sorting. The TCR-T cells and / or polynucleotides encoding the HPV-specific TCR are then manipulated to enhance or increase the recognition of HPV E6 and E7 proteins using retroviral / lentiviral transduction or TALEN / CRISPR genome editing techniques. The manipulated TCR-T cells or manipulated polynucleotides are cultured and then introduced into patients with cervical, anal, or head and neck cancer associated with HPV infection. The patients show a significant reduction in tumor size and an improvement in overall survival.

[0379] [Example 5] Combination therapy using TCR and chemotherapy T cells are isolated from pre-vaccinated HPV patients with HPV-associated cervical, anal, or head and neck cancer using density gradient centrifugation, magnetic cell separation, or flow cytometry sorting techniques. TCR sequences specific to HPV E6 and E7 proteins are identified, and polynucleotides encoding these TCRs are cloned into adenovirus vectors for genetic manipulation of T cells. These vectors are then used to induce HPV-specific TCR expression in the patients' T cells.

[0380] Patients are given a combination of TCR therapy and chemotherapy. TCR-modified T cells are injected into the patient, and the patient is then given chemotherapy. A synergistic effect is obtained with this combination therapy, resulting in a significant reduction in tumor size and improved overall survival compared to chemotherapy alone.

[0381] [Example 6] Combined therapy using TCR and radiation T cells are isolated from pre-vaccinated HPV patients with anal, cervical, or head and neck cancer associated with HPV infection using density gradient centrifugation, magnetic cell separation, or flow cytometry sorting techniques. TCR sequences specific to HPV E6 and E7 proteins are identified, and polynucleotides encoding these TCRs are cloned into adenovirus vectors for genetic manipulation of T cells. These vectors are then used to induce HPV-specific TCR expression in the patients' T cells.

[0382] The patient receives a combination of TCR therapy and radiotherapy. TCR-modified T cells are injected into the patient, and then the patient is given radiation therapy. This combination therapy produces a synergistic effect, resulting in a significant reduction in tumor size and improved overall survival compared to radiation therapy alone.

[0383] [Example 7] Combination therapy using TCR and debulking surgery T cells are isolated from pre-vaccinated HPV patients with anal, cervical, or head and neck cancer associated with HPV infection using density gradient centrifugation, magnetic cell separation, or flow cytometry sorting techniques. TCR sequences specific to HPV E6 and E7 proteins are identified, and polynucleotides encoding these TCRs are cloned into adenovirus vectors for genetic manipulation of T cells. These vectors are then used to induce HPV-specific TCR expression in the patients' T cells.

[0384] Patients receive a combination of TCR therapy and one or more debulking surgeries. TCR-modified T cells are injected into the patient either before or after one or more debulking surgeries. The combination therapy produces a synergistic effect, resulting in a significant reduction in tumor size and improved overall survival compared to debulking alone.

[0385] The above description and examples should not be considered limiting, and they may be modified in various ways within the scope of the present invention as defined by the appended claims.

[0386] [Example 8] Evaluation of TCR sequences after HPV6 / 11 vaccine treatment In the evaluation of the clonal nature of peripheral blood T cell responses to HPV6 / 11 vaccine treatment, as described in International Publication No. 2022 / 115470, T cell receptor (TCR) CDR3 sequences expressed on HPV-specific T cells were determined using TCRβ sequencing and FEST assays for subjects with sufficient clinical samples. This approach allows for the quantification of HPV-specific T cell frequencies in peripheral blood before and after vaccine treatment. In post-treatment PBMC samples stimulated with HPV6 and 11 peptides, the top 10 expanded CDR sequences determined to be HPV-specific represented a larger proportion of the TCRβ repertoire in responders compared to non-responders, as measured by the Simpson clonality index (P=0.02, unpaired two-sided t-test) (Figure 1C). This suggests that HPV-specific T cell clonal types are more expanded in the blood of patients who show clinical response after treatment compared to those who do not. Direct comparison of these HPV-specific CDR sequences in unstimulated pre- and post-treatment peripheral blood clearly demonstrated a consistent expansion in responders (Figure 2D). Conversely, expansion, no change, or decrease in HPV-specific CDR3 frequency after treatment compared to before was observed in non-responders. Overall, responders showed a larger expansion of peripheral blood HPV-specific CDR3 frequency compared to non-responders (Figure 1E, p<0.001, unpaired two-sided t-test). Both emerging and expanded HPV-specific CDR3 frequencies were detected in all patients analyzed, with CDR3 frequencies detectable after treatment but undetectable before treatment considered as emerging, and CDR3 frequencies detectable before treatment but expanded after treatment considered as expanded (Figure 1F).

[0387] The above description and examples should not be considered limiting, and they may be modified in various ways within the scope of the present invention as defined by the appended claims.

[0388] [Table 4-1]

[0389] Table 4-2

[0390] Table 4-3

[0391] Table 5-1

[0392] Table 5-2

[0393] Table 5-3

Claims

1. A T-cell receptor (TCR) produced as a result of treatment with an HPV vaccine, wherein the HPV vaccine is selected from an HPV quadrivalent recombinant vaccine; an HPV nonavalent recombinant vaccine; or an HPV bivalent recombinant vaccine.

2. The TCR according to claim 1, wherein the HPV vaccine is an HPV bivalent recombinant vaccine.

3. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

81.

4. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

81.

5. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:

81.

6. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:

81.

7. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:

81.

8. The TCR according to claim 2, wherein the HPV vaccine comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:

81.

9. The TCR according to claim 2, wherein the HPV vaccine comprises the nucleic acid sequence of Sequence ID No. 81 or a codon degenerate variant thereof.

10. The TCR according to claim 2, wherein the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO:

81.

11. The TCR according to claim 1, wherein the HPV vaccine is Gardasil.

12. The TCR according to claim 1, wherein the HPV vaccine is Gardasil 9 or Cervavac.

13. The TCR according to claim 1, wherein the HPV vaccine is Cervarix, Cecolin, or Wallinvax.

14. The TCR according to claim 1, comprising an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 1 to 80.

15. The TCR according to claim 1, comprising an amino acid sequence having at least 90% sequence identity with any one of sequence numbers 1 to 80.

16. The TCR according to claim 1, comprising an amino acid sequence having at least 95% sequence identity with any one of sequence numbers 1 to 80.

17. The TCR according to claim 1, comprising an amino acid sequence having at least 97% sequence identity with any one of sequence numbers 1 to 80.

18. The TCR according to claim 1, comprising an amino acid sequence having at least 98% sequence identity with any one of sequence numbers 1 to 80.

19. The TCR according to claim 1, comprising an amino acid sequence having at least 99% sequence identity with any one of sequence numbers 1 to 80.

20. The TCR according to claim 1, comprising one amino acid sequence from sequence numbers 1 to 80 or a conservatively substituted variant thereof.

21. The TCR according to claim 1, comprising one amino acid sequence from sequence numbers 1 to 80.

22. The TCR according to claim 1, which recognizes a specific antigen on cancer cells associated with HPV infection.

23. The TCR according to claim 22, wherein the specific antigen is the HPV E6 or E7 protein.

24. The TCR according to claim 1, further comprising modifications that enhance the effectiveness of the TCR against HPV infection.

25. The TCR according to claim 1, further comprising a modification that enhances the specificity of the TCR to an HPV epitope.

26. The TCR according to claim 1, which is linked to an effector molecule.

27. The TCR according to claim 26, wherein the effector molecule is selected from the group consisting of cytokines, toxins, radioisotopes, chemotherapeutic agents, antibodies, antibody fragments, and antibody-drug conjugates.

28. A method for treating a disease or disorder related to HPV infection in a subject requiring such treatment, comprising the step of introducing a TCR according to any one of claims 1 to 27 into the subject.

29. The method according to claim 28, wherein the TCR is introduced into the patient using adoptive cell transfer.

30. The method according to claim 28, wherein the disease or disorder is cancer.

31. The method according to claim 30, wherein the cancer is cervical cancer, anal cancer, or head and neck cancer.

32. The method according to claim 28, wherein the disease or disorder is recurrent respiratory papillomatosis.

33. Use of the TCR according to any one of claims 1 to 27 in the preparation of a pharmaceutical for the treatment of a disease or disorder related to HPV infection.

34. A method for preparing TCR-T cells for use in the treatment of a disease or disorder associated with HPV infection in a subject who has been administered an HPV vaccine, the method comprising the steps of isolating T cells from the subject, stimulating the T cells with an HPV antigen, and isolating HPV-specific TCR-T cells, wherein the HPV vaccine is selected from an HPV quadrivalent recombinant vaccine; an HPV nonavalent recombinant vaccine; or an HPV bivalent recombinant vaccine.

35. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO:

81.

36. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

81.

37. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 95% sequence identity with SEQ ID NO:

81.

38. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 97% sequence identity with SEQ ID NO:

81.

39. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 98% sequence identity with SEQ ID NO:

81.

40. The method according to claim 34, wherein the HPV vaccine comprises a nucleic acid sequence having at least 99% sequence identity with SEQ ID NO:

81.

41. The method according to claim 34, wherein the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO: 81 or a codon degenerate variant thereof.

42. The method according to claim 34, wherein the HPV vaccine comprises the nucleic acid sequence of SEQ ID NO:

81.

43. Manipulated or isolated cells comprising the TCR according to any one of claims 1 to 27.

44. The manipulated or isolated cells according to claim 43, which are mammalian cells.

45. The manipulated or isolated cells according to claim 43, which are human cells.

46. The manipulated or isolated cells according to claim 43, which are immune cells.

47. The manipulated or isolated cells according to claim 43, which are T cells.

48. A nucleic acid molecule encoding a TCR according to any one of claims 1 to 27.

49. A vector comprising the nucleic acid described in claim 48.

50. The vector according to claim 49, which is an adenovirus vector.

51. A pharmaceutical composition comprising manipulated or isolated cells as described in claim 43 and a pharmaceutically acceptable excipient.

52. The pharmaceutical composition according to claim 51, for use in the preparation of a pharmaceutical for use in the treatment of a disease or disorder related to HPV infection.

53. A method for producing manipulated cells expressing a TCR, comprising the step of introducing the nucleic acid according to claim 48 into the cells.

54. The method according to claim 53, wherein the cell is a T cell.

55. A kit comprising a TCR according to any one of claims 1 to 27 and instructions for use in the treatment of diseases or disorders associated with HPV infection.

56. A kit comprising manipulated or isolated cells as described in claim 43 and instructions for use in the treatment of diseases or disorders associated with HPV infection.

57. A kit comprising the nucleic acid molecule described in claim 48 and instructions for use in the treatment of a disease or disorder associated with HPV infection.

58. A method for treating a disease or disorder associated with HPV infection in a subject requiring such treatment, comprising the step of administering to the subject a TCR according to any one of claims 1 to 27 in combination with a therapy selected from chemotherapy, radiotherapy or checkpoint inhibitor therapy.

59. A method for treating a disease or disorder associated with HPV infection in a subject requiring such treatment, comprising the step of administering to the subject the manipulated or isolated cells described in claim 43 in combination with a therapy selected from chemotherapy, radiotherapy or checkpoint inhibitor therapy.

60. A method for treating a disease or disorder associated with HPV infection in a subject requiring such treatment, comprising the step of administering to the subject the manipulated or isolated cells described in claim 48 in combination with a therapy selected from chemotherapy, radiotherapy or checkpoint inhibitor therapy.

61. A method for detecting HPV infection in a subject, the method comprising the steps of contacting a sample from the subject with a TCR described in any one of claims 1 to 27, and detecting the binding of the TCR to the sample, wherein the binding of the TCR to the sample indicates the presence of HPV infection in the subject.

62. A method for producing manipulated cells containing a TCR, comprising the steps of culturing the cells and introducing a nucleic acid encoding a TCR according to any one of claims 1 to 27 into the cells under conditions sufficient for the expression of the TCR in the cells.

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