Validation of HPV16-derived stimulatory peptides

Abstract

The present invention provides an in vitro method for producing an immunoreactive substance against human cancer cells infected with an HPV16-related virus, the method comprising the step of expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide, wherein the stimulatory peptide (i) comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, wherein the HLA is derived from the HLA supertype HLA-A01, (ii) comprises the amino acid sequence of SEQ ID NO: 11, wherein the HLA is derived from the HLA supertype HLA-A02, or (iii) comprises an amino acid sequence selected from SEQ ID NOs: 34 to 37, wherein the HLA is derived from the HLA supertype HLA-A03. (iv) the HLA is derived from HLA supertype HLA-A24; (v) the HLA is derived from HLA supertype HLA-B07; or (vi) the HLA is derived from HLA supertype HLA-B15; and an HPV16-derived peptide consisting of the same amino acid sequence as the stimulatory peptide has been verified to be presented by human HPV16-positive cancer cells, as well as methods and uses related thereto.

Description

[Technical Field]

[0001] The present invention provides an in vitro method for producing an immunoreactive substance against human cancer cells infected with an HPV16-related virus, the method comprising the step of expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide, wherein the stimulatory peptide (i) comprises an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, wherein the HLA is derived from the HLA supertype HLA-A01, (ii) comprises the amino acid sequence of SEQ ID NO: 11, wherein the HLA is derived from the HLA supertype HLA-A02, or (iii) comprises an amino acid sequence selected from SEQ ID NOs: 34 to 37, wherein the HLA is derived from the HLA supertype HLA-A03. (iv) the HLA is derived from HLA supertype HLA-A24; (v) the HLA is derived from HLA supertype HLA-B07; or (vi) the HLA is derived from HLA supertype HLA-B15; and an HPV16-derived peptide consisting of the same amino acid sequence as the stimulatory peptide has been verified to be presented by human HPV16-positive cancer cells, as well as methods and uses related thereto. Summary of the Invention

[0002] At least 20% of human malignancies are the result of persistent infectious diseases. Cancers caused by infectious agents are attractive targets for cancer vaccination efforts because they offer the opportunity to target antigens that are immunologically non-self. Vaccinations can be either preventative, inducing immune responses that prevent infection in the first place, or therapeutic, stimulating the immune system to eradicate pre-existing disease. Preventative immunization against certain high-risk human papillomavirus (HPV) types or hepatitis B has become a paradigm for cancer immunoprophylaxis.

[0003] With regard to therapeutic vaccination, HPV-associated neoplasias may reemerge as a model case: the induction and maintenance of a malignant phenotype in response to two viral oncoproteins, E6 and E7. Studies of spontaneously resolving HPV-induced lesions and the prevalence of HPV in immunosuppressed and HIV-infected patients indicate that the cellular immune response is important in eliminating existing HPV infections. Various forms of vaccines, including peptide vaccines targeting T cells against E6 and E7, have already been explored.

[0004] Candidate peptides proposed for vaccination against HPV are abundant in the literature, e.g., Bonsack et al. (2019), Cancer Immunol Res;7(5):719; Blatnik (2018), Proteomics 18:1700390, DOI:10.1002 / pmic.201700390; Krishna et al. (2018) Cancer Res.2018;78(21):6159; Tsang et al. (2017), Vaccine 35(19):2605; Kast et al., J Immunol 1994;152(8):3904; Ressing et al., J Immunol 1995;154(11):5934; Bourgault Villada et al., Clin Exp Immunol 2010;159(1):45; Mizuuchi et al., Exp Mol Pathol 2012;92(1):185); Hara et al., Int J Oncol 2005;27(5):1371; Jang et al., Cancer 2012;118(8):2173, and Riemer et al., J Biol Chem 2010;285(38):29608. However, for many years, it has not been understood that the process of inducing an effective T cell response is complex and requires more than just the binding of peptides to MHC. In the case of MHC class I presentation, in order to first present the peptide, the precursor peptide must be cleaved by the proteasome of the presenting cell. Furthermore, T cell activation may require the peptide to be cross-presented to MHC class I by APC, which, unlike non-APC cells, can cleave the precursor peptide. Furthermore, T cells with a propensity to recognize the presented peptide must be present, which is critically dependent on the pool of available T cell receptors (see, e.g., Becker & Riemer (2022), Frontiers Immunol, doi:10.3389 / fimmu.2022.883989; Habib et al. (2022), Cells 6;11(3):421. doi:10.3390 / cells11030421).Furthermore, immune evasion mechanisms may prevent proper presentation of HPV-derived peptides in cancer cells (Steinbach & Riemer (2018), Int J Cancer 142:224). In conclusion, experimental binding of candidate peptides to MHC class I itself unfortunately does not yet indicate that this candidate peptide is a valid vaccine candidate.

[0005] Given the above, it is clear that validating candidate HPV peptides as suitable for vaccination is far more than routine. Presentation assays yield only minute amounts of peptide, and many peptides have complex mass spectrometry fragmentation patterns. Thus, in a 2010 study (Riemer et al. (2010), cited above), only one of 21 peptides predicted to be presented via HLA-A*0201 was confirmed as presented. Eight years after method development, 17 peptides were confirmed from a predicted set of 121 potential HLA-A2-binding peptides (Blatnik et al. (2018), cited above). Notably, cysteine-containing peptides were completely excluded, as they are prone to intramolecular and intermolecular reactions, which, as is typical in the art, complicate analysis and further reduce the amount of definition and detectable ions available for MS analysis. If the above in vitro methods fail to identify a suitable HPV peptide, the only remaining alternative is in vivo testing, which requires extensive animal and / or human testing with vaccination.

[0006] Furthermore, successful therapeutic vaccination against HPV requires not only that the vaccinated peptide be immunogenic, for example, by activating T cells when presented via MHC molecules, but also that the peptide must be presented by target cells, i.e., HPV-infected cells. As noted by Riemer et al. (2010, cited above), vaccination requires the definition of HPV-16 E6 and E7 T cell epitopes that are naturally processed and presented on the surface of virus-altered cells. Only such HPV peptide / MHC class I complexes can be recognized by cytolytic T lymphocytes and target transformed cells for destruction. Therefore, the success of a therapeutic HPV vaccine depends on the accurate identification of HPV epitopes presented on HPV-infected cells. Aside from direct evidence of MHC presentation by MS analysis, investigation of memory responses in healthy donors can serve as an alternative test of presentation. If a memory immune response is present, the epitope in question must have been presented during a previous encounter with the virus. However, the proven natural immunogenicity of a peptide is not sufficient to validate it in the context of limiting HLA complexes as potential targets. This is because APCs use a different proteasome, the immunoproteasome, compared with normal somatic cells, and antigen processing in HPV16-infected / transformed cells is significantly modified by the virus. Therefore, memory T cell responses only prove that a peptide was presented by antigen-presenting cells (APCs), but do not prove that this epitope will still be presented on HPV-dependent tumor cells. Therefore, not all possible HPV epitopes are presented on HPV-positive target cells.

[0007] Therefore, to be an effective target, HPV16-derived peptides must bind to HLA molecules, be immunogenic, and be presented on target cells, particularly HPV16-infected or transformed cells.

[0008] Thus, there is a need in the art for improved means and methods for immunoreactive substances and related reagents against HPV-transformed cells, e.g., HPV-positive cancer cells, that do not suffer from the above-mentioned drawbacks.

[0009] The technical problem underlying the present invention can be understood as providing means and methods for meeting the above-mentioned needs. This technical problem is solved by the claims and the embodiments characterized herein below.

[0010] Therefore, the present invention provides (A) contacting an immune cell with a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide; (B) thereby stimulating immune cells that specifically bind to HPV16-related virus-infected host cells; 1. A method for stimulating immune cells that specifically bind to human host cells infected with an HPV16-related virus, comprising: The method relates to a method in which the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1 to 111, and in a preferred embodiment, selected from SEQ ID NOs: 1 to 129.

[0011] The method for stimulating immune cells is preferably an in vitro method, and may preferably be carried out, for example, on a sample isolated from a subject. However, the method may also preferably be carried out in vivo, more preferably in a non-human experimental animal. The method may include additional steps other than those explicitly specified, and may be assisted or carried out by an automated device. DETAILED DESCRIPTION OF THE INVENTION

[0012] In general, terms used herein should be given their ordinary and customary meanings to those skilled in the art and should not be limited to specific or special meanings unless otherwise indicated. As used below, the terms "have," "comprise," or "include," or any grammatical variations thereof, are used non-exclusively. Thus, such terms can refer to both the absence of any additional features in the entity described in the context, and the presence of one or more additional features in addition to the feature suggested by such terms. For example, the expressions "A has B" and "A includes B" can refer to both the absence of any other elements in A in addition to B (i.e., A consists solely and exclusively of B), and the presence of one or more additional elements in entity A in addition to B, such as element C, element C and element D, or even further elements. Also, as will be understood by those skilled in the art, the phrases "comprising a" and "comprising an" preferably refer to "comprising one or more," i.e., are equivalent to "comprising at least one." Thus, unless otherwise indicated, references to a plurality of items preferably refer to at least one such item, more preferably a plurality thereof. Thus, for example, reference to the identification of "a cell" relates to the identification of at least one cell, preferably a plurality of cells.

[0013] Furthermore, as used hereinafter, the terms "preferably," "more preferably," "most preferably," "particularly," "more particularly," "particularly," "more particularly," or similar terms are used in combination with optional features without restricting further possibilities. Features advocated by such terms are therefore optional features and are not intended to limit the scope of the claims in any way. The present invention may be practiced by using alternative features, as will be recognized by those skilled in the art. Similarly, features advocated by "in one embodiment" or similar language are intended to be optional features, without any limitations on further embodiments of the invention, any limitations on the scope of the invention, or any limitations on the possibility of combining such advocated features with other optional or non-optional features of the invention.

[0014] The methods specified herein below are preferably in vitro methods. The method steps can in principle be performed in any order deemed suitable by a person skilled in the art, but are preferably performed in the order indicated, and one or more, preferably all, of the steps may also be assisted or performed by automated equipment. Furthermore, the method may include other steps in addition to those explicitly mentioned above.

[0015] As used herein, unless otherwise indicated, the term "about" refers to the indicated value with a technical precision generally accepted in the relevant field, preferably ±20%, more preferably ±10%, and most preferably ±5% of the indicated value. Furthermore, the term "essentially" indicates that there is no deviation that would have an effect on the indicated result or use, i.e., potential deviations deviate by more than ±20%, more preferably ±10%, and most preferably ±5% without producing the indicated result. Thus, "consisting essentially of" means including the specified component but excluding other components, excluding substances present as impurities, unavoidable substances present as a result of the process used to produce the component, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "consisting essentially of" encompasses all known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a set of components contains less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of the unspecified component(s).

[0016] The degree of identity (e.g., expressed as "% identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the sequence within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequences compared for optimal alignment. The percentage is preferably calculated by determining the number of positions where identical residues occur in both sequences over the entire length of the polynucleotide or polypeptide to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying this result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences in a comparison can be achieved by the Smith and Waterman (1981) local homology algorithm, the Needleman and Wunsch (1970) homology alignment algorithm, the Pearson and Lipman (1988) similarity search method, computer implementations of such algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or visual inspection. Given that two sequences are identified in a comparison, GAP and BESTFIT are preferably used to determine their optimal alignment and thereby the degree of identity. Preferably, default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used. In the context of biological sequences referred to herein, the term "essentially identical" refers to a percent identity value of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%. As will be understood, the term essentially identical includes 100% identity. The foregoing applies mutatis mutandis to the term "essentially complementary".

[0017] The term "fragment" of a biopolymer, preferably a polynucleotide or polypeptide, is used herein in a broad sense to refer to any subportion, preferably a subdomain, of the respective biopolymer, including the indicated sequence, structure, and / or function. Thus, the term encompasses not only subportions generated by actual fragmentation of the biopolymer, but also subportions obtained in an abstract manner, e.g., in silico, from the respective biopolymer. Thus, as used herein, not only Fc or Fab fragments, but also, for example, single-chain antibodies, bispecific antibodies, and nanobodies, may be referred to as fragments of immunoglobulins.

[0018] Unless otherwise specified in detail herein, the specified compounds, particularly polynucleotides and (poly)peptides, may be included in larger structures, for example, covalently or non-covalently bound to additional sequences, carrier molecules, retardants, and other excipients. In particular, the specified peptides and (poly)peptides may be included in fusion polypeptides, which contain additional peptides, for example, as purification and / or detection tags, as linkers, or to extend the in vivo half-life of the compound. The term "detectable tag" refers to a stretch of amino acids that is added to or introduced into a fusion polypeptide, and preferably, the tag is added to the C- or N-terminus of the fusion polypeptide. This stretch of amino acids preferably allows the polypeptide to be detected by an antibody that specifically recognizes the tag, or preferably allows the formation of a functional conformation, for example, a chelator, or preferably allows visualization, for example, in the case of a fluorescent tag. Preferred detectable tags are Myc tag, FLAG tag, 6-His tag, HA tag, GST tag or fluorescent protein tag, such as GFP tag.All such tags are well known in the art.Preferably, the other additional peptides contained in the fusion polypeptide comprise additional amino acids or other modifications that can act as secretion mediators, blood-brain barrier crossing mediators, cell-penetrating peptides, and / or immunostimulatory agents.The additional polypeptides or peptides that can be fused to polypeptides are signal sequences and / or transport sequences, such as IL-2 signal sequences, and linker sequences.

[0019] The term "polypeptide," as used herein, refers to a molecule consisting of several, typically at least 20, amino acids covalently linked to each other by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds are generally considered to be "peptides." The peptides referred to herein are disclosed as SEQ ID NOS: 1-111 and Table 1 herein below. As one of skill in the art will appreciate from this disclosure, the stimulating peptides described herein are preferably used with the HLA supertypes set forth in Table 1 herein. Thus, preferably, the immunizing peptide / HLA supertype combinations of Table 1(i)-(vi) are used.

[0020] The term "polynucleotide" is known to those skilled in the art. As used herein, this term includes nucleic acid molecules that contain or consist of one or more nucleic acid sequences specified herein and / or encode at least one stimulatory peptide. The polynucleotide of the present invention is preferably provided either as an isolated polynucleotide (i.e., isolated from its natural environment) or in a genetically modified form. The polynucleotide is preferably DNA, including cDNA, or RNA. This term encompasses not only single-stranded polynucleotides but also double-stranded polynucleotides, as well as closed circular and linear polynucleotides. Preferably, the polynucleotide is a chimeric molecule, i.e., preferably contains at least one nucleic acid sequence, preferably at least 20 bp, more preferably at least 100 bp, that is heterologous to the remaining nucleic acid sequence(s) or is an artificial nucleic acid sequence. Moreover, it also preferably includes chemically modified polynucleotides, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides.

[0021] The polynucleotide may be contained in an expression construct. The term "expression construct" as used herein refers to a heterologous polynucleotide comprising the above-mentioned polynucleotide and a nucleic acid sequence required for the expression of the polynucleotide. Preferably, the additional nucleic acid sequence heterologous to the polynucleotide encoding at least one stimulating peptide may typically be a promoter sequence, a regulatory sequence, and / or a transcription termination sequence, such as a terminator. Preferably, the expression construct is a eukaryotic expression construct, i.e., an expression construct that contains all the elements required for expression, preferably for inducible expression, in eukaryotic host cells. Suitable expression control sequences are well known in the art, and in particular include the CMV promoter or other constitutive promoters. However, inducible promoters and / or cell type-specific promoters may also be used.

[0022] The polynucleotide and / or expression construct may be contained in a vector. As used herein, the term "vector" refers to any polynucleotide configured to stably maintain the polynucleotide and / or expression construct specified herein above in a host cell. Thus, the term vector preferably includes phage, plasmid, and viral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Preferably, the vector is a plasmid or virus-derived vector, preferably a replication-incompetent viral vector. In addition, this term also refers to a targeting construct that allows random or site-specific integration of the targeting construct into the genomic DNA of a host cell. Such a targeting construct preferably contains DNA of sufficient length for homologous or heterologous recombination. A vector containing the polynucleotide and / or expression construct specified herein above preferably further contains at least one selectable marker for propagation and / or selection of the host cell. A vector can be incorporated into a host cell by various techniques well known in the art. For example, the plasmid vector can be introduced by precipitation, such as calcium phosphate precipitation or rubidium chloride precipitation, or complex with charged lipids or carbon-based clusters, such as fullerene. Alternatively, the plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before being applied to host cells. Preferably, the vector is a vertebrate vector, more preferably a mammalian vector or a shuttle vector. Preferably, the vector is an expression vector and / or a gene transfer or targeting vector.Methods well known to those skilled in the art can be used to construct recombinant polynucleotides and vectors, see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994). Preferably, the vector is an AAV vector or a lentiviral vector.

[0023] Methods for determining stimulation of immune cells are known in the art and are described elsewhere herein, particularly in the Examples. Preferably, stimulation is significant compared to untreated and / or vehicle controls.

[0024] The terms "human papillomavirus 16-related virus" and "HPV16-related virus" preferably relate to any virus that infects humans and encodes at least one amino acid sequence of SEQ ID NOs: 1 to 111. Preferably, the HPV16-related virus is a papillomavirus (PV), more preferably a human papillomavirus (HPV), even more preferably selected from the list consisting of HPV16, HPV31, HPV33, HPV35, HPV52, HPV58, and HPV67, and most preferably HPV16.

[0025] The term "subject" as used herein refers to a vertebrate, preferably a mammal, more preferably a human.Preferably, the subject comprises at least one HLA type as specified in Table 1 herein below.Preferably, the subject is infected with at least one HPV16-related virus.Preferably, the subject suffers from at least one HPV16-related virus-positive cellular proliferative disorder.

[0026] The term "cell proliferative disorder" refers to the abnormal proliferation of somatic cells in a subject, which may result in an imbalance in the cellular composition of body tissues and fluids and / or the formation of a tumor. Cell proliferative disorders may be induced by infectious agents, preferably viruses, more preferably HPV16-related viruses. Preferably, cell proliferative disorders are benign, i.e., preferably not threatening the health or life of a subject. Preferred benign cell proliferative disorders are warts, exophytic papillomas, condylomata, inverted papillomas, and HPV-induced precancerous lesions. Also preferably, cell proliferative disorders are premalignant or malignant, i.e., at least potentially threatening the health or life of a subject. Thus, preferably, the cell proliferative disorder is a premalignant or malignant proliferative disorder of the mucosa or skin, particularly a mucosa, such as the oropharynx, anogenital region, and / or genitalia, such as cervical intraepithelial neoplasia (CIN) or cancer, particularly cervical cancer and / or head and neck cancer. The term "cancer", as used herein, relates to a disease of animals, including humans, characterized by the uncontrolled proliferation of a group of somatic cells ("cancer cells"). This uncontrolled proliferation may involve the invasion and destruction of surrounding tissues, and in some cases the spread of cancer cells to other parts of the body. Preferably, the term cancer also includes recurrence. Thus, preferably, the cancer is a solid cancer, its metastasis or recurrence. Therefore, the induction of an immune response is preferably cancer prevention and / or cancer treatment.

[0027] The term "stimulatory peptide," as used herein, refers to any peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1-111. As will be understood by those skilled in the art from the examples provided herein, the aforementioned peptides have been verified to activate human T cells when presented to human T cells in a human leukocyte antigen (HLA) complex. Preferably, the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92, which have been additionally verified to be presented by human cancer cells infected with HPV16-related viruses, as shown in the examples herein below. Thus, stimulatory peptides consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92 have been verified to be presented by human cancer cells infected with HPV16-related viruses and to activate human T cells when presented to human T cells in an HLA complex. As those skilled in the art can understand, the human cancer cell that is infected with HPV16-related virus can lose part of HPV genome.Therefore, as used herein, any cancer cell that expresses at least HPV16-related E6 polypeptide and E7 polypeptide is preferably the human cancer cell that is infected with HPV16-related virus.

[0028] In a preferred embodiment, the immunizing peptide consists of an amino acid sequence containing at least one, preferably at least two, and more preferably three cysteine ​​residue(s). Thus, in a preferred embodiment, the immunizing peptide is selected from the group consisting of SEQ ID NOs: 1, 3-5, 7-12, 14, 16-25, 28, 30, 31, 34, 35, 37-40, 42-46, 48-50, 53, 55-57, 59, 60, 62-64, 68, 69, 71, 78, 80, 82-84, 86, 91-95, 99, 100, 104-106, 112-115, 117 to 120, 122 to 124, and 127, more preferably SEQ ID NOs: 4, 34, 35, 37, 38, 42 to 46, 48, 49, 55 to 57, 59, 62, 63, 68, 69, 84, 99, 106, 115, 118, and 120, and even more preferably SEQ ID NOs: 57, 62, 69, 84, and 120. In a preferred embodiment, the immunizing peptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A01, preferably selected from the list consisting of SEQ ID NOs: 1, 3 to 5, 7 to 10, and 112 to 115. In a further preferred embodiment, the immunizing peptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A02, preferably selected from the list consisting of SEQ ID NOs: 11, 12, 14, 16 to 25, 28, 30, and 31. In a further preferred embodiment, the immunizing peptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A03, preferably selected from the list consisting of SEQ ID NOs: 34, 37, 43, 46, 49, 50, 53, 56, 57, 117, and 118. In a further preferred embodiment, the immunizing peptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A11, preferably selected from the list consisting of SEQ ID NOs: 39, 40, 34, 35, 37, 38, and 42-45. In a further preferred embodiment, the immunizing peptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A24, preferably selected from the list consisting of SEQ ID NOs: 60, 62-64, 68, 69, 71, and 119.In a further preferred embodiment, the immunizing peptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-B07, preferably selected from the list consisting of SEQ ID NOs: 12, 78, 80, 82-84, and 122-124. In a further preferred embodiment, the immunizing peptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-B15, preferably selected from the list consisting of SEQ ID NOs: 9, 43, 64, 80, 86, 91-95, 99, 100, 104-106, and 127. In a further preferred embodiment, the immunizing peptide consists of an amino acid sequence containing at least one cysteine ​​residue and is a peptide verified herein to be presented on HPV16-infected cancer cells, i.e., preferably selected from the list consisting of SEQ ID NOs: 1, 11, 34, 35, 37, 60, 86, 91, and 92.

[0029] The term "human leukocyte antigen," which may also be referred to as "HLA," is understood by those skilled in the art to refer to a family of cell surface proteins that, in principle, present peptides to immune cells. HLA is also known as major histocompatibility complex, with HLA A, B, and C corresponding to MHC class I, and HLA DP, DM, DQ, and DR corresponding to MHC class II. As can be seen from Table 1 presented herein below, the stimulatory peptides referred to herein bind to HLA A, B, and C, and therefore the HLA is preferably HLA A, B, or C, i.e., MHC class I. The term HLA is used herein in a broad sense to refer to any HLA molecule or fragment thereof that has the activity of presenting stimulatory peptides to immune cells, such that cognate T cells are activated by a complex containing HLA and a stimulatory peptide. Thus, HLA may be, for example, soluble HLA or HLA multimers, and the term "HLA multimer" refers to soluble HLA in a multimeric form. In HLA multimers, soluble, non-membrane-bound HLA is used. Moreover, HLA is used as a multimer, and the term "multimer" preferably refers to at least a dimeric form, preferably at least a trimeric form. Preferably, the multimer consists of 2 to 8 HLA molecules, more preferably 3 to 6 HLA molecules, and most preferably 4 HLA molecules. Therefore, the HLA multimer is preferably an HLA tetramer. Preferably, the HLA molecules of the HLA multimer are bound via a biotin-streptavidin affinity pair. Preferably, the HLA multimer contains a detectable label, such as a fluorescent marker. Suitable methods for producing HLA multimers are known in the art, for example, by Altman et al. (1996), Science 274:94. Furthermore, HLA multimers are commercially available.

[0030] The term "stimulatory complex" as used herein refers to any complex containing a human leukocyte antigen (HLA) and a stimulatory peptide, both of which are identified herein above. The stimulatory complex may be generated as a soluble stimulatory complex, optionally containing additional components, such as costimulatory molecules, or may be contained on host cells, such as antigen-presenting cells, to which the stimulatory peptide has been added in vitro, or a synthetic APC or scaffold that mimics this may be used. Alternatively, immune complexes may be generated on host cells by contacting the host cells with the administered stimulatory peptide. Since the stimulatory peptide is preferably presented via MHC class I, in this case, the host cell can, in principle, be any host cell that expresses MHC class I. Preferred stimulatory complexes include those containing the stimulatory peptide / HLA combinations shown in Table 1, preferably those designated complexes Nos. 1, 2, 11, 34-38, 61, 62, 78-80, and 89-97.

[0031] The term "immune cell," as used herein, includes any and all cells capable of recognizing, i.e., preferably specifically binding to, any stimulatory complex specified herein. Thus, immune cells are preferably T cells expressing T cell receptors that specifically bind to immune complexes and / or B cells that specifically bind to immune complexes. The T cells are preferably CD8+ T cells, more preferably cytotoxic CD8+ T cells.

[0032] The term "stimulation of immune cells" is understood by those skilled in the art and preferably includes improving at least one characteristic of activated immune cells. Thus, this term includes initiating a new response as well as activating or enhancing an existing immune response. Stimulation of immune cells preferably involves inducing or enhancing a T cell response, most preferably a CD8+ T cell response, against a stimulating complex. Also preferably, stimulation of immune cells involves inducing or enhancing a B cell response, particularly the production of antibodies against the immune complex, against the stimulating complex. Thus, stimulation of immune cells preferably involves increasing the number of immune cells in a subject that specifically recognize the stimulating complex. Preferably, the immune cells are T cells, preferably CD8+ T cells, more preferably cytotoxic CD8+ T cells. Therefore, the stimulating peptide is preferably presented via a major histocompatibility complex (MHC) class I molecule, preferably on nucleated cells, more preferably on antigen-presenting cells and / or nucleated cells infected with HPV16-associated viruses, such as cells of HPV16-associated virus-positive cytoproliferative disorders, particularly HPV-associated lesions or HPV-associated cancers as specified hereinabove. Thus, stimulating immune cells preferably includes inducing an immune response against HPV16-associated virus-positive cytoproliferative disorders. Preferably, stimulating immune cells includes presenting the stimulating peptide to at least one HLA type belonging to one of the HLA supertypes A1, A2, A3 / A11, A24, B7, and B15 (Sidney et al. (2008), BMC Immunology 9 Art. No. 1, doi.org / 10.1186 / 1471-2172-9-1), preferably as assigned to a specific stimulating peptide in Table 1. Preferably, stimulating immune cells comprises administering to a subject an agent providing a stimulating peptide as specified herein above, wherein the subject is preferably a non-human animal and is preferably sacrificed after said stimulation, preferably after step (C) or (D) as specified herein below. However, stimulating immune cells also relates to in vitro activation and / or optionally subsequent enrichment of immune cells that recognize and / or bind to the stimulatory complex.

[0033] As used herein, the term "host cell" refers to any cell that can receive and present this stimulatory peptide via HLA. Preferably, the host cell is a eukaryotic cell, preferably an animal cell, such as an insect cell or a mammalian cell. More preferably, the host cell is a cell of livestock, pet, or laboratory animal. Most preferably, the host cell is a human cell.

[0034] Preferably, the method is for stimulating and identifying immune cells that specifically bind to a stimulatory complex, preferably an HPV16-related virus-infected human host cell. In such a case, the method preferably further comprises the step (C) of identifying the stimulated immune cells stimulated in step (B).

[0035] The term "identification" of immune cells is used herein in a broad sense to refer to any method for generating immune cells that recognize a stimulating complex and are distinguishable from immune cells that do not recognize the stimulating complex. Suitable methods are known in principle to those skilled in the art and preferably comprise the step of contacting the stimulating immune cells specified herein above with a derivative of the stimulating complex that carries a detectable label, where the term "derivative of the stimulating complex that carries a detectable label" preferably refers to the stimulating complex used in step (B) specified herein above, which is covalently or non-covalently bound to the detectable label. The detectable label may be any label deemed appropriate by those skilled in the art. Preferably, the "detectable label" is a label that can be detected by optical means and is known in principle in the art. More preferably, the detectable label is an optically detectable polypeptide, more preferably a fluorescent group, such as a fluorescent dye.

[0036] Also preferably, the method is for stimulating and enriching immune cells that specifically bind to stimulatory complexes, preferably HPV16-related virus-infected human host cells, and the method further comprises step (D) enriching the stimulated immune cells stimulated in step (B) and optionally identified in step (C).

[0037] The term "enrichment" as used herein refers to any method for increasing the number of immune cells that recognize immune complexes relative to the number of unprepared immune cells. Suitable methods are known in the art, and in particular include methods that involve contacting stimulated immune cells with a derivative of a stimulating complex bearing a detectable label as specified herein above, and enriching immune cells that bind to the stimulating complex via the detectable label. Therefore, the term "enrichment" is used herein in its conventional sense. In the context of enriching a cell type, the term preferably relates to increasing the fraction of the enriched cell type relative to other cell types present in the composition. Methods for enriching a specific cell type usually involve labeling the cell type of interest and applying an appropriate enrichment method, such as FACS and / or affinity-based binding to a solid surface. As those skilled in the art will understand, enrichment can also include labeling unwanted cell populations and removing such labeled cells. Moreover, similar methods can be used to enrich T cells that express CD3, CD8, CD137, and / or IFN-γ in addition to T cell receptors that recognize stimulating peptides.

[0038] Advantageously, in the research underlying the present invention, it has been found that the HLA presentation of HPV16-derived peptides by HPV16-positive cells can be evaluated by mass spectrometry, as disclosed in the examples, providing experimental evidence that the peptides are actually presented to the immune system on the intended target cells. Also, as shown in the examples, immunogenicity assays performed on peripheral blood lymphocytes (PBMCs) from various donors can be used to provide evidence that each peptide is actually active in inducing an immune response in vivo. Therefore, in order to generate a workable target structure for T cell epitope-centered immunotherapy for HPV16-mediated malignancies (therapeutic vaccines, adoptive transfer of T cells with transgenic TCRs that recognize verified HLA / peptide complexes, or antibody-based therapeutic agents that recognize verified HLA / peptide complexes (i.e., CAR-T cells, or bispecific antibodies that bring target cells and effector cells into close proximity), it has been found that it is important to verify that T cell epitope / HLA complexes are workable targets to ensure the effectiveness of the above-mentioned therapy. Rather, using HLA-binding prediction algorithms may not be sufficient to define potential targets because they are not sufficiently accurate and lack many binding peptides (see Table 2). Therefore, not only were the HLA binding of each stimulatory peptide mentioned herein experimentally verified, but the T cell memory responses of the stimulatory peptides were also evaluated using PBMCs from healthy human donors. The immunogenic peptides defined in this way must have been naturally processed and presented in previous HPV16 infections. It is also preferable to verify the HLA presentation of the putative stimulatory peptides directly on tumor (or other HPV-positive) cells. This can be achieved by immunopeptidomics, a mass spectrometry-based technique. Therefore, in particular, the combination of the two aforementioned methods can identify candidate peptides suitable for inducing cellular immune responses against cells infected with HPV16-related viruses and the resulting cell proliferation disorders.

[0039] The definitions given above apply mutatis mutandis below. Also, the further definitions and explanations given further below apply mutatis mutandis to all embodiments described herein. The present invention also relates to a method for determining the nucleic acid sequence of at least a portion of an immune reactant that specifically binds to a stimulating complex, preferably an HPV16-related virus-infected human host cell, comprising the steps of stimulating, identifying, and optionally enriching immune cells that specifically bind to a stimulating complex, preferably an HPV16-related virus-infected host cell, according to the method specified herein above, and the further step of sequencing at least a portion of at least one polynucleotide encoding said immune reactant of said immune cells.

[0040] The method for determining nucleic acid sequences of the present invention is an in vitro method. Moreover, the method may include other steps in addition to those explicitly mentioned above. In particular, the obtained sequence information may be used in the construction of an expression construct that expresses at least one chain of an immune reaction reagent that recognizes the stimulatory peptide. Moreover, one or more of the steps may be assisted or performed by an automated device.

[0041] The term "determining the sequence of at least a portion of an immunoreactive reagent" is understood by those skilled in the art. Sequencing can be accomplished by any sequencing method deemed appropriate by those skilled in the art. Preferably, the sequencing uses mRNA or cDNA encoding at least one polypeptide contained in the immunoreactive reagent as a template. As those skilled in the art will understand, an immunoreactive reagent may be composed of one or more polypeptides, preferably those identified herein above, and therefore it may be necessary to determine the sequence of more than one polynucleotide encoding the immunoreactive reagent. Preferably, the portion of the immunoreactive reagent, preferably a polynucleotide encoding a T cell receptor, to be sequenced includes at least CDR3, preferably at least the CDRs. Thus, preferably, at least CDRs 1-3 of each chain of the immunoreactive reagent are sequenced. However, sequencing the variable domain of the immunoreactive reagent or sequencing more than one complete chain of the immunoreactive agent is also contemplated.

[0042] The term "immunoreactive substance" as used herein refers to any and all molecules that recognize, i.e., preferably specifically bind to, stimulatory complexes, preferably HPV16-related virus-infected human host cells. Thus, the immunoreactive agent can be, for example, a T cell receptor or an immunoglobulin.

[0043] As used herein, the term "T cell receptor" refers to a receptor present on T cells that mediates antigen recognition by T cells, and is composed of an alpha chain and a beta chain or a gamma chain and a delta chain, preferably an alpha chain and a beta chain. The structure of a T cell receptor is known to those skilled in the art. Preferably, each chain of a T cell receptor comprises a cytoplasmic domain, a transmembrane domain, a constant domain, and a variable domain, wherein the variable domain confers specificity for an antigen. As used herein, when a cytoplasmic domain is present, both the transmembrane domain and the constant domain may also be referred to as the "non-variable domain" of the T cell receptor chain. In the variable domain, antigen recognition is essentially determined by the complementarity-determining regions (CDRs) designated CDR1, CDR2, and CDR3. As known to those skilled in the art, in contrast to, for example, immunoglobulins, in T cell receptors, only the CDR3s of two TCR chains, i.e., the CDR3 of the alpha chain and the CDR3 of the beta chain, contact the epitope presented by the MHC, while the other two CDRs only essentially contact the MHC. Therefore, to obtain information about the structural basis of the antigen specificity of a T cell receptor, it is preferable to obtain sequence information for the CDR3s of each of the two chains of the T cell receptor. As will be understood by those skilled in the art, the T cell receptor on a T cell is typically part of a polypeptide complex containing additional polypeptides, referred to as the "T cell receptor complex." In addition to the T cell receptor, the T cell receptor complex preferably includes at least one of CD3 delta, CD3 gamma, CD3 epsilon, and CD3 zeta. Preferably, the T cell receptor complex binds to CD8 as a co-receptor. The numbering and structural identification of TCR amino acids preferably follows the suggestions of the International Immunogenetics (ImMunoGeneTics, IMGT) database (Lefranc et al. (2003), Dev. Comp. Immunol., 27:55-77), which identifies conserved amino acids that essentially always occupy the same position within the TCR sequence.

[0044] As used herein, the term "immunoglobulin," which may also be referred to as "antibody," refers to any cell-bound or soluble immunoglobulin or fragment thereof from any of the IgA, IgD, IgE, IgG, or IgM classes that recognizes the stimulatory complexes identified herein above. Antibodies against a predetermined antigen can be prepared by well-known methods. Preferably, the immunoglobulin contains six complementarity-determining regions. The complementarity-determining regions of an antibody are preferably distinct from those of a TCR and are preferably regions within the variable domains of the heavy and light chains of the antibody that define the binding affinity and specificity of the antibody. In the heavy chain, there are three CDRs: CDR1-H, CDR2-H, and CDR3-H; and in the light chain, there are three CDRs: CDR1-L, CDR2-L, and CDR3-L. Preferably, determining the nucleic acid sequence of at least a portion of the immunoglobulin includes determining the sequences encoding these CDRs, preferably all six CDRs.

[0045] As will be understood by those skilled in the art, sequence information about the CDRs of an antibody, e.g., a monoclonal antibody, can be used to generate primatized, chimeric, or humanized antibodies or fragments thereof. Single-chain antibodies, single-domain antibodies, nanobodies, or antibody fragments, e.g., Fab, scFab, etc., can also be generated. Antibodies of the present invention also include bispecific or trispecific antibodies, synthetic antibodies, or chemically modified derivatives of any of the aforementioned antibodies. The aforementioned information can also be used to generate CAR T cells carrying sequences that recognize a stimulatory complex, for example, as single-chain antibodies.

[0046] The present invention also provides a method for producing an immunoreactive substance against a human host cell infected with an HPV16-related virus, comprising the steps of: (i) stimulating immune cells that specifically bind to HPV16-related virus-infected human host cells according to the methods specified herein above; (ii) identifying and optionally enriching the stimulated immune cells of step (i), preferably according to the methods specified herein above; (iii) determining the nucleic acid sequence of at least a portion of the immune reactants expressed by the immune cells identified and optionally enriched in step (ii); (iv) producing the immunoreactant by expressing at least a partial nucleic acid sequence encoding the immunoreactant, preferably in an expression system; The present invention relates to a method, comprising:

[0047] Accordingly, the present invention provides a method for producing an immunoreactive substance against human host cells infected with an HPV16-related virus, comprising the step of expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex), The present invention also relates to a method wherein the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1 to 111, and in a preferred embodiment, SEQ ID NOs: 1 to 129.

[0048] Accordingly, the present invention provides an in vitro method for producing an immunoreactive substance against human cancer cells infected with an HPV16-related virus, comprising the step of expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide, wherein the stimulatory peptide is (i) an HLA consisting of an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, wherein the HLA is derived from the HLA supertype HLA-A01; (ii) consisting of the amino acid sequence of SEQ ID NO: 11, and this HLA is derived from the HLA supertype HLA-A02, (iii) consisting of an amino acid sequence selected from SEQ ID NOs: 34 to 37, and the HLA is derived from the HLA supertype HLA-A03 / A11; (iv) consisting of an amino acid sequence selected from SEQ ID NO: 60 and SEQ ID NO: 61, wherein the HLA is derived from the HLA supertype HLA-A24, or (v) consisting of an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 76, and SEQ ID NO: 77, wherein the HLA is derived from the HLA supertype HLA-B07; or (vi) consisting of an amino acid sequence selected from SEQ ID NO: 61 and SEQ ID NOs: 85 to 92, and this HLA is derived from the HLA supertype HLA-B15; The present invention also relates to a method in which it has been verified that an HPV16-derived peptide having the same amino acid sequence as this stimulatory peptide is presented by human HPV16-positive cancer cells.

[0049] The term "expression" is understood by those skilled in the art in the context of expression of a nucleic acid sequence. Thus, expression of at least a partial nucleic acid sequence encoding an immunoreactive substance preferably relates to causing expression of this nucleic acid sequence, for example, in a suitable expression cell. The term "expressing cell," as used herein, relates to any cell capable of expressing at least a partial nucleic acid sequence encoding an immunoreactive substance, for example, encoded by an expression construct or vector as specified herein above. Thus, the expressing cell is preferably a bacterial cell, for example, an E. coli cell, a unicellular eukaryotic cell, for example, a yeast cell, a fungal or insect cell, or a mammalian cell, preferably cultured in vitro. Also preferably, expression is in vitro expression in an in vitro translation system. More preferably, expression is in vitro expression in a cell.

[0050] The present invention also relates to immune cells expressing an immune reactant that specifically binds to HPV16-related virus infected host cells, the immune cells being obtained or obtainable by the methods specified herein above.

[0051] The present invention also relates to an immunoreactive agent against human cancer cells infected with an HPV16-related virus, which specifically binds to a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex), wherein the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1-111, and the immunoreactive agent is preferably produced or producible according to any of the methods specified herein. Preferably, the immunoreactive agent specifically binds to an immune complex selected from any one of Tables 1(i)-1(vi) identified herein below, preferably stimulatory complexes designated Complex Nos. 1, 2, 34-38, 61, 62, 78-80, and 89-97, and in preferred embodiments, stimulatory complexes designated Complex Nos. 1, 2, 34-38, 61, 62, 77-80, 89-97, 132, and 136.

[0052] The present invention also relates to the use, preferably in vitro, of a stimulatory peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92, and in preferred embodiments, SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 75-77, 85-92, 121, and 136, which has been verified to be presented by human HPV16-positive cancer cells and to stimulate human anti-stimulatory peptide immune cells when presented in a complex with human leukocyte antigen (HLA), for stimulating, identifying, and optionally enriching immune cells that specifically bind to HPV16-related virus-infected human host cells.

[0053] The present invention further relates to immune cells and / or immune reactants as specified herein above for use in medicine, preferably for use in the treatment of cell proliferative disorders associated with HPV16-related viruses.

[0054] The terms "treating" and "treatment" refer to a significant improvement of the disease or disorder referred to herein or symptoms associated therewith. As used herein, this treatment also includes overall restoration of health related to the disease or disorder referred to herein. When this term is used herein, it should be understood that treatment may not be effective in all subjects treated. However, this term preferably requires that a statistically significant proportion of subjects suffering from the disease or disorder referred to herein can be successfully treated. Whether a proportion is statistically significant can be determined by those skilled in the art using various well-known statistical evaluation tools, for example, as described herein below, without going into detail. Preferably, cancer treatment reduces tumor burden in a subject. As will be understood by those skilled in the art, the effectiveness of cancer treatment depends on various factors, including, for example, the stage and type of cancer. Preferably, the treatment causes the subject's T cells to recognize inappropriately proliferating cells, preferably neoplastic cells, more preferably cancer cells. Thus, preferably, the treatment has the effect of killing tumor cells and causing cessation of tumor growth, in particular cessation of tumor cell proliferation, more preferably tumor regression, more preferably tumor resolution. As used herein, the above applies mutatis mutandis to the treatment of other HPV16-related virus-positive cellular proliferative disorders.

[0055] Whether a proportion is statistically significant can be determined by those skilled in the art using various well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, or Mann-Whitney test, without going into detail. A preferred confidence interval is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment is effective in at least 60%, at least 70%, at least 80%, or at least 90% of the subjects in a given cohort or population.

[0056] In view of the above, the following embodiments are particularly contemplated. Embodiment 1: (A) contacting an immune cell with a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex); (B) thereby stimulating immune cells that specifically bind to HPV16-related virus-infected host cells; A method for stimulating immune cells that specifically bind to human host cells infected with an HPV16-related virus, comprising: the stimulatory peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1 to 111, and in a preferred embodiment, SEQ ID NOs: 1 to 129.

[0057] Embodiment 2: The method of embodiment 1, wherein the stimulatory peptide has been validated to stimulate human anti-stimulatory peptide immune cells when presented in a complex with human leukocyte antigen (HLA).

[0058] Embodiment 3: The method of embodiment 1 or 2, wherein the human host cells are human cancer cells infected with an HPV16-related virus, and an HPV16-derived peptide consisting of an amino acid sequence identical to the stimulatory peptide has been verified to be presented by human HPV16-positive cancer cells, and the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92.

[0059] Embodiment 4: The method of any one of embodiments 1 to 4, wherein said stimulation is activation of said immune cells and / or an increase in the number of immune cells that specifically bind to said stimulatory complex. Embodiment 5: The method of any one of embodiments 1 to 4, wherein the stimulatory complex is contained on an antigen-presenting cell (APC) or artificial APC that presents at least one of the stimulatory complexes, or is an HLA multimer that includes the stimulatory peptide.

[0060] Embodiment 6: The method of any one of embodiments 1 to 5, which is a method for stimulating and identifying immune cells that specifically bind to the stimulatory complex, further comprising the step of (C) identifying the stimulated immune cells stimulated in step (B).

[0061] Embodiment 7: The method of any one of embodiments 1 to 6, which is a method for stimulating and enriching immune cells that specifically bind to human host cells infected with an HPV16-related virus, further comprising step (D) enriching the stimulated immune cells stimulated in step (B) and optionally identified in step (C).

[0062] Embodiment 8: The method of embodiment 6 or 7, further comprising contacting the immune cells of step (B) with a derivative of the stimulating complex that carries a detectable label, and identifying and optionally enriching immune cells that bind to the stimulating complex by means of the detectable label.

[0063] Embodiment 9: The method of any one of embodiments 1 to 8, which is an in vitro method. Embodiment 10: The method of any one of embodiments 1 to 8, wherein step (A) comprises administering the stimulatory complex to a subject.

[0064] Embodiment 11: The method of embodiment 10, wherein the subject is a non-human animal, and the subject is preferably sacrificed after step (C) or (D). Embodiment 12: A method for determining the nucleic acid sequence of at least a portion of an immune reactant that specifically binds to human host cells infected with an HPV16-related virus, comprising stimulating, identifying, and optionally enriching immune cells that specifically bind to HPV16-related virus-infected host cells according to the method of any one of embodiments 1 to 11, and the further step of sequencing at least a portion of at least one polynucleotide encoding said immune reactant in said immune cells.

[0065] Embodiment 13: A method for producing an immune reactant against a human host cell infected with an HPV16-related virus, comprising: (i) stimulating immune cells that specifically bind to HPV16-related virus-infected human host cells according to the method of any one of embodiments 1 to 5 or 9 to 11; (ii) identifying and optionally enriching the stimulated immune cells of step (i), preferably according to the method of any one of embodiments 6 to 10; (iii) determining the nucleic acid sequence of at least a portion of the immune reactants expressed by the immune cells identified and optionally enriched in step (ii); (iv) producing the immunoreactant by expressing at least a partial nucleic acid sequence encoding the immunoreactant, preferably in an expression system; A method comprising:

[0066] Embodiment 14: A method for producing an immunoreactive substance against a human host cell infected with an HPV16-related virus, comprising expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex), The stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1 to 111.

[0067] Embodiment 15: Expressing at least a partial nucleic acid sequence encoding an immune response substance obtained from immune cells stimulated with a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex). 1. An in vitro method for producing an immunoreactive substance against human cancer cells infected with an HPV16-related virus, comprising: (i) an HLA consisting of an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, wherein the HLA is derived from the HLA supertype HLA-A01; (ii) consisting of the amino acid sequence of SEQ ID NO: 11, and this HLA is derived from the HLA supertype HLA-A02, (iii) consisting of an amino acid sequence selected from SEQ ID NOs: 34 to 37, and the HLA is derived from the HLA supertype HLA-A03 / A11; (iv) consisting of an amino acid sequence selected from SEQ ID NO: 60 and SEQ ID NO: 61, wherein the HLA is derived from the HLA supertype HLA-A24, or (v) consisting of an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 76, and SEQ ID NO: 77, wherein the HLA is derived from the HLA supertype HLA-B07; or (vi) consisting of an amino acid sequence selected from SEQ ID NO: 61 and SEQ ID NOs: 85 to 92, and this HLA is derived from the HLA supertype HLA-B15; The method verifies that an HPV16-derived peptide having the same amino acid sequence as this stimulatory peptide is presented by human HPV16-positive cancer cells.

[0068] Embodiment 16: The method of embodiment 14 or 15, having at least one further feature of embodiments 1 to 13. Embodiment 17: The method of any one of embodiments 1 to 16, wherein the immune cell is a T cell expressing a T cell receptor that specifically binds to the immune complex and / or a B cell that specifically binds to the immune complex.

[0069] Embodiment 18: The method according to any one of embodiments 1 to 17, wherein the immune cells are T cells, preferably CD8+ T cells, more preferably cytotoxic CD8+ T cells. Embodiment 19: The method of any one of embodiments 1 to 16, wherein the HPV16-related virus is HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 or HPV67, more preferably HPV16.

[0070] Embodiment 20: An immune cell expressing an immunoreactive substance that specifically binds to an HPV16-related virus-infected host cell, wherein the immune cell is obtained or obtainable by the method of any one of embodiments 1 to 11.

[0071] Embodiment 21: The immune cell of embodiment 13, wherein the immune cell is a T cell and the immune reactant is a T cell receptor, or the immune reactant is a B cell and the immune reactant is an immunoglobulin.

[0072] Embodiment 22: An immunoreactive substance against human cancer cells infected with an HPV16-related virus that specifically binds to a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide, wherein the stimulatory peptide consists of an amino acid sequence selected from SEQ ID NOs: 1 to 111.

[0073] Embodiment 23: An immunoreactant according to embodiment 22, produced or producible according to the method of any one of embodiments 13 to 19. Embodiment 24: Use, preferably in vitro, of a stimulatory peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92, validated to be presented by human HPV16-positive cancer cells and validated to stimulate human anti-stimulatory peptide immune cells when presented in complex with human leukocyte antigen (HLA), to stimulate, identify, and optionally enrich immune cells that specifically bind to HPV16-associated virus-infected human host cells.

[0074] Embodiment 25: The subject matter of any one of the preceding embodiments, wherein the immune reactant is a TCR, an anti-immune complex antibody, or a fragment or derivative thereof. Embodiment 26: The subject matter of any one of the preceding embodiments, wherein the immunoreactive reagent is comprised in a T cell.

[0075] Embodiment 27: An immune cell and / or immune reactant according to any one of embodiments 20 to 23 for use in medicine, preferably for use in the treatment of a cell proliferative disorder associated with an HPV16-related virus.

[0076] All references cited herein are hereby incorporated by reference in their entirety and with respect to the disclosures specifically referred to herein. The following examples are intended to be merely illustrative of the present invention and should not be construed as limiting the scope of the invention. [Example]

[0077] Example 1: Methods for assessing T cell reactivity Interferon-γ ELISpot assay Prior to the ELISpot assay, ELISpot plates (Millipore Multiscreen-HA membrane sterile plates) were coated with 100 μl of 2 μg / mL anti-human IFN-γ (1-D1K) antibody in sterile PBS and incubated at 4°C overnight or for up to 3 days.

[0078] Before setting up the ELISpot assay, the coating antibody was discarded, the wells were washed three times with 200 μL of sterile PBS, and blocked with 200 μL of ELISpot medium under standard incubation conditions for 1–1.5 hours. After blocking, the ELISpot medium was discarded, and the wells were filled with 100 μL of antigen solution.

[0079] A total of 8 wells of each (short-term) T cell line (see below) were stimulated: 4 wells with the respective antigen (10 μg / mL single peptide, 0.1% DMSO or CEF peptide pool (1 μg / mL of each peptide)), 2 wells with concanavalin A (2 μg / mL) as a non-specific mitogen stimulation positive control, and 2 wells with 0.1% (v / v) DMSO as a background control. 1-2 × 10 short-term antigen-specific T cell lines (described in 1.4) were stimulated in 100 μL per well. 5 The cells were added at a concentration of 1 x 10 cells (total volume 200 μL / well). The cell numbers of three representative wells (CEF, DMSO, peptide stimulated) were assessed and the final cell number was 1 x 10 cells. 6This allowed for the calculation of spot-forming units (SFU) per cell. ELISpot plates were incubated for 20–24 h under standard culture conditions. Moving the plate or incubator during incubation was strictly avoided to prevent smeared spots.

[0080] The remaining cell suspension was discarded, and the ELISpot plate was washed twice with PBS and twice with ELISpot wash buffer. All washes were performed with 200 μL / well. Sterile anti-human IFNγ biotin (7 B6-1) antibody at 1 ng / mL in sterile PBS was then added at 100 μL / well. After 2 hours of incubation in the dark at room temperature, the antibody was discarded, and the plate was washed four times with ELISpot wash buffer. Sterile streptavidin-alkaline phosphatase was diluted 1:2000 in sterile PBS, and 100 μL of this dilution was dispensed per well.

[0081] After 1.5 hours of incubation in the dark at room temperature, the antibody was discarded and the plate was washed four times with ELISpot wash buffer. BCIP / NBT-Plus substrate was then filtered through a 0.22 μm filter and dispensed at 100 μL per well. The plate was covered with aluminum foil and left at room temperature for 18–20 minutes until spots developed in the positive control wells. The reaction was stopped by washing the ELISpot plate with tap water and allowing the plate to air-dry for a minimum of 1.5 hours.

[0082] Analysis of ELISpot plates To obtain objective results for the number of SFU per well, ELISpot plates were counted using an automated ELISpot plate reader from CTL. The SmartCount™ counting mode in ImmunoSpot 5.1.36 Professional DC software was used for analysis. Settings were individually adjusted for each donor to account for donor-specific variance. Typically, background balance was set to a value between 0 and 10, and spot separation was set to a value between 1 and 4. The automatically determined minimum spot size was adjusted to exclude small background spots based on negative control wells. Similarly, the maximum spot size was automatically determined based on positive control wells. Quality control of each well was performed manually to detect and correct errors in the counting algorithm caused by uneven spreading or fibers. Based on the counting results, a 1 × 10 count was used. 6 The mean SFU per cell and the stimulation index (SI), defined as the fold change in mean SFU in peptide-stimulated wells relative to DMSO background wells, were calculated for each peptide-specific cell line. 6 Responses with SFU>100 per cell and responses with SI>4 and SFU>50 were considered positive.

[0083] T cell phenotyping and intracellular cytokine staining To characterize and quantify responding cells upon peptide stimulation, peptide-specific T cell cultures with positive ELISpot results were further analyzed, and T cell phenotyping and intracellular cytokine staining (ICS) were assessed by flow cytometry.

[0084] Cells from each subsequent short-term T cell culture, as well as DMSO-stimulated and non-responder T cell lines, were collected into 2 mL reaction tubes and centrifuged at 400 × g for 10 minutes. The supernatant was discarded, and the pellet from the non-responder T cell line was resuspended in 100 μL of ELISpot medium to serve as an unstimulated and unstained control. The pellets from the responder and DMSO-stimulated T cell lines were resuspended in 200 μL of ELISpot medium and equally divided into two wells of a V-bottom 96-well plate. One well of the responder T cell line was restimulated with 10 μg / mL of the specific peptide, while the other well was mock-stimulated with 0.1% (v / v) DMSO. As a positive control, one well of the DMSO-stimulated T cell line was activated by treatment with 20 ng / mL of phorbol 12-myristate 13-acetate and 1 μM ionomycin. A 1:10 dilution of GolgiStop (containing monensin) and a 1:15 dilution of GolgiPlug (containing brefeldin A) were then added to all wells to inhibit intracellular transport processes. Monensin inhibits trans-Golgi transport, and brefeldin A inhibits transport between the ER and the Golgi apparatus (Chardin and McCormick, 1999; Mollenhauer et al., 1990). Stimulated cells were incubated for 5 hours under standard culture conditions.

[0085] After incubation, the cells were resuspended and centrifuged at 400xg for 5 minutes, and the supernatant was discarded. The cells were then resuspended in 50µL of cold staining buffer (unstained) or 50µL of cold staining buffer (unstimulated and stimulated) containing phenotyping antibodies against CD3, CD4, and CD8 and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain and incubated at 4°C for 30 minutes. After surface staining, the cells were washed twice with 200µL of staining buffer, centrifuged at 1400rpm, and then fixed with 1% paraformaldehyde (PFA) solution for 15 minutes at 4°C. All subsequent centrifugation steps were performed at 1400rpm for 5 minutes. After fixation, the cells were again washed twice with 200µL of cold staining buffer and then resuspended in 100µL of 1x perm / wash buffer diluted in PBS (prepared at 10x) for 15 minutes at 4°C to permeabilize the cell membrane. The plate was then centrifuged and the supernatant discarded. The cells were resuspended in 50 μL of perm / wash buffer (unstained) diluted in PBS or 50 μL of perm / wash buffer (unstimulated and stimulated) containing intracellular cytokine antibodies against IFNγ, TNFα, and granzyme B for 30 minutes at 4°C. After staining for intracellular cytokines, the cells were washed twice with 200 μL of 1× perm / wash buffer. Then, 100 μL of fix / perm solution (provided in the BD Cytofix / Cytoperm kit) was added per well. After 20 minutes of incubation at 4°C, the cells in each well were washed twice with 200 μL of 1× perm / wash buffer, resuspended in 100 μL of staining buffer, and stored at 4°C overnight.

[0086] OneComp eBeads™ (eBeads) and ArC™ Amine Reactive Compensation beads (ArC beads) were stained as compensation controls. For each antibody, 50 μL of eBeads suspension was stained with the same dilution used for cell staining. ArC beads were stained with 1 μL of LIVE / DEAD™ Fixable Near-IR Dead Cell Stain stock solution in 50 μL of staining buffer. The beads were incubated at 4°C for 30 minutes, then washed twice with 1 mL of staining buffer and centrifuged at 400 x g. Finally, the stained beads were resuspended in 400 μL of staining buffer, and one drop of ArC™ negative beads was added to the labeled Arc beads. All beads were stored overnight at 4°C.

[0087] The next day, samples and single-stain compensation controls were acquired on a BD FACS Canto™ II analyzer using BD FACS Diva Software Version 6. The resulting data were analyzed using FlowJo Software Version 10 by applying the same gating strategy to all samples.

[0088] Short-term T cell line To expand antigen-specific memory T cells previously induced by natural HPV16 infection, short-term antigen-specific T cell lines were generated from PBMCs. To do this, T cells were stimulated with HLA-matched HPV16 E6 or E7-derived HLA ligands. To establish short-term T cell lines, human PBMCs were thawed and resuspended in T cell medium supplemented with recombinant human interleukin-7 (rhIL-7) (10 ng / mL) and recombinant human interleukin-15 (rhIL-15) (20 ng / mL). Cells were counted and 1–2 × 10 cells were used for each round of antigen stimulation. 6Cells were seeded into wells of a 24-well plate in a total volume of 2 mL per T cell line. To obtain antigen-specific T cell lines, 10 μg / mL of HLA-binding HPV16 E6 / E7-derived peptides was added to each well. As a peptide-specific positive control, one cell line was stimulated with a peptide pool (CEF peptide pool) consisting of 23 well-described HLA class I-restricted epitopes (Currier et al., 2002) from the widespread viruses human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza A, at a concentration of 1 μg / mL for each peptide. As single-peptide controls, two cell lines were stimulated with 10 μg / mL of HLA-matched EBV- and CMV-derived peptides. As a peptide-specific negative control, cell lines were stimulated with 10 μg / mL of HLA-matched human immunodeficiency virus (HIV)-derived peptide (10 μg / mL) and treated with 0.1% (v / v) dimethyl sulfoxide (DMSO, peptide solvent) to serve as a nonspecific negative control. Stimulated PBMCs were cultured under standard culture conditions.

[0089] On day 3, the cells were supplemented with recombinant human interleukin-2 (rhIL-2) (20 U / mL) and rhIL-15 (20 ng / mL) to promote T cell proliferation. Seven days after the culture step, a half-medium exchange was performed. To do this, cells were pelleted to the bottom of the wells by centrifugation at 300 x g for 5 minutes, and 1 mL of supernatant was carefully removed from each well. Then, 1 mL of fresh T cell medium supplemented with rhIL-2 (final 20 U / mL) and rhIL-15 (final 20 ng / mL) was added, and the cells were resuspended.

[0090] After 12 days of culture, half of the cells from each culture were used to set up an ELISpot assay as described in 1.1. The remaining cells were fed with 1 mL of ELISpot medium and cultured for an additional 2 days before being used for T cell phenotyping and intracellular cytokine staining (ICS) as described in 1.2.

[0091] Cytotoxicity assay A flow cytometry-based VITAL FR cytotoxicity assay was used as published by Stanke et al. (Stanke et al., 2010) to assess whether peptide-specific T cells could specifically kill HPV-transformed target cells. Briefly, two HLA-A2 + Cell line, HPV16 negative (HPV16 - ) control cell line (C33A) and HPV16-positive (HPV16 + ) The target cell line (CaSki) was fluorescently labeled with either FarRed or CFSE, and peptide-specific CD8 + After 48 hours of co-incubation, fluorescently labeled cells were analyzed by flow cytometry to determine viable cells, and the specific cytolytic function of effector cells was calculated as a ratio of target cells to control cells.

[0092] CaSki and C33A cells were fluorescently labeled with CFSE and FarRed, respectively. Cells of both cell lines were harvested and cultured at 1 × 10 6 The cells were resuspended in unsupplemented RPMI medium at a concentration of 100 cells / mL. For cell labeling, 5 μM CSFE was added to CaSki cells and 0.25 μM FarRed was added to C33A cells. The cells were incubated at 37°C, and after 10 minutes, the reaction was stopped by adding 50 mL of RPMI supplemented with 10% FBS. The cells were centrifuged at 300 x g for 5 minutes, the supernatant was discarded, and the cells were resuspended in cell culture medium. Labeled cells were added at a concentration of 1–2 × 10 6 Cells / flask were seeded into T25 cell culture flasks.

[0093] The next day, labeled CaSki and C33A and epitope-specific semi-long-term T cell lines (described in 1.6) were collected. MACS CD8 + CD8 T cells were isolated from epitope-specific semi-long-term T cell lines using a T cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol for the LS column. + T cells were isolated. CD8 +T cells were resuspended in T cell medium and added to wells of an F-bottom 96-well plate in serial dilutions, 6 x 10 per well. 4 cells, 3 x 10 4 cells, 1.5 x 10 4 cells, 0.75×10 4 cells, and 0.375 × 10 4 Cell triplicates were generated. CaSki and C33A cells were mixed 1:1 in T cell medium, and 3 × 10 3 Cells were added to each well containing T cells, resulting in effector:target (E:T) ratios of 1:20, 1:10, 1:5, 1:2.5, and 1:1.25. In addition, triplicates of mixed target cells were seeded in the absence of T cells, designated E:T 0. After 48 hours of incubation at 37°C and 5% CO2, cells were harvested and analyzed on a BD FACS Canto™ II analyzer using BD FACS Diva Software Version 6. The resulting data were analyzed using FlowJo Software Version 10 by applying the same gating strategy to all samples. Specific killing was then calculated using the following formulas (1) and (2) in Excel (Microsoft Office 2016):

[0094]

number

[0095] Semi-long term T cell line Sub-long-term T cell lines were established to obtain the large numbers of T cells required for analysis of epitope-specific cytotoxic T cell responses by the VITAL FR cytotoxicity assay.

[0096] Autologous DCs were generated from PBMCs as described in 1.7 and supplemented with the desired HPV16 peptide. PBMCs from the same donor were thawed and resuspended in T cell medium supplemented with rhIL-7 (10 ng / mL) and rhIL-15 (20 ng / mL). 1 × 10 7PBMCs were seeded into 24-well plates in a total volume of 1 mL per well. Peptide-loaded DCs were added at a ratio of 50:1 (PBMC:DC) to 1 mL of T cell medium supplemented with rhIL-7 (10 ng / mL) and rhIL-15 (20 ng / mL) for a final volume of 2 mL. Cell cultures were fed with rhIL-2 (final concentration: 40 U / mL) every other day. When the medium turned yellow, half-medium replacement with 1 mL of fresh T cell medium supplemented with IL-2 was performed. After 8 days, peptide stimulation with peptide-loaded DCs was repeated, and the culture was continued. On day 15, T cell lines were collected and CD8 + T cells were isolated and used for cytotoxicity assays.

[0097] Generation of autologous DCs To generate autologous DCs, frozen human PBMCs were thawed. After thawing, cells were counted and 5 × 10 6 From this suspension, the cells were resuspended in DC medium to reach a density of 1 x 10 cells / mL in 2 mL of DC medium. 7 Cells were seeded per well of a 6-well plate.

[0098] After 3 hours of incubation at 37°C and 5% CO2, the supernatant was removed and transferred to a 50 mL tube. The wells were gently washed with 1 mL of warm DC medium to remove non-adherent cells, and the wash medium was collected along with the supernatant. The remaining adherent cells were further cultured in 2 mL of fresh DC medium supplemented with 100 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng / mL interleukin-4 (IL-4). The collected non-adherent cells, including T cells and B cells, were counted and frozen for later use.

[0099] After 3 days of culture, cells were fed with 0.5 mL of fresh DC medium supplemented with 100 ng / mL GM-CSF and 50 ng / mL IL-4. On day 6, DCs were matured by adding 1000 U / mL tumor necrosis factor α (TNFα), 10 ng / mL interleukin-1β (IL-1β), 10 ng / mL interleukin-6 (IL-6), 1 μM prostaglandin E2 (PGE2), and 1 μg / mL lipopolysaccharide (LPS) to each well. Cells were incubated with this maturation cocktail for 48 hours and then collected by gentle scraping with a cell scraper.

[0100] Live-cell imaging-based cytotoxicity assay In addition to the VITAL FR assay described above, we applied a newly established cytotoxicity assay based on live-cell imaging. In this assay, transiently red-labeled HPV16-transformed target cells were co-incubated with either HPV16 peptide-specific or non-specific CD8+ T cells. Apoptotic cells were stained with green caspase dye. The frequency of apoptotic target cells (stained red and green) was analyzed over time by live-cell imaging. Peptide-specific killing was detected by the increased frequency of apoptotic target cells in co-cultures with HPV16 peptide-specific CD8+ T cells compared with co-cultures with non-specific CD8+ T cells. This highly sensitive assay was used to detect killing mediated by low-frequency CTL populations, which, in contrast to general endpoint assays, allows for the time-course analysis of CTL-mediated cytotoxicity. Furthermore, this assay is highly flexible and can be easily adapted to other target cell lines of interest.

[0101] Example 2: Immunoprecipitation (IP) and LC / MS analysis of peptides presented by cultured cells Immunoprecipitation of HLA-presented peptides HLA immunoprecipitation (IP) was performed according to previously published protocols (Bassani-Sternberg et al., 2016; Chong et al., 2018). Briefly, HPV16+ cells were lysed in lysis buffer containing 1% N-octyl-β-D glucopyranoside, 0.25% Na-deoxycholate, protease inhibitor cocktail (Sigma-Aldrich, Mannheim, Germany) / PMSF (Carl Roth, Karlsruhe, Germany) in PBS. After centrifugation at 40,000 × g for 30 min at 4°C, HLA-peptide complexes were immunoprecipitated by incubation with mouse anti-human HLA-A, B, C monoclonal antibody (clone W6 / 32, Biolegend, San Diego, CA, USA) cross-linked to Protein G Sepharose beads (Cytiva, Marlborough, MA, USA) or Protein A-Sepharose™ 4B (Invitrogen Corporation, Camarillo, CA, USA) or, in some cases, HLA type-specific antibodies (HLA-A2: clone BB7.2, HLA-A3: clone GAP A3, HLA-A11 / A24: clone Hb 164, HLA-B7: clone BB7.1) on a rotating wheel with constant mixing for 4 h at 4°C. After centrifugation at 3200 × g for 3 min at room temperature, the supernatant was discarded. The pelleted HLA-peptide complexes bound to the antibody-beads were washed three times: first with 20 mM ice-cold Tris-HCl pH 8 containing 150 mM NaCl, then with the same buffer but supplemented with 400 mM NaCl, and finally with 20 mM Tris-HCl alone. Peptides were eluted from the HLA-bound antibody-beads with 0.3% TFA. At this stage, optional alkylation of cysteine-containing peptides was performed. One ml of the IP eluate was reduced by incubation with 100 μl of HEPES / 50 mM TCEP at room temperature for 10 minutes. Alkylation was performed by incubation with 100 μl of 400 mM iodoacetamide (IAA) at room temperature for 20 minutes in the dark. Alkylation was quenched by incubation with 100 μl of 1 M HEPES / 50 mM TCEP at room temperature for 3 minutes.The sample was re-acidified with 60 μl of 10% TFA. The resulting peptides were desalted using a SepPak 96-well plate (Waters, Milford, MA, USA) by reverse-phase purification. The oxidation reaction was carried out simultaneously with loading onto the sorbent. Performic acid was prepared by mixing 25 μl of 30% HO and 45 μl of 10% formic acid in a glass vial and allowing to stand for 5 minutes. Oxidation of the sample was carried out by briefly applying 1 ml of performic acid solution to the well, which was then washed with 1 ml of 0.1% TFA. The peptides eluted in 28% ACN in 0.1% TFA were dried by vacuum centrifugation (Concentrator plus, Eppendorf, Hamburg, Germany).

[0102] Direct injection and LC-MS A series of predicted target peptides were obtained from commercial sources as stable isotope-labeled (SIL) peptides (crude synthetic products: isotopic purity >99 atom%). 13 C and 15 N). To alkylate cysteine-containing peptides, 2 ng / peptide of vacuum-dried peptide mixture was resolubilized in 20 μl of 100 mM HEPES by 3 min of sonication in a 0.5 μl Protein LoBind microcentrifuge tube. 5 μl of sample (500 pmol / peptide) was reduced by adding 2 μl of HEPES / 50 mM TCEP and incubating for 10 min at room temperature. Alkylation was performed by adding 2 μl of 400 mM IAA and incubating for 20 min at room temperature in the dark. Alkylation was quenched by adding 2 μl of 100 mM HEPES / 50 mM TCEP. Samples were acidified by adding 0.3% TFA to a volume of 500 μl and desalted using 100 mg of absorbent wells in a 96-well SepPak plate (Waters, Milford, MA, USA). The elution step was performed with 80% ACN / 0.1% TFA.

[0103] Direct infusion analysis was first performed to optimize the normalized collision energy (NCE) for each single peptide in the mixture. NCE values ​​ranged from 4% to 42%, with each value measured four times for each peptide's four charge states (1+ to 4+) in 2% increments. An Orbitrap Exploris 480 (Thermo Fisher Scientific) was operated with targeted MS2 scans (PRM) at 200 m / z with a resolution of 30K, a standard automatic gain control (AGC) target, a maximum injection time (IT) of 350 ms, and one microscan in centroid data acquisition mode. For all measurements, the isolation window was set to 0.4, 0.7, 1, or 1.5 m / z, depending on the precursor mass, as recommended by the manufacturer. Ion chromatograms of all predicted transitions were extracted from the centroid spectrum with a mass tolerance of 12 ppm and deconvoluted using a custom-written R script. The highest NCE was chosen to maximize the probability of detecting at least five transitions by maximizing the overall intensity of the fifth most intense transition. Retention times (RT) for each peptide were determined by LC-MS in DDA mode using an integrated list of target peptides in all four charge states. MS resolution was set to 120K, AGC target 3e6, and maximum IT 50 ms. LC was operated as follows:

[0104] For LC-MS, samples were dissolved in 2.5–5 μl of 5% ACN in 0.1% TFA, and 50 fmol of Peptide Retention Time Calibration (PRTC) Mixture (88321, Pierce™) was added, followed by a 3-minute ultrasonic bath. All samples were analyzed by liquid chromatography (U-3000, Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 (Thermo Fisher Scientific). Due to the potential for carryover of photocontamination from previous quality control tests, such as SIL peptides (Salek et al. 2022), 150 fmol of BSA digest (88341, Pierce™) and 50 fmol of PRTC Mixture (without target peptide) were systematically injected, followed by an IP sample as a negative control, to test for the absence of any artifacts. The LC gradient consisted of multiple segments. First, solvent B (100% ACN, 0.1% FA) was increased from 2% to 6% within 5 min, followed by A (0.1% FA in HO) at 94% and then B at 28.5% within 75 min. This reached 80% within 4 min, followed by a 5-min wash at 80% B. Finally, the column was equilibrated at 2% B for 11 min.

[0105] For LC-MS experiments, MS was acquired at 60K resolution with m / z at 200, a mass range of 150–1450 m / z, an AGC target of 3e6, and a maximum IT of 25 ms. MS2 data were acquired by PRM scans using either 60K or 120K resolution with m / z at 200. The target precursor list presented preselected charge states, corresponding m / z values, and optimized collision energy values ​​for each target, along with their expected retention times (+ / - 1–3 min) predefined by the SIL peptide. The normalized AGC target was set to 1000% (or 1e6). The maximum injection time mode was set to dynamic, allowing sampling of a minimum of five points across the chromatographic peak. Dynamic RT characteristics using Pierce PRTC mixtures were active.

[0106] Data analysis LC-MS data were analyzed using Skyline software v.20.2 (MacLean et al., 2010). A minimum of the top five most intense product ions were extracted with a mass tolerance of 7 ppm. Detected peaks were manually curated. Light peaks were discarded if their retention time or shape did not match the heavy reference, if their normalized spectral contrast angle (NSCA) (Toprak et al., 2014) was low, or if too few transitions were detected.

[0107] The findings of the present invention are also summarized in Table 2 below, which shows that there are a huge number of peptides (approximately 9000) that can be derived from HPV16 E6 and E7. The prediction algorithm identifies approximately one-third of the peptides that actually bind to HLA, and in about 50% of cases, the binding peptides are incorrectly predicted. Moreover, only one-half of the immunogenic peptides are correctly predicted, and only approximately one-quarter of the peptides actually presented on HPV16-positive tumor cells are correctly predicted.

[0108] Detection of cysteine-containing peptides For the detection of HPV16-derived T cell epitopes, an immunopeptidomics workflow was established, in which scientists individually evaluated each candidate epitope and devised an optimized targeting strategy. Because HPV16 proteins E6 and E7 are rich in cysteine, 148 of the 242 candidate peptides contained cysteine ​​(see Table 3 below). Contrary to common practice in the art (see, e.g., Blatnik et al. (2018), cited above), cysteine-containing peptides were not excluded from the analysis. Detection of cysteine-containing peptides is particularly challenging in MS experiments due to the propensity of thiol side chains to undergo oxidative modification, necessitating an adapted strategy. The resulting dispersion of cysteine-containing peptides in various forms does not allow for the sensitive detection required for targeted experiments. For this reason, previous studies of HPV16 did not target any cysteine-containing peptides; in this study, we overcame this limitation by incorporating a cysteine ​​alkylation reaction.

[0109] Table 1

[0110] Table 2

[0111] Table 3

[0112] Table 4-1

[0113] Table 4-2

[0114] Table 5

[0115] Table 6

[0116] Table 7-1

[0117] Table 7-2

[0118] Table 8

[0119] References Altman et al. (1996), Science 274:94 Bassani-Sternberg et al. (2016), Nat Commun, 7, 13404. doi:10.1038 / ncomms13404 Becker & Riemer (2022), Frontiers Immunol, doi: 10.3389 / fimmu.2022.883989 Blatnik (2018), Proteomics 18:1700390, DOI: 10.1002 / pmic.201700390 Bonsack et al. (2019), Cancer Immunol Res; 7(5):719 Bourgault Villada et al., Clin Exp Immunol 2010;159(1):45 Chardin & McCormick (1999), Cell 97, 153-155. Chong et al. (2018), Mol Cell Proteomics, 17(3), 533-548. doi:10.1074 / mcp.TIR117.000383 Currier et al. (2002), J Immunol Methods 260, 157-172. Habib et al. (2022), Cells 6;11(3):421. doi:10.3390 / cells11030421 Hara et al., Int J Oncol 2005;27(5):1371 Jang et al., Cancer 2012;118(8):2173 Kast et al., J Immunol 1994;152(8):3904 Krishna et al. (2018) Cancer Res.2018;78(21):6159 Lefranc et al. (2003), Dev. Comp. Immunol., 27:55 MacLean et al. (2010), Bioinformatics, 26(7), 966-968. doi:10.1093 / bioinformatics / btq054 Mizuuchi et al., Exp Mol Pathol 2012;92(1):185 Mollenhauer et al. (1990), Biochim Biophys Acta 1031, 225-246. Ressing et al., J Immunol 1995;154(11):5934 Riemer et al., J Biol Chem 2010;285(38):29608 Salek et al. (2022), Analyt Bioanalyt Chem, 414(8),2545-2552. doi:10.1007 / s00216-022-03931-w Sydney et al. (2008), BMC Immunology 9 Art. No. 1, doi.org / 10.1186 / 1471-2172-9-1 Stanke et al. (2010), J Immunol Methods 360, 56-65. Steinbach & Riemer (2018), Int J Cancer 142:224 Toprak et al. (2014), Mol Cell Proteomics, 13(8), 2056-2071. doi:10.1074 / mcp.O113.036475 Tsang et al. (2017), Vaccine 35(19):2605 Ugel et al., Cancer Res. 2009;69(24):9376

Claims

1. 1. An in vitro method for producing an immunoreactive substance against human cancer cells infected with an HPV16-related virus, comprising expressing at least a partial nucleic acid sequence encoding an immunoreactive substance obtained from immune cells stimulated with a complex comprising a human leukocyte antigen (HLA) and a stimulatory peptide (stimulatory complex), The stimulatory peptide (i) consisting of an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, wherein the HLA is derived from HLA supertype HLA-A01; (ii) consisting of the amino acid sequence of SEQ ID NO: 11, wherein the HLA is derived from HLA supertype HLA-A02; (iii) consisting of an amino acid sequence selected from SEQ ID NOs: 34 to 37, wherein the HLA is derived from the HLA supertype HLA-A03 / A11; (iv) consisting of an amino acid sequence selected from SEQ ID NO: 60 and SEQ ID NO: 61, wherein the HLA is derived from HLA supertype HLA-A24; (v) consisting of an amino acid sequence selected from SEQ ID NO: 61, SEQ ID NO: 76, and SEQ ID NO: 77, wherein the HLA is derived from the HLA supertype HLA-B07; or (vi) SEQ ID NO: 61, and an amino acid sequence selected from SEQ ID NOs: 85 to 92, wherein the HLA is derived from the HLA supertype HLA-B15; The method further comprises verifying that an HPV16-derived peptide having the same amino acid sequence as the stimulatory peptide is presented by human HPV16-positive cancer cells.

2. 2. The method of claim 1, wherein the stimulatory peptide has been validated to stimulate human anti-stimulatory peptide immune cells when presented in a complex with a human leukocyte antigen (HLA).

3. The method of claim 1 or 2, wherein the stimulating complex is contained on an antigen-presenting cell (APC) or artificial APC that presents at least one of the stimulating complexes, or is an HLA multimer that contains the stimulating peptide.

4. The method according to any one of claims 1 to 3, wherein the immune cells are T cells that express a T cell receptor that recognizes the stimulatory complex and / or B cells that recognize the stimulatory complex.

5. The method of any one of claims 1 to 4, wherein the T cells are CD4+ or CD8+ T cells, preferably CD8+ T cells, preferably CD8+ effector memory T cells.

6. The method according to any one of claims 1 to 5, wherein the HPV16-related virus is HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 or HPV67, more preferably HPV16.

7. (A) contacting an immune cell with a complex (stimulatory complex) comprising a human leukocyte antigen (HLA) and a stimulatory peptide; (B) thereby stimulating immune cells that specifically bind to the human host cells. A method for stimulating immune cells that specifically bind to human host cells infected with an HPV16-related virus, comprising: the stimulating peptide selected from the list consisting of SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92; and it has been verified that an HPV16-derived peptide consisting of the same amino acid sequence as the stimulating peptide is presented by human HPV16-positive cancer cells.

8. The method of claim 7, which is a method for stimulating and identifying immune cells that specifically bind to the human host cells, further comprising the step of (C) identifying the stimulated immune cells stimulated in step (B).

9. 9. The method of claim 7 or 8, wherein the method is for stimulating and enriching immune cells that specifically bind to the human host cells, further comprising step (D) enriching the stimulated immune cells stimulated in step (B) and optionally identified in step (C).

10. 10. The method of claim 8 or 9, further comprising contacting the immune cells of step (B) with a derivative of the stimulating complex that carries a detectable label, and identifying and optionally enriching immune cells that bind to the stimulating complex by use of the detectable label.

11. The method of any one of claims 8 to 10, wherein (i) the method is an in vitro method, or (ii) step (A) comprises a step of administering the stimulatory complex to a subject, the subject is a non-human animal, and the subject is sacrificed after step (C) or (D).

12. 12. A method for determining the nucleic acid sequence of at least a portion of an immune reactant that specifically binds to a human host cell infected with an HPV16-related virus, the method comprising the steps of stimulating, identifying and optionally enriching immune cells that specifically bind to said human host cells according to the method of any one of claims 1 to 11, and the further step of sequencing at least a portion of at least one polynucleotide encoding said immune reactant of said immune cells.

13. 1. A method for producing an immunoreactive substance against a human host cell infected with an HPV16-related virus, comprising: (i) stimulating immune cells that specifically bind to human host cells infected with HPV16-related viruses according to the method of any one of claims 7 to 11; (ii) identifying and optionally enriching the stimulated immune cells of step (i), preferably according to the method of claim 12; (iii) determining the nucleic acid sequence of at least a portion of the immune reactants expressed by the immune cells identified and optionally enriched in step (ii); (iv) producing the immunoreactant by expressing at least a partial nucleic acid sequence encoding said immunoreactant, preferably in an expression system; A method comprising:

14. In vitro use of a stimulatory peptide consisting of an amino acid sequence selected from SEQ ID NOs: 1, 2, 11, 34-37, 60, 61, 76, 77, and 85-92, which has been validated to be presented by human HPV16-positive cancer cells and to stimulate human anti-stimulatory peptide immune cells when presented in a complex with human leukocyte antigen (HLA), for stimulating, identifying, and optionally enriching immune cells that specifically bind to HPV16-associated virus-infected human host cells.

15. The method of any one of claims 1 to 6 or 11 to 13, wherein the immunoreactive substance is a TCR, an anti-immune complex antibody, or a fragment or derivative thereof.

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