Validated HPV16-derived stimulating peptides

Abstract

The present invention relates to an in vitro method for preparing an immunoreactive agent against human cancer cells infected with an HPV16-associated virus, said method comprising expressing at least part of a nucleic acid sequence encoding an immunoreactive agent obtained from an immune cell stimulated by a complex comprising a human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), wherein the stimulating peptide (i) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and the HLA is from an HLA supertype HLA-A01; (ii) consists of an amino acid sequence of SEQ ID NO: 11, and the HLA is from an HLA supertype HLA-A02; (iii) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 34 to SEQ ID NO: 37, and the HLA is from an HLA supertype HLA-A03 / A11; (iv) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 60 and SEQ ID NO: 61, and the HLA is from an HLA supertype HLA-A24; (v) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 76 and SEQ ID NO: 77, and the HLA is from an HLA supertype HLA-B07; or (vi) consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 61 and SEQ ID NO: 85 to SEQ ID NO: 92, and the HLA is from an HLA supertype HLA-B15; and wherein HPV16-derived peptides consisting of the same amino acid sequence as the stimulating peptides have been demonstrated to be presented by human HPV16-positive cancer cells, and methods and uses related thereto.

Description

[0001] This invention relates to an in vitro method for preparing an immunoreactive agent against human cancer cells infected with HPV16-associated virus, the method comprising expressing at least a portion of a nucleic acid sequence encoding an immunoreactive agent, the immunoreactive agent being obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), wherein the stimulating peptide (i) comprises an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and the HLA is derived from HLA supertype HLA-A01; (ii) comprises an amino acid sequence selected from SEQ ID NO:11, and the HLA is derived from HLA supertype HLA-A02; (iii) comprises an amino acid sequence selected from SEQ ID NO:34 to SEQ ID NO:37, and the HLA is derived from HLA supertype HLA-A03 / A11; (iv) comprises an amino acid sequence selected from SEQ ID NO:60 and SEQ ID NO:61, and the HLA is derived from HLA supertype HLA-A24; (v) comprises an amino acid sequence selected from SEQ ID NO:61, SEQ ID NO:12, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, and the HLA is derived from HLA supertype HLA-A03 / A11; (iv) comprises an amino acid sequence selected from SEQ ID NO:60 and SEQ ID NO:61, and the HLA is derived from HLA supertype HLA-A24; (v) comprises an amino acid sequence selected from SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:35, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:60, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, SEQ ID NO:69, The HLA consists of the amino acid sequences of NO:76 and SEQ ID NO:77, and the HLA is derived from the HLA supertype HLA-B07; or (vi) it consists of the amino acid sequences selected from SEQ ID NO:61 and SEQ ID NO:85 to SEQ ID NO:92, and the HLA is derived from the HLA supertype HLA-B15; and it has been confirmed that the HPV16-derived peptide, which consists of the same amino acid sequence as the stimulating peptide, is presented by human HPV16-positive cancer cells, and methods and uses relating thereto are also involved.

[0002] At least 20% of malignant tumors are the result of persistent infections. Cancers caused by infectious agents are an attractive target for cancer vaccination methods because they offer the opportunity to target the immune system with non-self antigens. Vaccinations can be preventative, inducing an immune response to prevent infection in the first place, or therapeutic, stimulating the immune system to eradicate established disease. Prophylactic immunization against certain high-risk human papillomavirus (HPV) types or against hepatitis B has become a paradigm for cancer immunoprophylaxis.

[0003] Regarding therapeutic vaccination, HPV-associated tumor formation may again serve as a model case: the induction and maintenance of a malignant phenotype depends on two viral oncoproteins, E6 and E7. Studies of spontaneously regressing HPV-induced lesions, and the prevalence of HPV in immunosuppressed and HIV-infected patients, suggest that cell-mediated immune responses are crucial for clearing established HPV infection. Various forms of vaccines, including peptide vaccines, that target E6 and E7 with T cells have been explored.

[0004] The literature presents a rich list of candidate peptides for anti-HPV vaccination, such as those from 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.

[0005] 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, regarding peptide vaccines, it must be recognized over the years that the process leading to an effective T cell response is complex and requires more than just the potential binding of the peptide to the MHC. In the case of MHC class I molecule presentation, the precursor peptide must be cleaved by the proteasome of the presenting cell to provide the peptide in the first place; in addition, T cell activation may require cross-presentation of peptides on MHC class I molecules via APCs, which may cleave the precursor peptide in a manner different from that of non-APC cells. Furthermore, the presence of T cells with a predisposition to recognize the presented peptide is essential, which hinges on the availability of a T cell receptor repertoire (see, for example, Becker & Riemer (2022), Frontiers Immunol, doi:10.3389 / fimmu.2022.883989; Habib et al. (2022), Cells 6; 11(3):421. doi:10.3390 / cells11030421.). Similarly, in cancer cells, immune evasion mechanisms may prevent the proper presentation of HPV-derived peptides (Steinbach & Riemer (2018), Int J Cancer 142:224). In conclusion, unfortunately, the experimental binding of a candidate peptide to an MHC class I molecule does not alone make the candidate peptide a plausible candidate for a vaccine.

[0006] Given the above, it is clear that validating candidate HPV peptides is far from suitable for vaccination: the amount of peptides available in presentation assays is extremely limited, and for many peptides, the fragmentation pattern in mass spectrometry is complex. Thus, in a 2010 study (Riemer et al. (2010), loc.cit.), only one of the 21 peptides predicted to be presented via HLA-A*0201 was confirmed. After eight years of method development, 17 peptides were confirmed from a predicted pool of 121 potential HLA-A2 binding peptides (Blatnik et al. (2018), loc.cit.). Notably, cysteine-containing peptides were completely excluded, as is common in the art, because they readily undergo intramolecular and intermolecular reactions, complicating the analysis and further reducing the amount of defined and detectable ions available for MS analysis. If HPV peptides cannot be identified as suitable by the aforementioned in vitro methods, the only remaining alternative is in vivo testing, which requires extended animal and / or human testing via vaccination.

[0007] Furthermore, for successful therapeutic HPV vaccination, not only must the injected peptide be immunogenic, such as activating T cells upon presentation via MHC molecules, but the peptide must also be presented by target cells (i.e., cells infected with HPV). As Riemer et al. ((2010), loc.cit.) pointed out, defining the HPV-16E6 and E7 T cell epitopes that are naturally processed and presented on the surface of virus-modified cells is crucial for vaccination. Only those HPV peptide / MHC class I complexes that can be recognized by cytotoxic T lymphocytes can target and destroy transformed cells. Therefore, the success of therapeutic HPV vaccines depends on the accurate identification of HPV epitopes displayed on HPV-infected cells. In addition to directly demonstrating MHC presentation through MS analysis, studying memory responses in healthy donors can serve as an alternative test for presentation. If a memory immune response is present, the epitope in question must have been presented during a previous exposure to the virus. However, even demonstrating innate immunogenicity is insufficient to validate the peptide as an actionable target within the context of its restricted HLA complex. This is because APCs use a different proteasome than normal somatic cells, the immunoproteasome, and antigen processing in HPV16-infected / transformed cells is heavily regulated by the virus. Therefore, a memory T cell response only demonstrates that the peptide was previously presented by antigen-presenting cells (APCs), and does not prove that the epitope will still be presented on HPV-dependent tumor cells. Thus, not all possible HPV epitopes will be presented on HPV-positive target cells.

[0008] Therefore, in order to become an effective target, HPV16-derived peptides must bind to HLA molecules, be immunogenic, and be presented on target cells, especially on cells infected or transformed by HPV16.

[0009] Therefore, there is a need in the art for improved means and methods for immune responses against cells transformed by HPV (e.g., HPV-positive cancer cells), and related reagents, without the disadvantages described above.

[0010] The technical problem upon which this invention is based can be viewed as providing means and methods to satisfy the aforementioned needs. This technical problem is solved through the claims and the embodiments described below.

[0011] Accordingly, the present invention relates to a method for stimulating immune cells that specifically bind to human host cells infected with HPV16-associated viruses, the method comprising:

[0012] (A) Contacting a complex containing human leukocyte antigen (HLA) and a stimulating peptide (the stimulating complex) with immune cells, and

[0013] (B) This stimulates immune cells that specifically bind to host cells infected with HPV16-associated viruses.

[0014] The stimulating peptide is composed of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111, and in a preferred embodiment, is composed of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:129.

[0015] The method for stimulating immune cells is preferably an in vitro method, which can, preferably, be performed on a separated sample of the subject. However, the method can also, preferably, be performed in vivo, and more preferably in non-human laboratory animals. The method may include further steps in addition to those explicitly defined, and may be assisted or performed by automated equipment.

[0016] Generally, the terms used herein should be given their common and customary meanings to those skilled in the art, and should not be limited to specific or customary meanings unless otherwise stated. As used below, the terms “have,” “comprise,” or “include,” or any variations thereof, are used in a non-exclusive manner. Thus, these terms can refer either to a situation where no other features exist in the entity described in this context besides the features introduced by these terms, or to a situation where one or more other features exist. For example, the statements “A has B,” “A contains B,” and “A includes B” can refer either to a situation where no other element exists in A besides B (i.e., A consists only of B), or to a situation where one or more other elements exist in entity A besides B, such as element C, elements C and D, or even more elements. Similarly, as those skilled in the art will understand, elements appearing in the context of “comprise” and “include” without a quantifier preferably mean “comprises one or more,” which is equivalent to “comprises at least one.” Accordingly, unless otherwise stated, a statement involving one of a plurality of items preferably involves at least one such item, more preferably involving a plurality of such items; thus, for example, identifying a “cell” involves identifying at least one type of cell, preferably identifying a plurality of types of cells.

[0017] Furthermore, as used below, the terms “preferred,” “more preferably,” “most preferably,” “particularly,” “more particularly,” “specifically,” “more specifically,” or similar terms are used in combination with optional features without limiting further possibilities. Therefore, features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As those skilled in the art will recognize, the invention can be practiced by using alternative features. Similarly, features introduced by “in one embodiment” or similar expressions are intended to be optional features, without limiting other embodiments of the invention, without limiting the scope of the invention, and without limiting the possibility of combining features introduced in this way with other optional or non-optional features of the invention.

[0018] The methods described below are preferably in vitro methods. These method steps can, in principle, be performed in any arbitrary order that a person skilled in the art deems suitable, but are preferably performed in an indicated order; and one or more, preferably all, of the steps can be assisted or performed by automated equipment. Furthermore, the methods may include steps other than those explicitly mentioned above.

[0019] As used herein, unless otherwise stated, the term "about" refers to an indication value having technical precision recognized in the relevant art, preferably ±20%, more preferably ±10%, and most preferably ±5%. Furthermore, the term "substantially" means that there is no deviation affecting the indication result or its use, i.e., potential deviations will not cause the indication result to deviate from the specified value by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially composed of" means including the specified components but excluding other components, except for substances present as impurities, substances unavoidably present due to the process used to provide the components, and components added for purposes other than achieving the technical effects of the invention. For example, compositions defined using the phrase "substantially composed of" encompass any known and acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition substantially composed of a group of components will contain 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 one or more unspecified components.

[0020] The degree of identity between two biological sequences (e.g., expressed as "% identity"), preferably between DNA sequences, RNA sequences, or amino acid sequences, can be determined using algorithms known in the art. Preferably, the degree of identity is determined by comparing two sequences that are optimally aligned on a comparison window, wherein fragments of the sequence in the comparison window may contain additions or deletions (e.g., vacancies or single-stranded overhangs) compared to the sequence being compared for optimal alignment. This percentage is calculated by determining, preferably over the entire length of the polynucleotide or polypeptide, the number of positions of the same residues appearing in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), the computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given two identified sequences for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment, thereby determining the degree of identity. Preferably, a default value of 5.00 for the vacancy weight and 0.30 for the vacancy weight length is used. In the context of biological sequences discussed herein, the term "substantially identical" means a % 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, "substantially identical" means 100% identical. The above, with necessary modifications, applies to the term "substantially complementary".

[0021] The term "fragment" is used herein in its broad sense when referring to a biomacromolecule, preferably a polynucleotide or polypeptide, to refer to any sub-part, preferably a subdomain, of the corresponding biomacromolecule that contains the indicated sequence, structure, and / or function. Thus, the term includes sub-parts resulting from the actual fragmentation of a biomacromolecule, but also includes sub-parts derived from the corresponding biomacromolecule in an abstract manner, such as in silico. Therefore, as used herein, Fc fragments or Fab fragments, and for example, single-chain antibodies, bispecific antibodies, and nanobodies, may be referred to as fragments of immunoglobulins.

[0022] Unless otherwise specifically stated herein, the defined compounds, particularly polynucleotides and (poly)peptides, may be contained in larger structures, for example, covalently or non-covalently linked to other sequences, carrier molecules, blocking agents, and other excipients. In particular, the defined peptides and (poly)peptides may be contained in fusion polypeptides comprising other peptides, which may be used, for example, as tags for purification and / or detection, as linkers, or for extending the in vivo half-life of the compound. The term "detectable tag" refers to an amino acid segment added to or introduced into a fusion polypeptide; preferably, the tag is added to the fusion polypeptide at the C-terminus or N-terminus. The amino acid segment preferably allows detection of the polypeptide by an antibody that specifically recognizes the tag; or it preferably allows the formation of a functional conformation, such as a chelating agent; or it preferably allows visualization, such as in the case of a fluorescent tag. Preferred detectable tags are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags, or fluorescent protein tags, such as GFP tags. These tags are well known in the art. Other peptides preferably included in the fusion polypeptide include other amino acids or other modifications that can function as secretion mediators, blood-brain barrier channel mediators, cell-penetrating peptides, and / or immunostimulants. Other polypeptides or peptides that the polypeptide may fuse with are signaling sequences and / or transport sequences, such as IL-2 signaling sequences and adapter sequences.

[0023] The term "peptide," as used herein, refers to a molecule consisting of several amino acids, typically at least 20 amino acids, covalently linked together by peptide bonds. Molecules consisting of fewer than 20 amino acids covalently linked by peptide bonds are generally considered "peptides." The peptides mentioned herein are disclosed as SEQ ID NO:1 to SEQ ID NO:111, and as shown in Table 1 below. As understood by those skilled in the art from the disclosure herein, the stimulating peptides described herein are preferably used in conjunction with the HLA supertypes shown in Table 1. Therefore, the immune peptide / HLA supertype combinations of Tables 1(i) to 1(vi) are preferred.

[0024] The term "polynucleotide" is known to those skilled in the art. As used herein, the term includes nucleic acid molecules comprising or constituting one or more nucleic acid sequences as specified herein and / or encoding at least one stimulating peptide. The polynucleotides of the present invention should preferably be provided as isolated polynucleotides (i.e., isolated from their natural environment) or in genetically modified form. Polynucleotides are preferably DNA, including cDNA, or RNA. The term encompasses single-stranded and double-stranded polynucleotides, as well as circularly closed and linear polynucleotides. Preferably, the polynucleotide is a chimeric molecule, i.e., preferably comprising at least one nucleic acid sequence, preferably at least 20 bp, more preferably at least 100 bp, which is heterologous to or an artificial nucleic acid sequence with the remaining one or more nucleic acid sequences. Furthermore, preferably, it also includes chemically modified polynucleotides, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides such as biotinylated polynucleotides.

[0025] Polynucleotides may be included in the expression construct. The term "expression construct," as used herein, refers to a heterologous polynucleotide containing the aforementioned polynucleotide and the nucleic acid sequence required for expression of that polynucleotide. Typically, this additional nucleic acid sequence, preferably heterologous to the polynucleotide encoding the at least one stimulating peptide, can 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 containing all elements required for expression, preferably induced expression, in a eukaryotic host cell. Suitable expression control sequences are well known in the art and particularly include CMV promoters or other constitutive promoters. However, inducible and / or cell-type-specific promoters may also be used.

[0026] Polynucleotides and / or expression constructs may be contained in vectors. The term "vector," as used herein, refers to any polynucleotide suitable for stably maintaining the polynucleotides and / or expression constructs as defined above in host cells. Therefore, the term vector preferably encompasses bacteriophage, plasmid, and viral vectors, as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Preferably, the vector is a plasmid or viral-derived vector, preferably a replication-defective viral vector. Furthermore, the term also refers to a targeting construct that allows random or site-specific integration of the targeting construct into the host cell's genomic DNA. Such a targeting construct preferably contains DNA of sufficient length for homologous or heterologous recombination. Vectors encompassing polynucleotides and / or expression constructs as defined above preferably also contain at least one selective marker for host cell proliferation and / or selection. Vectors can be incorporated into host cells using various techniques known in the art. For example, plasmid vectors can be introduced in precipitates such as calcium phosphate or rubidium chloride precipitates, in complexes with charged lipids, or in carbon-based clusters such as fullerenes. Alternatively, plasmid vectors can be introduced using heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line before application 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.

[0027] Methods for determining immune cell stimulation are known in the art and are described elsewhere herein, particularly in the examples. Preferably, the stimulation is a significant stimulus compared to an untreated control and / or a vector control.

[0028] The terms "human papillomavirus 16-associated virus" and "HPV16-associated virus" refer to any virus, preferably a human-infecting virus, that encodes at least one amino acid sequence from SEQ ID NO:1 to SEQ ID NO:111. Preferably, the HPV16-associated virus is a papillomavirus (PV), more preferably a human papillomavirus (HPV), and even more preferably selected from HPV16, HPV31, HPV33, HPV35, HPV52, HPV58, and HPV67, with HPV16 being the most preferred.

[0029] The term "object," as used herein, refers to vertebrates, preferably mammals, more preferably humans. Preferably, the object contains at least one HLA type as specified in Table 1 below. Preferably, the object is infected with at least one HPV16-associated virus. Preferably, the object suffers from at least one HPV16-associated virus-positive abnormal cell proliferation.

[0030] The term "abnormal cell proliferation" refers to the abnormal proliferation of somatic cells in a subject; its consequences may include an imbalance in the cellular composition of body tissues and fluids and / or tumor formation. Abnormal cell proliferation may be induced by infectious agents, preferably viruses, more preferably HPV16-associated viruses. Preferably, the abnormal cell proliferation is benign, i.e., preferably does not threaten the health or life of the subject. Preferred benign abnormal cell proliferations are warts, exophytic papillomas, condyloma, inverted papillomas, and pre-neoplastic HPV-induced lesions. Also preferably, the abnormal cell proliferation is pre-malignant or malignant, i.e., at least potentially threatening the health or life of the subject; therefore, preferably, the abnormal cell proliferation is pre-malignant or malignant abnormal proliferation of mucous membranes or skin, especially mucous membranes, such as the oropharynx, anogenital region, and / or reproductive organs, such as cervical intraepithelial neoplasia (CIN) or cancer, especially cervical cancer and / or head and neck cancer. The term "cancer," as used herein, refers to a disease in animals (including humans) characterized by the uncontrolled growth of a group of somatic cells ("cancer cells"). This uncontrolled growth may be accompanied by invasion and destruction of surrounding tissues and may spread the cancer cells to other parts of the body. Preferably, the term cancer also includes recurrence. Thus, preferably, cancer is a solid tumor, metastasis, or recurrence thereof. Thus, preferably, inducing an immune response is cancer prevention and / or cancer treatment.

[0031] The term "stimulating peptide," as used herein, refers to any peptide consisting of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111. As those skilled in the art will understand from the examples provided herein, the aforementioned peptides have been shown to activate human T cells when presented as a human leukocyte antigen (HLA) complex to human T cells. Preferably, the stimulating peptide consists of an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:85 to SEQ ID NO:92, which, as shown in the examples below, have been additionally demonstrated to be presented by human cancer cells infected with HPV-16-associated virus. Therefore, the stimulating peptide composed of the amino acid sequences selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:85 to SEQ ID NO:92 has been shown to be presented by human cancer cells infected with HPV-16-associated virus and has been shown to activate human T cells when presented as an HLA complex. As will be understood by those skilled in the art, human cancer cells infected with HPV-16-associated virus may lose a portion of the HPV genome; therefore, as used herein, preferably, any cancer cell expressing at least the HPV16-associated E6 and E7 peptides is a human cancer cell infected with HPV-16-associated virus.

[0032] In a preferred embodiment, the immune peptide consists of an amino acid sequence containing at least one, preferably at least two, more preferably three cysteine ​​residues.Therefore, in a preferred embodiment, the immune peptide is selected from SEQ ID NO:1, SEQ ID NO:3 to SEQ ID NO:5, SEQ ID NO:7 to SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 to SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37 to SEQ ID NO:40, SEQ ID NO:42 to SEQ ID NO:46, SEQ ID NO:48 to SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:55 to SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:62 to SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82 to SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82 to SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82 to SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:89 ... NO:91 to SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:104 to SEQ ID NO:106, SEQ ID NO:112 to SEQ ID NO:115, SEQ ID NO:117 to SEQ ID NO:120, SEQ ID NO:122 to SEQ ID NO:124, and SEQ ID NO: 127, more preferably selected from SEQ ID NO: 4, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 42 to SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 55 to SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:84, SEQ ID NO:99, SEQ ID NO:106, SEQ ID NO:115, SEQ ID SEQ ID NO:118 and SEQ ID NO:120, or even more preferably selected from SEQ ID NO:57, SEQ ID NO:62, SEQ ID NO:69, SEQ ID NO:84 and SEQ ID NO:120.In a preferred embodiment, the immunopeptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A01, preferably selected from SEQ ID NO:1, SEQ ID NO:3 to SEQ ID NO:5, SEQ ID NO:7 to SEQ ID NO:10, and SEQ ID NO:112 to SEQ ID NO:115; in an even preferred embodiment, the immunopeptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A02, preferably selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 to SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, and SEQ ID NO:31; in an even more preferred embodiment, the immunopeptide comprises an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A03, preferably selected from SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:115. SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:117 and SEQ ID NO:118; In a further preferred embodiment, the immunopeptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A11, preferably selected from SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:42 to SEQ ID NO:45; In a further preferred embodiment, the immunopeptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-A11.

[0033] HLA-A24 presentation, preferably selected from SEQ ID NO:60, SEQ ID NO:62 to SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:71 and SEQ ID NO:119; in a further preferred embodiment, the immunopeptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-B07, preferably selected from SEQ ID NO:12, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82 to SEQ ID NO:84, and SEQ ID NO:122 to SEQ ID NO:124; in a further preferred embodiment, the immunopeptide consists of an amino acid sequence containing at least one cysteine ​​residue and is presented via HLA-B15, preferably selected from SEQ ID NO:9, SEQ ID NO:43, SEQ ID NO:64, SEQ ID NO:80, SEQ ID NO:86, SEQ ID NO:91 to SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:104 to SEQ ID NO:119. NO:106 and SEQ ID NO:127. In a further preferred embodiment, the immunopeptide consists of an amino acid sequence containing at least one cysteine ​​residue and is a peptide demonstrated herein to be present on cancer cells infected with HPV16, preferably selected from SEQ ID NOs:1, SEQ ID NO:11, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:86, SEQ ID NO:91 and SEQ ID NO:92.

[0034] The term "human leukocyte antigen," also known as "HLA," is understood by those skilled in the art to refer in principle to a family of cell surface proteins involved in the presentation of peptides to immune cells. HLA is also referred to as the major histocompatibility complex (MHC), with HLA A, HLA B, and HLAC corresponding to MHC class I, and HLA DP, DM, DQ, and DR corresponding to MHC class II. As will be understood from Table 1 provided below, the stimulating peptides mentioned herein bind to HLA A, HLA B, and HLA C, so HLA is preferably HLA A, HLA B, or HLA C, i.e., MHC class II. The term HLA is used herein in its broad sense, referring to any HLA molecule or fragment thereof having the activity of presenting stimulating peptides to immune cells, preferably such that homologous T cells are activated by a complex containing the HLA and the stimulating peptide. Thus, HLA can be, for example, soluble HLA or HLA-oligomers, with the term "HLA-oligomer" referring to the oligomeric form of soluble HLA. In HLA-oligomers, a soluble, non-membrane-bound form of HLA is used; furthermore, HLA is used in oligomeric form, and the term "oligomer" preferably refers to at least a dimer form, more preferably at least a trimer form; preferably, the oligomer contains two to eight HLA molecules, more preferably three to six HLA molecules, and most preferably four HLA molecules. Therefore, HLA oligomers are preferably HLA-tetramers. Preferably, the HLA molecules in the HLA-oligomer are linked by a biotin-streptavidin affinity pair. Preferably, the HLA-oligomer contains a detectable marker, such as a fluorescent marker. Suitable methods for providing HLA-oligomers are known in the art, for example from Altman et al. (1996), Science 274:94. Furthermore, HLA-oligomers are commercially available.

[0035] As used herein, the term "stimulatory complex" refers to any complex comprising human leukocyte antigen (HLA) and a stimulating peptide, both as specified above. Stimulatory complexes may be provided as soluble stimulatory complexes, which may optionally contain other components, such as co-stimulatory molecules, or may be contained on host cells, such as antigen-presenting cells loaded with the stimulating peptide in vitro; synthetic APCs or scaffolds mimicking the same function may also be used. Immune complexes may also be generated on host cells by contacting the host cells with the administered stimulating peptide. Since the stimulating peptide is preferably presented via MHC class I, the host cell in this case can, in principle, be any host cell expressing MHC class I. Preferred stimulatory complexes are those comprising the stimulating peptide / HLA combination as shown in Table 1, and preferably those designated as complex ID NO:1, complex ID NO:2, complex ID NO:11, complex ID NO:34 to complex ID NO:38, complex ID NO:61, complex ID NO:62, complex ID NO:78 to complex ID NO:80, and complex ID NO:89 to complex ID NO:97.

[0036] The term "immune cell," as used herein, includes any and all cells capable of recognizing, and preferably specifically binding, the stimulatory complexes specified elsewhere herein. Therefore, 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, said T cells being preferably CD8+ T cells, more preferably cytotoxic CD8+ T cells.

[0037] The term "stimulation of immune cells" is understood by those skilled in the art and preferably includes an increase in at least one characteristic of activated immune cells. Thus, the term encompasses both initiating a de novo response and activating or enhancing an existing immune response. Stimulation of immune cells preferably involves inducing or enhancing a T-cell response to a stimulating complex, most preferably a CD8+ T-cell response; also preferably, stimulation of immune cells involves inducing or enhancing a B-cell response to a stimulating complex, particularly the production of antibodies against said immune complex. Accordingly, stimulation of immune cells preferably includes increasing the number of immune cells in the subject that specifically recognize said stimulating complex, wherein; preferably, said immune cells are T cells, preferably CD8+ T cells, more preferably cytotoxic CD8+ T cells. Therefore, the stimulating peptide is preferably presented via major histocompatibility complex (MHC) class I molecules, preferably on nucleated cells, more preferably on antigen-presenting cells and / or on nucleated cells infected with HPV16-associated virus, such as improperly proliferating HPV16-associated virus-positive cells, particularly HPV-associated lesions or HPV-associated cancers as defined above. Therefore, stimulating immune cells preferably includes inducing an immune response to the proliferation of inappropriate HPV16-associated virus-positive cells. Preferably, stimulating immune cells includes presenting a stimulating peptide on at least one HLA type belonging to one of 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 specific stimulating peptides in Table 1. Preferably, stimulating immune cells includes administering an agent to a subject that provides the stimulating peptide as specified above, wherein the subject is preferably a non-human animal and preferably euthanized after the stimulation, preferably after step (C) or step (D) specified below. However, stimulating immune cells may also involve in vitro activation and / or enrichment of immune cells that recognize and / or bind to the stimulating complex, optionally followed by further action.

[0038] As used herein, the term "host cell" refers to any cell capable of receiving and presenting the stimulating peptide via HLA. Preferably, the host cell is a eukaryotic cell, more preferably an animal cell, such as an insect cell or a mammalian cell. More preferably, the host cell is a cell of a livestock, companion animal, or laboratory animal. Most preferably, the host cell is a human cell.

[0039] Preferably, the method is a method for stimulating and recognizing immune cells that specifically bind to the stimulating complex, preferably specifically bind to human host cells infected with HPV16-associated virus; in this case, the method preferably further includes (C) recognizing the stimulated immune cells stimulated in step (B).

[0040] The term "recognition" of immune cells is used herein in its broad sense, referring to any method that distinguishes immune cells that recognize the stimulating complex from those that do not. Suitable methods are known in principle to those skilled in the art, and preferably include contacting immune cells stimulated as defined above with a derivative of the stimulating complex carrying a detectable label, wherein the term "derivative of the stimulating complex carrying a detectable label" refers to the stimulating complex, preferably the stimulating complex used in step (B) as defined above, which is covalently or non-covalently linked to the detectable label. The detectable label can be any label that those skilled in the art deem suitable. Preferably, the "detectable label" is a label detectable by optical means, which are known in principle to the art. More preferably, the detectable label is an optically detectable polypeptide, more preferably a fluorescent group, such as a fluorescent dye.

[0041] Preferably, the method is a method for stimulating and enriching immune cells that specifically bind to the stimulating complex, preferably specifically bind to human host cells infected with HPV16-associated virus, and wherein the method further includes step (D) enriching the stimulated immune cells stimulated in step (B) and optionally identified in step (C).

[0042] The term "enrichment," as used herein, refers to any method of increasing the number of immune cells in a formulation that recognize immune complexes relative to those that do not. Suitable methods are known in the art and particularly include methods comprising contacting stimulated immune cells with a derivative of a stimulating complex carrying a detectable label as specified above and enriching immune cells bound to the stimulating complex by said detectable label. Therefore, the term enrichment is used herein in its conventional sense. In the context of enriching cell types, the term preferably refers to increasing the proportion of cell types enriched in the composition relative to other present cell types. Methods for enriching specific cell types generally involve labeling the cell type of interest and applying appropriate enrichment methods, such as FACS and / or affinity-based binding to a solid surface. As will be understood by those skilled in the art, enrichment may also include labeling undesired cell populations and removing such labeled cells. Using similar methods, T cells expressing CD3, CD8, CD137, and / or IFN-γ in addition to expressing T cell receptors that recognize stimulating peptides can also be enriched.

[0043] Advantageously, in the work upon which this invention is based, it was discovered that HLA presentation of HPV16-derived peptides by HPV16-positive cells can be assessed by mass spectrometry as disclosed in the examples, providing experimental evidence that the peptides are indeed presented to the immune system on the intended target cells. Furthermore, immunogenicity assays performed on peripheral blood lymphocytes (PBMCs) from various donors, as shown in the examples, can provide evidence that the corresponding peptides are indeed active in inducing an immune response in vivo. Therefore, in order to provide actionable target structures for T-cell epitope-based immunotherapies against HPV16-mediated malignancies (therapeutic vaccines, adoptive transfer of T cells with transgenic TCRs that recognize validated HLA / peptide complexes, or therapies based on antibodies that recognize validated HLA / peptide complexes (i.e., CAR-T cells or bispecific antibodies that bring target and effector cells into close proximity)), it has been found meaningful to validate T-cell epitope / HLA complexes as actionable targets to ensure the efficacy of the aforementioned therapies. Preferably, using HLA binding prediction algorithms to define actionable targets may be insufficient, as these algorithms are not precise enough and may miss many binding peptides (see Table 2). Therefore, not only has the HLA binding of each stimulating peptide mentioned herein been experimentally validated, but the T-cell memory response of the stimulating peptides has also been assessed using PBMCs from healthy human donors. Immunogenic peptides thus defined must have undergone natural processing and presentation in a prior HPV16 infection. Furthermore, it is preferable to validate the HLA presentation of the putative stimulating peptides directly on tumor (or other HPV-positive) cells. This can be achieved using mass spectrometry-based techniques, immunopeptidomics. Therefore, in particular, the combination of the above two methods can identify promising candidate peptides for inducing cellular immune responses against cells infected with HPV16-associated viruses and the resulting inappropriate cell proliferation.

[0044] The definitions made above, with necessary modifications, apply hereinafter. Additional definitions and interpretations made hereinafter, with necessary modifications, also apply to all embodiments described in this specification.

[0045] The present invention also relates to a method for determining at least a portion of the nucleic acid sequence of an immune response agent that specifically binds to a stimulating complex, preferably specifically binds to human host cells infected with HPV16-associated virus, the method comprising the further steps of stimulating, identifying and optionally enriching immune cells that specifically bind to a stimulating complex, preferably specifically bind to host cells infected with HPV16-associated virus, according to the methods specified above, and sequencing at least a portion of at least one polynucleotide encoding the immune response agent in the immune cells.

[0046] The method of the present invention for determining nucleic acid sequences is an in vitro method. Furthermore, it may include additional steps beyond those explicitly mentioned above. In particular, the obtained sequence information can be used to construct expression constructs expressing at least one strand of an immunoreaction reagent that recognizes the stimulating peptide. Moreover, one or more of the steps may be assisted or performed by automated equipment.

[0047] The term "determining the sequence of at least a portion of the immunoreaction reagent" is understood by those skilled in the art. Sequencing can be performed by any sequencing method deemed suitable by those skilled in the art. Preferably, the sequencing uses mRNA or cDNA encoding at least one polypeptide contained in the immunoreaction reagent as a template. As is known to those skilled in the art, the immunoreaction reagent may consist of one or more polypeptides, preferably as specified above, and therefore it may be necessary to determine more than one polynucleotide encoding the immunoreaction reagent. Preferably, the sequenced portion of the polynucleotide encoding the immunoreaction reagent, preferably a T-cell receptor, contains at least CDR3, preferably at least CDR; therefore, preferably, at least CDR1 to CDR3 of each strand of the immunoreaction reagent are sequenced. However, sequencing of variable domains of the immunoreaction reagent, or sequencing of two or more complete strands of the immunoreaction reagent, is also contemplated.

[0048] The term "immunoreactive agent," as used herein, refers to every molecule that recognizes, i.e., preferably specifically binds to the stimulating complex, and more preferably specifically binds to human host cells infected with HPV16-associated virus. Therefore, an immunoreactive agent can be, for example, a T-cell receptor or an immunoglobulin.

[0049] As used herein, the term "T-cell receptor" refers to a receptor present on T cells that mediates T-cell antigen recognition, consisting of an α-chain and a β-chain or a γ-chain and a δ-chain, preferably consisting of an α-chain and a β-chain. The structure of the T-cell receptor is known to those skilled in the art; preferably, each chain of the T-cell receptor comprises a cytoplasmic domain, a transmembrane domain, a constant domain, and a variable domain, wherein the variable domain confers specificity for antigens. As used herein, if a cytoplasmic domain is present, it may be referred to together with the transmembrane domain and the constant domain as the "non-variable domain" of the T-cell receptor chain. Within the variable domain, antigen recognition is essentially determined by complementarity-determining regions (CDRs), referred to as CDR1, CDR2, and CDR3. As is known to those skilled in the art, unlike, for example, immunoglobulins, in the T-cell receptor, only the CDR3s of two TCR chains, namely, for example, the CDR3 of the α-chain and the CDR3 of the β-chain, contact the epitope presented by the MHC, while the other two CDRs contact essentially only the MHC. Therefore, to obtain information about the structural basis of T cell receptor antigen specificity, it is preferably sufficient to obtain the sequence information of CDR3 for 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 other polypeptides, referred to as the "T cell receptor complex". The T cell receptor complex preferably contains at least one of CD3δ, CD3γ, CD3ε, and CD3ζ in addition to the T cell receptor. Preferably, the T cell receptor complex is associated with CD8 as a co-receptor. The amino acid numbering and substructure identification of the TCR preferably follow the recommendations of the international ImMunoGeneTics (IMGT) database (Lefranc et al. (2003), Dev. Comp. Immunol., 27:55-77), which identifies conserved amino acids that are substantially always in the same position in the TCR sequence.

[0050] As used herein, the term "immunoglobulin," also known as "antibody," refers to any cell-binding or soluble immunoglobulin or fragment thereof from any class of IgA, IgD, IgE, IgG, or IgM that recognizes the stimulatory complex as defined above. Antibodies against a predetermined antigen can be prepared by known methods. Preferably, the immunoglobulin comprises six complementarity-determining regions (CDRs). The CDRs of the antibody are preferably different from those of the TCRs and are preferably regions in the variable structural domains of the antibody heavy and light chains that determine antibody binding affinity and specificity. The heavy chain has three CDRs, CDR1-H, CDR2-H, and CDR3-H, and the light chain has three CDRs, CDR1-L, CDR2-L, and CDR3-L. Preferably, determining at least a portion of the nucleic acid sequence of the immunoglobulin includes determining the sequence encoding at least the CDRs, preferably all six CDRs.

[0051] As those skilled in the art will understand, sequence information regarding the CDR of an antibody (e.g., a monoclonal antibody) can be used to provide primate-derived, chimeric, or humanized antibodies, or fragments thereof. Single-chain antibodies, single-domain antibodies, nanobodies, or antibody fragments such as Fab, scFab, etc., can also be provided. The antibodies of this invention also include bispecific or trispecific antibodies, synthetic antibodies, or any chemically modified derivatives of the aforementioned antibodies. The above information can also be used to provide CAR T cells carrying sequences that recognize the stimulus complex, for example, as single-chain antibodies.

[0052] The present invention also relates to a method for preparing an immunoreactive agent against human host cells infected with HPV16-associated virus, the method comprising:

[0053] (i) Stimulate immune cells that specifically bind to human host cells infected with HPV16-associated viruses according to the methods specified above;

[0054] (ii) Identify and optionally enrich the stimulated immune cells of step (i), preferably according to the method specified above;

[0055] (iii) Determine at least a portion of the nucleic acid sequence of the immune response agent expressed by the immune cells identified and optionally enriched in step (ii), and

[0056] (iv) The immune response agent is prepared by expressing at least a portion of the nucleic acid sequence encoding the immune response agent, preferably in an expression system.

[0057] Therefore, the present invention also relates to a method for preparing an immunoreactive agent against human host cells infected with HPV16-associated virus, the method comprising expressing at least a portion of a nucleic acid sequence encoding the immunoreactive agent, said immunoreactive agent being obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), wherein

[0058] The stimulating peptide consists of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111, and in a preferred embodiment, consists of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:129.

[0059] Therefore, the present invention also relates to an in vitro method for preparing an immunoreactive agent against human cancer cells infected with HPV16-associated virus, the method comprising expressing at least a portion of a nucleic acid sequence encoding the immunoreactive agent, said immunoreactive agent being obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), wherein said stimulating peptide

[0060] (i) It consists of an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and the HLA is derived from the HLA supertype HLA-A01;

[0061] (ii) Composed of the amino acid sequence of SEQ ID NO:11, and the HLA is derived from the HLA supertype HLA-A02.

[0062] (iii) Composed of an amino acid sequence selected from SEQ ID NO:34 to SEQ ID NO:37, and the HLA is derived from the HLA supertype HLA-A03 / A11;

[0063] (iv) Composed of an amino acid sequence selected from SEQ ID NO:60 and SEQ ID NO:61, and the HLA is derived from the HLA supertype HLA-A24;

[0064] (v) Composed of an amino acid sequence selected from SEQ ID NO:61, SEQ ID NO:76, and SEQ ID NO:77, and the HLA is derived from the HLA supertype HLA-B07; or

[0065] (vi) It consists of an amino acid sequence selected from SEQ ID NO:61 and SEQ ID NO:85 to SEQ ID NO:92, and the HLA is derived from the HLA supertype HLA-B15;

[0066] Furthermore, it has been confirmed that HPV16-derived peptides, consisting of the same amino acid sequence as the stimulating peptide, are presented by human HPV16-positive cancer cells.

[0067] In the context of expressing a nucleic acid sequence, the term "expression" will be understood by those skilled in the art. Therefore, expressing at least a portion of a nucleic acid sequence encoding an immunoreactive agent preferably involves causing the expression of said nucleic acid sequence, for example, causing the expression of said nucleic acid sequence in suitable expression cells. The term "expression cell," as used herein, refers to any cell capable of expressing at least a portion of a nucleic acid sequence encoding an immunoreactive agent, for example, encoded by the expression construct or vector defined above. Therefore, expression cells are preferably bacterial cells, such as E. coli cells, cells of single-celled eukaryotes such as yeast cells, fungal cells, or insect cells, or mammalian cells, preferably cultured in vitro. Also preferably, expression is in vitro expression in an in vitro translation system. More preferably, expression is in vivo expression in cells.

[0068] The present invention also relates to immune cells expressing an immune response agent that specifically binds to host cells infected with HPV16-associated virus, wherein the immune cells are obtained by or can be obtained by the method specified above.

[0069] The present invention also relates to an immunoreactive agent for human cancer cells infected with HPV16-associated virus, said immunoreactive agent specifically binding to a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), said stimulating peptide comprising an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111, said immunoreactive agent preferably prepared according to methods specified elsewhere herein or may be prepared according to methods specified elsewhere herein. Preferably, the immune reactant specifically binds to the immune complexes specified in any one of Tables 1(i) to 1(vi) below, preferably to the complexes selected from those designated as complex ID NO:1, complex ID NO:2, complex ID NO:34 to complex ID NO:38, complex ID NO:61, complex ID NO:62, complex ID NO:78 to complex ID NO:80 and complex ID NO:89 to complex ID NO:97, and in a preferred embodiment to the complexes selected from those designated as complex ID NO:1, complex ID NO:2, complex ID NO:34 to complex ID NO:38, complex ID NO:61, complex ID NO:62, complex ID NO:77 to complex ID NO:80, complex ID NO:89 to complex ID NO:97, complex ID NO:132 and complex ID NO:136.

[0070] The present invention also relates to the use, preferably in vitro use, of a stimulating peptide for stimulating, identifying, and optionally enriching immune cells that specifically bind to human host cells infected with HPV16-associated virus, said stimulating peptide comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:85 to SEQ ID NO:92. In a preferred embodiment, said stimulating peptide comprises an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:75 to SEQ ID NO:77, SEQ ID NO:85 to SEQ ID NO:92, SEQ ID NO:121, and SEQ ID NO:92. The amino acid sequence of NO:136 has been shown to be presented by human HPV16-positive cancer cells and has been shown to stimulate human anti-stimulatory peptide immune cells when presented as a human leukocyte antigen (HLA) complex.

[0071] The present invention also relates to immune cells and / or immune response agents as specified above, for medical use, preferably for the treatment of inappropriate cell proliferation associated with HPV16-associated virus.

[0072] The terms "treatment" and "therapy" refer to a significant improvement in the disease or condition mentioned herein or its accompanying symptoms. Treatment as used herein also includes complete recovery of health for the disease or condition mentioned herein. It should be understood that, as used herein, treatment may not be effective for all subjects. However, the term requires, preferably, that a statistically significant proportion of subjects suffering from the disease or condition mentioned herein be successfully treated. Whether a proportion is statistically significant can be easily determined by those skilled in the art using various well-known statistical assessment tools (e.g., statistical assessment tools described below). Preferably, treating cancer involves reducing the tumor burden of the subject. As those skilled in the art will understand, the effectiveness of treatment, such as cancer treatment, depends on a variety of factors, including, for example, cancer stage and cancer type. Preferably, treatment results in the recognition of inappropriately proliferating cells, preferably neoplasmic cells, more preferably cancer cells, by the subject's T cells. Therefore, preferably, treatment has the effect of killing tumor cells, causing tumor growth to stop, particularly causing tumor cell proliferation to stop, more preferably causing tumor regression, and more preferably causing complete tumor resolution. As used in this article, the above content has been modified as necessary to address inappropriate cell proliferation in the treatment of other HPV16-related virus-positive cases.

[0073] Whether the proportion is statistically significant can be easily determined by those skilled in the art using various well-known statistical evaluation tools (e.g., determination of confidence intervals, p-value determination, Student's test, Mann-Whitney test, etc.). Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. Preferred p-values ​​are 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.

[0074] In view of the above, the following implementation plan is specifically proposed:

[0075] Implementation Scheme 1: A method for stimulating immune cells that specifically bind to human host cells infected with HPV16-associated virus, the method comprising:

[0076] (A) Contacting a complex containing human leukocyte antigen (HLA) and a stimulating peptide (the stimulating complex) with immune cells, and

[0077] (B) This stimulates immune cells that specifically bind to host cells infected with HPV16-associated viruses.

[0078] The stimulating peptide is composed of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111, and in a preferred embodiment, is composed of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:129.

[0079] Implementation Scheme 2: The method according to Implementation Scheme 1, wherein the stimulating peptide has been shown to stimulate human anti-stimulating peptide immune cells when presented as a human leukocyte antigen (HLA) complex.

[0080] Implementation Scheme 3: The method according to Implementation Scheme 1 or 2, wherein the human host cell is a human cancer cell infected with HPV16-associated virus, wherein an HPV16-derived peptide consisting of the same amino acid sequence as the stimulating peptide has been demonstrated to be presented by human HPV16-positive cancer cells, and wherein the stimulating peptide consists of an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:85 to SEQ ID NO:92.

[0081] Implementation Scheme 4: The method according to any one of Implementation Schemes 1 to 4, wherein the stimulation is to activate the immune cells and / or increase the number of immune cells that specifically bind to the stimulation complex.

[0082] Implementation Scheme 5: The method according to any one of Implementation Schemes 1 to 4, wherein the stimulating complex is contained on an antigen-presenting cell (APC) or an artificial APC that presents the at least one stimulating complex, or on an HLA oligomer containing the stimulating peptide.

[0083] Implementation Scheme 6: The method according to any one of Implementation Schemes 1 to 5, wherein the method is a method for stimulating and identifying immune cells that specifically bind to the stimulating complex, and wherein the method further comprises (C) identifying the stimulated immune cells stimulated in step (B).

[0084] Implementation Scheme 7: The method according to any one of Implementation Schemes 1 to 6, wherein the method is a method for stimulating and enriching immune cells that specifically bind to human host cells infected with HPV16-associated virus, and wherein the method further comprises step (D) enriching the stimulated immune cells stimulated in step (B) and optionally identified in step (C).

[0085] Implementation Scheme 8: The method according to Implementation Scheme 6 or 7, wherein the method further comprises contacting the immune cells of step (B) with a derivative of the stimulating complex carrying a detectable label and identifying and optionally enriching the immune cells bound to the stimulating complex by the detectable label.

[0086] Implementation Scheme 9: The method according to any one of Implementation Schemes 1 to 8, wherein the method is an in vitro method.

[0087] Implementation Scheme 10: The method according to any one of Implementation Schemes 1 to 8, wherein step (A) includes applying the stimulating complex to the subject.

[0088] Implementation Scheme 11: The method according to Implementation Scheme 10, wherein the object is a non-human animal, and wherein the object is preferably euthanized after step (C) or step (D).

[0089] Implementation Scheme 12: A method for determining at least a portion of the nucleic acid sequence of an immune response agent that specifically binds to human host cells infected with HPV16-associated virus, the method comprising the further steps of stimulating, identifying and optionally enriching immune cells that specifically bind to host cells infected with HPV16-associated virus according to any one of Implementation Schemes 1 to 11, and sequencing at least a portion of at least one polynucleotide encoding the immune response agent in the immune cells.

[0090] Implementation Scheme 13: A method for preparing an immunoreactive agent against human host cells infected with HPV16-associated virus, the method comprising:

[0091] (i) Stimulating immune cells that specifically bind to human host cells infected with HPV16-associated virus according to any one of embodiments 1 to 5 or 9 to 11;

[0092] (ii) Identify and optionally enrich the stimulated immune cells of step (i), preferably by means of any one of embodiments 6 to 10;

[0093] (iii) Determine at least a portion of the nucleic acid sequence of the immune response agent expressed by the immune cells identified and optionally enriched in step (ii), and

[0094] (iv) The immune response agent is prepared by expressing at least a portion of the nucleic acid sequence encoding the immune response agent, preferably in an expression system.

[0095] Implementation Scheme 14: A method for preparing an immunoreactive agent against human host cells infected with HPV16-associated virus, the method comprising expressing at least a portion of a nucleic acid sequence encoding the immunoreactive agent, said immunoreactive agent being obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), wherein

[0096] The stimulating peptide consists of an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111.

[0097] Implementation Scheme 15: An in vitro method for preparing an immunoreactive agent against human cancer cells infected with HPV16-associated virus, the method comprising:

[0098] The expression of at least a portion of a nucleic acid sequence encoding an immune response agent obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (the stimulating complex), wherein the stimulating peptide...

[0099] (i) It consists of an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and the HLA is derived from the HLA supertype HLA-A01;

[0100] (ii) Composed of the amino acid sequence of SEQ ID NO:11, and the HLA is derived from the HLA supertype HLA-A02.

[0101] (iii) Composed of an amino acid sequence selected from SEQ ID NO:34 to SEQ ID NO:37, and the HLA is derived from the HLA supertype HLA-A03 / A11;

[0102] (iv) Composed of an amino acid sequence selected from SEQ ID NO:60 and SEQ ID NO:61, and the HLA is derived from the HLA supertype HLA-A24;

[0103] (v) Composed of an amino acid sequence selected from SEQ ID NO:61, SEQ ID NO:76, and SEQ ID NO:77, and the HLA is derived from the HLA supertype HLA-B07; or

[0104] (vi) It consists of an amino acid sequence selected from SEQ ID NO:61 and SEQ ID NO:85 to SEQ ID NO:92, and the HLA is derived from the HLA supertype HLA-B15;

[0105] Furthermore, it has been confirmed that HPV16-derived peptides, consisting of the same amino acid sequence as the stimulating peptide, are presented by human HPV16-positive cancer cells.

[0106] Implementation Scheme 16: The method according to Implementation Scheme 14 or 15, wherein the method has a further feature of at least one of Implementation Schemes 1 to 13.

[0107] Implementation Scheme 17: The method according to any one of Implementation Schemes 1 to 16, wherein the immune cells are T cells expressing T cell receptors that specifically bind to immune complexes and / or B cells that specifically bind to immune complexes.

[0108] Implementation Scheme 18: The method according to any one of Implementation Schemes 1 to 17, wherein the immune cells are T cells, preferably CD8+ T cells, and more preferably cytotoxic CD8+ T cells.

[0109] Implementation Scheme 19: The method according to any one of Implementation Schemes 1 to 16, wherein the HPV16-related virus is HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 or HPV67, more preferably HPV16.

[0110] Implementation Scheme 20: An immune cell expressing an immune response agent that specifically binds to a host cell infected with HPV16-associated virus, wherein the immune cell is obtained by or can be obtained by any one of the methods described in Implementation Scheme 1 to 11.

[0111] Implementation Scheme 21: The immune cell according to Implementation Scheme 13, wherein the immune cell is a T cell and the immune response agent is a T cell receptor, or wherein the immune cell is a B cell and the immune response agent is an immunoglobulin.

[0112] Implementation Scheme 22: An immunoreactive agent for human cancer cells infected with HPV16-associated virus, said immunoreactive agent specifically binding to a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (stimulating complex), said stimulating peptide comprising an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:111.

[0113] Implementation Scheme 23: The immune response agent according to Implementation Scheme 22 is prepared according to any one of Implementation Schemes 13 to 19 or can be prepared according to any one of Implementation Schemes 13 to 19.

[0114] Implementation Scheme 24: Use, preferably in vitro use, of a stimulating peptide for stimulating, identifying, and optionally enriching immune cells that specifically bind to human host cells infected with HPV16-associated virus, said stimulating peptide comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:85 to SEQ ID NO:92, which has been shown to be presented by human HPV16-positive cancer cells and has been shown to stimulate human anti-stimulating peptide immune cells when presented as human leukocyte antigen (HLA) complexes.

[0115] Implementation Scheme 25: The subject matter of any one of the preceding implementation schemes, wherein the immune response agent is a TCR, an anti-immune complex antibody, or a fragment or derivative thereof.

[0116] Implementation Scheme 26: The subject matter of any one of the preceding implementation schemes, wherein the immune response reagent is contained in T cells.

[0117] Implementation Scheme 27: An immune cell and / or immune response agent according to any one of Implementation Schemes 20 to 23, for medical use, preferably for treating inappropriate cell proliferation associated with HPV16-associated virus.

[0118] All references cited in this specification are incorporated herein by reference in their entirety, including any disclosures specifically mentioned herein.

[0119] The following examples are for illustrative purposes only. In no way should they be construed as limiting the scope of the invention.

[0120] Example 1: A method for assessing T cell responsiveness

[0121] Interferon-γ ELISpot assay

[0122] Prior to the ELISpot assay, the ELISpot plate (Millipore Multiscreen-HA membrane sterile plate) was 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.

[0123] Before setting up the ELISpot assay, discard the coating antibody, wash the wells three times with 200 μl of sterile PBS, and block with 200 μl of ELISpot medium under standard culture conditions for 1 to 1.5 hours. After blocking, discard the ELISpot medium and add 100 μl of antigen solution to the wells.

[0124] Each (short-term) T cell line (see below) was stimulated in a total of 8 wells: four wells were infused with the corresponding antigen (10 μg / mL single peptide, 0.1% DMSO or CEF peptide pool (1 μg / mL for each peptide)), two wells were infused with concanavalin A (2 μg / mL) as a positive control for nonspecific mitogen stimulation, and two wells were infused with 0.1% (v / v) DMSO as a background control. Short-term antigen-specific T cell lines (described in 1.4) were stimulated with 1 × 10n mitogens per 100 μL well. 5 Up to 2×10 5 A concentration of cells was added (total volume 200 μl / well). Cell counts were assessed in three representative wells (CEF-stimulated, DMSO-stimulated, and peptide-stimulated wells) to allow for subsequent calculation of cell counts per 1 × 10⁻⁶ cells. 6 Spot-forming units (SFU) per cell. Incubate ELISpot plates under standard culture conditions for 20 to 24 hours. Strictly avoid moving the plates or incubator during incubation to prevent the formation of blurry spots.

[0125] Discard the remaining cell suspension, wash the ELISpot plate twice with PBS, and then twice with ELISpot wash buffer. All washes were performed using 200 μl / well. Then, add 1 ng / mL of sterile anti-human IFNγ biotin (7B6-1) antibody to 100 μl / well of sterile PBS. Incubate in the dark at room temperature for two hours, then discard the antibody and wash the plate four times with ELISpot wash buffer. Dilute sterile streptavidin-alkaline phosphatase 1:2000 in sterile PBS and dispense 100 μl / well.

[0126] After incubating in the dark at room temperature for 1.5 hours, discard the antibody and wash the plate four times with ELISpot wash buffer. Then, filter the BCIP / NBT-Plus substrate through a 0.22 μm filter and dispense 100 μl / well. Cover the plate with aluminum foil and incubate at room temperature for 18 to 20 minutes, or until spots appear in the positive control wells. Wash the ELISpot plate with tap water to stop the reaction and air dry the plate for at least 1.5 hours.

[0127] Analysis of ELISpot plates

[0128] To obtain objective results regarding the number of SFUs per well, the ELISpot boards were counted using a CTL automated ELISpot board reader. SmartCount was implemented using ImmunoSpot 5.1.36 Professional DC software. TM The counting mode was analyzed. Settings were individually adjusted for each donor to account for donor-specific differences. Typically, background balance was set to values ​​from 0 to 10, and spot separation to values ​​from 1 to 4. The automatically determined minimum spot size was adjusted based on the negative control wells to exclude small background spots. Similarly, the maximum spot size was automatically determined based on the positive control wells. Manual quality control was performed on each well to detect and correct errors in the counting algorithm caused by uneven staining or fibrils. Based on the counting results, the average spot size per 1 × 10⁻⁶ cells for each peptide-specific cell line was calculated. 6 SFU and Stimulation Index (SI) per cell, where SI is defined as the fold change in mean SFU from peptide-stimulated wells relative to DMSO background wells. SI > 2 and per 1 × 10 6 A response of SFU > 100 per cell, and a response of SI > 4 and SFU > 50 per cell, are considered positive.

[0129] T cell phenotype analysis and intracellular cytokine staining

[0130] To characterize and quantify responsive cells after peptide stimulation, peptide-specific T cell cultures with positive ELISpot results were further evaluated by flow cytometry for T cell phenotypic analysis and intracellular cytokine staining (ICS).

[0131] Collect continued short-term T cell cultures, along with DMSO-stimulated and non-responsive T cell lines, into 2 mL reaction tubes and centrifuge at 400 × g for 10 min. Discard the supernatant, resuspend the pellet of the non-responsive T cell line in 100 μl ELISpot medium, and use it as an unstimulated and unstained control. Resuspend the pellets of the responsive and DMSO-stimulated T cell lines in 200 μl ELISpot medium and aliquot them into two wells of a V-bottom 96-well plate. One well of the responsive T cell line was restimulated with 10 μg / mL of its specific peptide, while the other well was subjected to simulant stimulation 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 phorbol 12-tetradecanoate 13-acetate and 1 μM iomycin. Next, 1:10 dilutions of GolgiStop (containing monensin) and 1:15 dilutions of GolgiPlug (containing brefidobacterium A) were added to all wells to inhibit intracellular transport. Monensin inhibits trans-Golgi transport, while brefidobacterium A inhibits transport between the ER and the Golgi apparatus (Chardin and McCormick, 1999; Mollenhauer et al., 1990). The stimulated cells were incubated under standard culture conditions for 5 hours.

[0132] After incubation, resuspend the cells and centrifuge at 400×g for 5 minutes, discarding the supernatant. Then, resuspend the cells in 50 μl of cold staining buffer (unstained) or 50 μl of a mixture containing phenotypic analysis antibodies against CD3, CD4, and CD8 and LIVE / DEAD. TMCells were incubated in cold staining buffer (unstimulated and stimulated) at 4°C for 30 minutes after surface staining. Following staining, cells were washed twice with 200 μl of staining buffer and centrifuged at 1400 rpm, then fixed with 1% paraformaldehyde (PFA) solution at 4°C for 15 minutes. All subsequent centrifugation steps were performed at 1400 rpm for 5 minutes. After fixation, cells were washed twice again with 200 μl of cold staining buffer and resuspended in 100 μl of 1×perm / wash buffer diluted with PBS (10× was provided), and permeabilized at 4°C for 15 minutes. The plate was then centrifuged and the supernatant was discarded. Cells were resuspended in 50 μl of perm / wash buffer diluted with PBS (unstained) or 50 μl of perm / wash buffer containing antibodies against intracellular cytokines targeting IFNγ, TNFα, and granzyme B (unstimulated and stimulated), and incubated at 4°C for 30 minutes. After intracellular cytokine staining, 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 to each well. After incubation at 4°C for 20 min, cells in each well were washed twice with 200 μl of 1×perm / wash buffer, resuspended in 100 μl of staining buffer, and stored overnight at 4°C.

[0133] OneComp eBeads TM (eBeads) and ArC TM Amine-reactive compensating beads (ArC beads) were used as a compensating control for staining. For each antibody, 50 μl of eBeads suspension was stained at the same dilution as for cell staining. ArC beads were stained with 1 μl of LIVE / DEAD. TM Fixable Near-IR Dead Cell Stain stock solution was stained in 50 μl of staining buffer. The beads were incubated at 4 °C for 30 min, then washed twice with 1 mL of staining buffer and centrifuged at 400 × g. Finally, the stained beads were resuspended in 400 μl of staining buffer, and one drop of ArC was added to the labeled ArC beads. TM Negative compensating beads were used, and all beads were stored overnight at 4°C.

[0134] The next day, BD FACSDiva software version 6 was used in BD FACS Canto TM Samples and single-stained compensation controls were acquired using the II analyzer. The acquired data were analyzed using FlowJo software version 10, with the same gating strategy applied to all samples.

[0135] Short-term T cell lines

[0136] Short-term antigen-specific T cell lines were generated from PBMCs to expand antigen-specific memory T cells induced by prior natural HPV16 infection. For this purpose, T cells were stimulated with HLA-matched HPV16 E6 or E7-derived HLA ligands. To establish the short-term T cell lines, human PBMCs were thawed and subsequently resuspended in T cell culture 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 × 10⁶ cells were stimulated per antigen. 6 to 2×10 6 Cells were seeded into the wells of a 24-well plate, with a total volume of 2 mL per T cell line. To obtain antigen-specific T cell lines, 10 μg / mL of HLA-bound HPV16 E6 / E7-derived peptides were added to each well. As a peptide-specific positive control, one cell line was stimulated with a peptide pool (CEF peptide pool) consisting of HLA class I restrictive epitopes of 23 widely described viruses, including human cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza A virus (Currier et al., 2002), at a concentration of 1 μg / mL for each peptide. As a single peptide control, two cell lines were stimulated with 10 μg / mL of HLA-matched EBV-derived peptides and CMV-derived peptides. As a peptide-specific negative control, the cell lines were stimulated with 10 μg / mL of HLA-matched human immunodeficiency virus (HIV)-derived peptides, while treatment with 0.1% (v / v) dimethyl sulfoxide (DMSO, peptide solvent) served as a non-specific negative control. Stimulated PBMCs were cultured under standard culture conditions.

[0137] On day 3, cells were replenished with recombinant human interleukin-2 (rhIL-2) (20 U / mL) and rhIL-15 (20 ng / mL) to promote T cell proliferation. After seven days of culture establishment, a half-medium change was performed. For this, cells were centrifuged at 300 × g for 5 minutes to collect the pellet at the bottom of each well, and 1 mL of supernatant was carefully removed from each well. Then, 1 mL of fresh T cell culture medium was added, supplemented with rhIL-2 (final concentration 20 U / mL) and rhIL-15 (final concentration 20 ng / mL), and the cells were resuspended.

[0138] After 12 days of culture, half of the cells from each culture were used to establish the ELISpot assay, as described in 1.1. The remaining cells were replenished with 1 mL of ELISpot medium and cultured for another two days until the cells were used for T cell phenotype analysis and intracellular cytokine staining (ICS), as described in 1.2.

[0139] Cytotoxicity assay

[0140] The VITAL FR cytotoxicity assay, based on flow cytometry as described by Stanke et al. (Stanke et al., 2010), was used to assess whether peptide-specific T cells could specifically kill HPV-transformed target cells. In short, two HLA-A2... + Cell line, HPV16 negative (HPV16) - Control cell line (C33A) and HPV16 positive (HPV16) + Target cell lines (CaSki) were labeled with FarRed or CFSE fluorescence and associated with the peptide-specific CD8+ that serves as effector cells. + T cells were co-incubated. After 48 hours of co-incubation, fluorescently labeled cells were analyzed by flow cytometry to identify surviving cells, and the specific cell lysis function of effector cells was calculated by the ratio of target cells to control cells.

[0141] CaSki cells and C33A cells were labeled with CFSE and FarRed, respectively. Cells from both cell lines were harvested and resuspended in standard RPMI medium at a concentration of 1 × 10⁻⁶. 6 Cells / mL. For cell labeling, 5 μm CSFE was added to CaSki cells and 0.25 μm FarRed was added to C33A cells. Cells were incubated at 37°C for 10 minutes, and the reaction was terminated by adding 50 mL of RPMI containing 10% FBS. Cells were centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the cells were resuspended in their cell culture medium. The labeled cells were then incubated at 1 × 10⁻⁶ cells / mL. 6 to 2×10 6 Cells were seeded into T25 cell culture flasks at a density of cells / flask.

[0142] The following day, labeled CaSki and C33A cells, along with epitope-specific half-life T cell lines (as described in 1.6), were harvested. MACS CD8 was used. + The T-cell isolation kit (Miltenyi Biotec) isolates CD8+ cells from epitope-specific half-long-term T-cell lines according to the manufacturer's protocol for LS columns. + T cells. CD8 + T cells were resuspended in T cell culture medium and added serially diluted to the wells of an F-bottom 96-well plate to achieve 6 × 10⁶ cells per well. 4 1, 3×10 4 1.5 × 10 4 0.75×10 4 The sum is 0.375 × 10 4Three replicates of 100 cells were prepared. CaSki cells and C33A cells were mixed at a 1:1 ratio in T cell culture medium, and 3 × 10⁶ cells were added to each well containing T cells. 3 The cells were divided into 1:20, 1:10, 1:5, 1:2.5, and 1:1.25 effector cell:target cell (E:T) ratios. Additionally, three replicates of a mixture of target cells without T cells were seeded, termed E:T 0. After incubation at 37°C and 5% CO2 for 48 hours, the cells were harvested and analyzed using BD FACS Diva software version 6 in BD FACS Canto. TM The data were analyzed on the II analyzer. The obtained data were analyzed using FlowJo software version 10, with the same gating strategy applied to all samples. Subsequently, specific kills were calculated in Excel (Microsoft Office 2016) using equations (1) and (2) given below.

[0143]

[0144] Half-life T cell lines

[0145] Establishing a semi-long-term T cell line to obtain a large number of T cells is necessary for analyzing epitope-specific cytotoxic T cell responses in the VITAL FR cytotoxicity assay.

[0146] Autologous DCs were generated from PBMCs as described in 1.7 and loaded with the desired HPV16 peptide. PBMCs from the same donor were thawed and resuspended in T cell culture medium supplemented with rhIL-7 (10 ng / mL) and rhIL-15 (20 ng / mL). 1 × 10⁻⁶ 7 One PBMC / well was seeded into a 24-well plate, with a total volume of 1 mL / well. The peptide-loaded DCs were added to 1 mL of T-cell culture medium supplemented with rhIL-7 (10 ng / mL) and rhIL-15 (20 ng / mL) at a ratio of 50:1 (PBMC:DC), to a final volume of 2 mL. The cell culture was replenished with rhIL-2 (final concentration: 40 U / mL) every other day. If the medium turned yellow, it was replaced halfway with 1 mL of fresh IL-2-supplemented T-cell culture medium. After eight days, peptide stimulation with peptide-loaded DCs was repeated, and the culture continued. On day 15, the T-cell line was harvested and used for CD8 cell culture. + T cell isolation and cytotoxicity assay.

[0147] Generation of autologous DC

[0148] To generate autologous dendritic cells (DCs), frozen human PBMCs were thawed. After thawing, the cells were counted and resuspended in DC culture medium to reach a density of 5 × 10⁶ cells / year. 6Cell density per mL. From this suspension, in 6-well plates, 1 × 10⁻⁶ cells / mL were added to each well in DC medium at a density of 2 mL / well. 7 Individual cells were inoculated.

[0149] After incubation at 37°C and 5% CO2 for 3 hours, the supernatant was removed and transferred to 50 mL tubes. The wells were gently washed with 1 mL of warm DC medium to remove non-adherent cells, and the washing medium was collected along with the supernatant. The remaining adherent cells were further cultured with 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 containing T and B cells were counted and frozen for future use.

[0150] After three days of culture, cells were replenished with 0.5 mL of fresh DC medium supplemented with 100 ng / mL GM-CSF and 50 ng / mL IL-4. Six days after the start of culture, 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 mixture for 48 hours and harvested by gently scraping with a cell scraper.

[0151] Cytotoxicity assay based on live cell imaging

[0152] In addition to the VITAL FR assay described above, a newly established live-cell imaging-based cytotoxicity assay was also applied. In this assay, transiently red-labeled HPV16-transformed target cells were co-incubated with either HPV16 peptide-specific CD8+ T cells 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 using live-cell imaging. Peptide-specific killing was detected by the increased frequency of apoptotic target cells in co-culture with HPV16 peptide-specific CD8+ T cells compared to co-culture with non-specific CD8+ T cells. This highly sensitive assay is used to detect low-frequency CTL population-mediated killing and, unlike common endpoint assays, enables time-course analysis of CTL-mediated cytotoxicity. Furthermore, it is highly flexible and can be easily adapted to other target cell lines of interest.

[0153] Example 2: Immunoprecipitation (IP) and LC / MS analysis of peptides presented by cultured cells

[0154] Immunoprecipitation of HLA-presenting peptides

[0155] HLA immunoprecipitation (IP) was performed according to previously published protocols (Bassani-Sternberg et al., 2016; Chong et al., 2018). In short, HPV16+ cells were lysed using a lysis buffer containing 1% N-octyl-β-D-glucopyranoside, 0.25% sodium deoxycholate, and a mixture of protease inhibitors (Sigma-Aldrich, Mannheim, Germany) / PMSF (Carl Roth, Karlsruhe, Germany). After centrifugation at 40,000 × g for 30 min at 4°C, cells were lysed using protein G Sepharose beads (Cytiva, Marlborough, MA, USA) or Protein A-Sepharose. TMMouse anti-human HLA-A, B, C monoclonal antibodies (clone W6 / 32, Biolegend, San Diego, CA, USA) from 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) were continuously mixed on a rotating wheel at 4°C and incubated for 4 hours to immunoprecipitate HLA-peptide complexes. The supernatant was discarded after centrifugation at 3200×g for 3 minutes at room temperature. The HLA-peptide complexes precipitated onto the antibody beads were washed three times in each of the following steps: first with ice-cold 20 mM pH 8 Tris-HCl containing 150 mM NaCl, followed by washing with the same buffer but containing 400 mM NaCl, and finally washing alone with 20 mM Tris-HCl. The peptide was eluted from the antibody-bead bound to HLA using 0.3% TFA. Alkylation of the cysteine-containing peptide was optionally performed at this stage. 1 mL of IP eluent was reduced with 100 μl of HEPES / 50 mM TCEP and incubated at room temperature for 10 min. Alkylation was then performed by incubation in the dark at room temperature for 20 min with 100 μl of 400 mM iodoacetamide (IAA). Alkylation was quenched by incubation at room temperature for 3 min with 100 μl of 1 M HEPES / 50 mM TCEP. The sample was re-acidified with 60 μl of 10% TFA. The resulting peptide was desalted by reverse-phase purification using a SepPak 96-well plate (Waters, Milford, MA, USA). Oxidation was performed while the sample was loaded onto an adsorbent. Performic acid was prepared by mixing 5 μl of 30% H2O2 and 45 μl of 10% formic acid in a glass vial and allowing it to stand for 5 min. Samples were oxidized by briefly applying 1 ml of performic acid solution to the wells, followed by washing with 1 ml of 0.1% TFA. Peptides eluted in 28% ACN in 0.1% TFA were dried by vacuum centrifugation (Concentrator plus, Eppendorf, Hamburg, Germany).

[0156] Direct injection and LC-MS

[0157] The predicted target peptide set (crude synthetic product; isotopic purity > 99 atomic%) was obtained from a commercial supplier in the form of a stable isotope-labeled (SIL) peptide. 13 C and 15To alkylate cysteine-containing peptides, a 2 ng vacuum-dried peptide mixture was resuspended in 20 μl of 100 mM HEPES and sonicated for 3 min in a 0.5 μl protein LoBind microcentrifuge tube. A 5 μl sample (500 pmol per peptide) was reduced by adding 2 μl of HEPES / 50 mM TCEP and incubating at room temperature for 10 min. Alkylation was then performed by adding 2 μl of 400 mM IAA and incubating in the dark at room temperature for 20 min. Alkylation was quenched by adding 2 μl of 100 mM HEPES / 50 mM TCEP. The sample was acidified by adding 0.3% TFA to a volume of 500 μl and desalted using 100 mg adsorbent wells in a 96-well SepPak plate (Waters, Milford, MA, USA). Elution was performed with 80% ACN / 0.1% TFA.

[0158] First, direct injection analysis was performed to optimize the normalized collision energy (NCE) for each monopeptide in the mixture. NCE values ​​ranged from 4% to 42%, with each value measured four times for each peptide across its four charge states (1+ to 4+) in 2% steps. An Orbitrap Exploris 480 (Thermo Fisher Scientific) was run in targeted MS2 scan (PRM) mode with 30K resolution at 200 m / z, standard automatic gain control (AGC) targeting, a maximum injection time (IT) of 350 ms, 1 microscan, and centroid data acquisition mode. For all measurements, isolation windows were set at 0.4 m / z, 0.7 m / z, 1 m / z, or 1.5 m / z, depending on the precursor quality, as recommended by the manufacturer. Ion chromatograms of all anticipated transitions were extracted from the centroid spectra with a 12 ppm quality tolerance and deconvolved using a custom R script. The optimal NCE value was selected to maximize the intensity of any fifth strongest transition, thereby maximizing the chance of detecting at least five transitions. The retention time (RT) of each peptide was determined by LC-MS in DDA mode using an inclusion list of the target peptides containing all four charge states. The MS resolution was set to 120 K, 3e6 AGC target, 50 ms maximum IT, and the LC operation was as described below.

[0159] For LC-MS, the sample was dissolved in 2.5 μl to 5 μl of 0.1% TFA in 5% ACN and calibrated with a 50 fmol peptide retention time calibration (PRTC) mixture (88321, Pierce). TMAs a spiked control, samples were then subjected to 3-minute sonication in a water bath. All samples were analyzed by liquid chromatography (U-3000, Thermo Fisher Scientific) coupled with an Orbitrap Exploris 480 (Thermo Fisher Scientific). 150 fmol of BSA digest (88341, Pierce) was systematically injected before the IP sample. TM A mixture of 50 fmol PRTC (without the target peptide) and 94% A (0.1% FA in H2O) was used as a negative control to test for any artifacts, such as mild contamination that the SIL peptide might carry after previous quality control tests (Salek et al. 2022). The LC gradient consisted of multiple segments. First, solvent B (100% ACN, 0.1% FA) was added from 2% to 6%, followed by 94% A (0.1% FA in H2O) over 5 minutes, then to 28.5% B over 75 minutes. It reached 80% in 4 minutes, followed by washing for 5 minutes at 80% B. Finally, the column was equilibrated at 2% B for 11 minutes.

[0160] For LC-MS experiments, MS was acquired at 200 m / z at 60 K resolution, with a mass range from 150 m / z to 1450 m / z, 3e6 AGC target, and a maximum IT of 25 ms. MS² data were acquired via PRM scans at 200 m / z using either 60 K or 120 K resolution. A list of target precursors was provided, including preselected charge states, corresponding m / z values, optimized collision energies for each target, and their expected retention times (+ / - 1 min to 3 min) predefined with SIL peptides. The normalized AGC target was set to 1000% (or 1e6). The maximum injection time mode was set to dynamic, allowing a minimum of 5 points to be sampled across the chromatographic peak. Dynamic RT characteristics using the Pierce PRTC mixture were active.

[0161] Data Analysis

[0162] LC-MS data were analyzed using Skyline software v.20.2 (MacLean et al., 2010). The top five product ions with a minimum mass tolerance of 7 ppm were extracted. Detected peaks were manually screened. Light peaks were discarded when their retention time or shape did not match the heavy reference, when the normalized spectral contrast angle (NSCA) (Toprak et al., 2014) was low, or when too few transitions were detected.

[0163] The findings of this invention are also summarized in Table 2 below; as shown in the table, there are a large number of peptides (approximately 9000) that can be derived from HPV 16E6 and E7. The prediction algorithm identified approximately one-third of the peptides that actually bind to HLA and incorrectly predicted the binding in approximately 50% of the cases. Furthermore, for immunogenic peptides, only half were correctly predicted, and for peptides actually presented on HPV16-positive tumor cells, only approximately one-quarter were correctly predicted.

[0164] Detection of cysteine-containing peptides

[0165] To detect HPV16-derived T-cell epitopes, an immunopeptidomics workflow was established, in which each individual candidate epitope was evaluated by scientists and optimized targeting strategies were designed. Since HPV16 proteins E6 and E7 are cysteine-rich, 148 of the 242 candidate peptides contained cysteine ​​(see Table 3 below). Contrary to common practice in existing techniques, such as see Blatnik et al. (2018, loc. cit.), cysteine-containing peptides were not excluded from the analysis. This necessitates an adaptive strategy, as MS assays for detecting cysteine-containing peptides are particularly challenging due to the tendency for oxidative modification of thiol side chains. The resulting cysteine-containing peptides are dispersed in various forms, preventing the required sensitive detection in targeting experiments. Therefore, previous studies on HPV16 did not target any cysteine-containing peptides; this limitation was overcome by incorporating cysteine ​​alkylation reactions into the experiments.

[0166] Table 3: Number of candidate peptides containing cysteine.

[0167] Number of candidate peptides total 242 Free of cysteine 148(61％) Contains cysteine 94(39％)

[0168] Table 1: Preferred stimulating peptide / HLA combinations; Protein / Location: HPV16 protein and amino acid position from which the peptide is derived, indicating deviation from the HPV16 reference sequence, if applicable; Presentation: HLA presentation of the peptide by HPV16-positive cells as shown by MS; ELISPOT: Immunogenicity of the peptide as shown by IFN-γ ELISPOT analysis; Cytokines: Immunogenicity of the peptide as shown by intracellular cytokine staining (IFN-γ, TNF-α); Cytotoxicity: Ability of peptide-specific T cells to kill HPV16-positive cells. *Data from published resources are highlighted.

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] References

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Claims

1. An in vitro method for preparing an immunoreactive agent against human cancer cells infected with HPV16-associated virus, the method comprising: The expression of at least a portion of a nucleic acid sequence encoding an immune response agent obtained from immune cells stimulated by a complex comprising human leukocyte antigen (HLA) and a stimulating peptide (the stimulating complex), wherein the stimulating peptide... (i) It consists of an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2, and the HLA is derived from the HLA supertype HLA-A01; (ii) It consists of the amino acid sequence of SEQ ID NO:11, and the HLA is derived from the HLA supertype HLA-A02; (iii) Composed of an amino acid sequence selected from SEQ ID NO:34 to SEQ ID NO:37, and the HLA is derived from the HLA supertype HLA-A03 / A11; (iv) Composed of an amino acid sequence selected from SEQ ID NO:60 and SEQ ID NO:61, and the HLA is derived from the HLA supertype HLA-A24; (v) Composed of an amino acid sequence selected from SEQ ID NO:61, SEQ ID NO:76, and SEQ ID NO:77, and the HLA is derived from the HLA supertype HLA-B07; or (vi) It consists of an amino acid sequence selected from SEQ ID NO:61 and SEQ ID NO:85 to SEQ ID NO:92, and the HLA is derived from the HLA supertype HLA-B15; Furthermore, it has been confirmed that HPV16-derived peptides, consisting of the same amino acid sequence as the stimulating peptide, are presented by human HPV16-positive cancer cells.

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

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

4. The method according to any one of claims 1 to 3, wherein the immune cells are T cells expressing T cell receptors that recognize the stimulating complex and / or B cells that recognize the stimulating complex.

5. The method according to any one of claims 1 to 4, wherein the T cell is a CD4+ T cell or a CD8+ T cell, preferably a CD8+ T cell, and more preferably a CD8+ effector memory T cell.

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 method for stimulating immune cells that specifically bind to human host cells infected with HPV16-associated virus, the method comprising: (A) Contacting a complex containing human leukocyte antigen (HLA) and a stimulating peptide (the stimulating complex) with immune cells, and (B) thereby stimulating immune cells that specifically bind to the human host cells, wherein the stimulating peptide is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:85 to SEQ ID NO:92, and wherein HPV16-derived peptides consisting of the same amino acid sequence as the stimulating peptide have been shown to be presented by human HPV16-positive cancer cells.

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

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

10. The method of claim 8 or 9, wherein the method further comprises contacting the immune cells of step (B) with a derivative of the stimulating complex carrying a detectable label, and identifying and optionally enriching immune cells bound to the stimulating complex by using the detectable label.

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

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

13. A method for preparing an immunoreactive agent against human host cells infected with HPV16-associated virus, the method comprising: (i) The method according to any one of claims 7 to 11 stimulates immune cells that specifically bind to human host cells infected with HPV16-associated virus; (ii) Identify and optionally enrich the stimulated immune cells of step (i), preferably by means of the method according to claim 12; (iii) Determine at least a portion of the nucleic acid sequence of the immune response agent expressed by the immune cells identified and optionally enriched in step (ii), and (iv) The immune response agent is prepared by expressing at least a portion of the nucleic acid sequence encoding the immune response agent, preferably in an expression system.

14. The in vitro use of a stimulating peptide comprising an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:11, SEQ ID NO:34 to SEQ ID NO:37, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:76, SEQ ID NO:77 and SEQ ID NO:85 to SEQ ID NO:92 for stimulating, identifying and optionally enriching immune cells that specifically bind to human host cells infected with HPV16-associated virus, said stimulating peptide having been demonstrated to be presented by human HPV16-positive cancer cells and to stimulate human anti-stimulating peptide immune cells when presented as human leukocyte antigen (HLA) complexes.

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

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