Predicting immunogenicity of t cell epitopes

Abstract

The invention relates to a method for predicting T cell epitopes. In particular, the present invention relates to methods of predicting whether modifications in peptides or polypeptides, such as tumor-associated neoantigens, are immunogenic. The methods of the present invention are particularly useful in the provision of vaccines specific to patient tumors, and thus in the context of personalized cancer vaccines.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511277379.5 (22) Application Date 2014.05.07 (30) Priority Data PCT / EP2013 / 001400 2013.05.10 EP (62) Divisional Application Data 201480026604.1 2014.05.07 (71) Applicant Biotech Europe AG Address Mainz, Germany Applicant TRON-Mainz Johannes Gutenberg University Translational Oncology Medical School Public Welfare Company (72) Inventors Jürg Sain Abel David Tadmore Johann Christopher Cassel Sebastian Bogir Martin Lohr (74) Patent Agency Beijing Ying Sai Jia Hua Intellectual Property Agency Co., Ltd. 11204 Patent Attorney Wang Dazuo Hong Xin (51) Int.Cl. A61K 39 / 00 (2006.01) A61P 35 / 00 (2006.01) C07K 7 / 06 (2006.01) C07K 7 / 08 (2006.01) C12N 15 / 11 (2006.01) (54) Invention Title Predicting the Immunogenicity of T Cell Epitopes (57) Abstract This invention relates to a method for predicting T cell epitopes. In particular, this invention relates to a method for predicting whether modifications in peptides or polypeptides, such as tumor-associated neoantigens, are immunogenic. The method of this invention is particularly suitable for providing tumor-specific vaccines to patients, and therefore applicable to the case of personalized cancer vaccines. Claims (2 pages), Description (48 pages), Sequence List (electronic publication), Drawings (13 pages), CN 121130065 A 2025.12.16 CN 1 21 13 00 65 A 1. A pharmaceutical composition comprising one or more modified peptides, wherein each modified peptide is immunogenic, wherein each modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a designated position relative to a corresponding unmodified peptide, wherein each modified peptide and the corresponding unmodified peptide are identical except for the one or more amino acid modifications, wherein the amino acid modifications are impossible to occur in nature, and each modified amino acid and the corresponding unmodified amino acid are chemically and physically distinct, wherein each modified peptide and the corresponding unmodified peptide are 8 to 15 amino acids in length, and wherein each modified peptide and the corresponding unmodified peptide bind to one or more MHC molecules.2. An immunogenic modified peptide, wherein the immunogenic modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a designated position relative to a corresponding unmodified peptide, wherein the immunogenic modified peptide and the corresponding unmodified peptide are identical except for the one or more amino acid modifications, wherein the amino acid modifications are impossible to occur in nature, and each modified amino acid and the corresponding unmodified amino acid are chemically and physically distinct, wherein each immunogenic modified peptide and the corresponding unmodified peptide has a length of 8 to 15 amino acids, and wherein each immunogenic modified peptide and the corresponding unmodified peptide binds to one or more MHC molecules. 3. Use of the immunogenic peptide of claim 2 in the preparation of a medicament for treating cancer. 4. A nucleic acid sequence encoding an immunogenic modified peptide, wherein the immunogenic modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a designated position relative to a corresponding unmodified peptide, wherein the immunogenic modified peptide and the corresponding unmodified peptide are identical except for the one or more amino acid modifications, wherein the amino acid modifications are impossible to occur in nature, and each modified amino acid and the corresponding unmodified amino acid are chemically and physically distinct, wherein each immunogenic modified peptide and the corresponding unmodified peptide is 8 to 15 amino acids in length, and wherein each immunogenic modified peptide and the corresponding unmodified peptide binds to one or more MHC molecules. 5. Use of the nucleic acid sequence of claim 4 in the preparation of a medicament for treating cancer. 6. A vaccine comprising: one or more modified peptides; one or more nucleic acids encoding said one or more modified peptides; or multiple cells expressing said one or more modified peptides, wherein each modified peptide is immunogenic, wherein each modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a designated position relative to a corresponding unmodified peptide, wherein each modified peptide and the corresponding unmodified peptide are identical except for said one or more amino acid modifications, wherein said amino acid modifications are impossible to occur in nature, and each modified amino acid and the corresponding unmodified amino acid are chemically and physically distinct, wherein each modified peptide and the corresponding unmodified peptide are 8 to 15 amino acids in length, and wherein each modified peptide and the corresponding unmodified peptide bind to one or more MHC molecules. Claims 1 / 2 page 2 CN 121130065 A 7. The vaccine of claim 6, wherein said one or more modified peptides comprise one or more non-synonymous mutations corresponding to tumor-specific cancer mutations. 8. The vaccine of claim 7, wherein said vaccine is a personalized cancer vaccine designed to target a patient's tumor-specific cancer mutation.9. A method for predicting the immunogenicity of a modified peptide comprising an amino acid modification, wherein the modification is a substitution, the method comprising the steps of: a) determining a binding score of the modified peptide to one or more MHC molecules, b) determining a binding score of an unmodified peptide to one or more MHC molecules, and c) determining a chemical and physical similarity score between the unmodified amino acid and the modified amino acid using an evolution-based log-odds matrix for determining chemical and physical similarity, wherein the chemical and physical similarity score is determined based on the probability of amino acid interchange in the native state, wherein a higher frequency of amino acid interchange in the native state is considered a higher amino acid similarity, wherein the modified peptide is predicted to be immunogenic if (i) the binding score of the unmodified peptide to one or more MHC molecules satisfies a threshold indicating binding to one or more MHC molecules, (ii) the binding score of the modified peptide to one or more MHC molecules satisfies a threshold indicating binding to one or more MHC molecules, and (iii) the chemical and physical similarity score of the unmodified amino acid to the modified amino acid satisfies a threshold indicating chemical and physical dissimilarity. Claims 2 / 2 Page 3 CN 121130065 A Predicting the Immunogenicity of T Cell Epitopes

[0001] This application is a divisional application of Chinese Patent Application No. 202110057195.3.

[0002] Technical Field of the Invention

[0003] The present invention relates to a method for predicting T cell epitopes. In particular, the present invention relates to a method for predicting whether modifications in peptides or polypeptides, such as tumor-associated neoantigens, are immunogenic. The method of the present invention is particularly suitable for providing tumor-specific vaccines to patients, and therefore applicable to the case of personalized cancer vaccines. Background of the Invention

[0005] Personalized cancer vaccines are therapeutic vaccines tailored to target tumor-specific mutations unique to a given patient. Because such treatments do not harm healthy cells and have the potential to provide lifelong remission, they offer great hope to cancer patients. However, not every mutation expressed by a cancer can be used as a target for a vaccine. In fact, most somatic mutations in cancers do not elicit an immune response when targeted by vaccines (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)). Since tumors can encode up to 100,000 somatic mutations (M. R. Stratton, Science Signalling 331, 1553 (2011)), and vaccines target only a small number of epitopes, it is clear that a key objective of cancer immunotherapy is to identify which mutations are likely to be immunogenic.

[0006] From a biological perspective, for somatic mutations to elicit an immune response, several criteria must be met: the cell must express an allele containing the mutation; the mutation must be located in a protein-coding region and be non-synonymous; the translated protein must be cleaved by the proteasome; the epitope containing the mutation must be presented by the MHC complex; the presented epitope must be recognized by the T cell receptor (TCR); and finally, the TCR-pMHC complex must initiate a signaling cascade that activates T cells (S. Whelan, N. Goldman, Molecular biology and evolution 18, 691 (2001)). To date, no algorithm has been proposed that can predict with high certainty which mutations are likely to meet all these criteria. In this report, we consider several factors that may contribute to immunogenicity, compare these factors with experimental data, and propose a simple model for identifying immunogenic mutations.

[0007] MHC Binding Prediction: Current Status in the Field

[0008] More than 20 years ago, it was determined that there are sites on MHC-binding peptides that are more conducive to binding than other sites (e.g., A. Sette et al., Proceedings of the National Academy of Sciences 86, 3296 (1989)). The identification and description of those anchor sites enabled the discovery of patterns of MHC-binding peptides, and thus formed the basis for the development of prediction methods. In recent years, significant progress has been made in the field of in-silico models of antigen processing mechanisms. Two pioneering methods developed in the late 1990s, BIMAS (KC Parker, MA Bednarek, JE Coligan, The Journal of Immunology 152, 163 (1994)) and SYFPEITHI (H.-G. Rammensee, J. Bachmann, N. P. N. Emmerich, O. A. Bachor, S. Stevanovi, Immunogenetics 50, 213 (1999)), were based on knowledge of anchor sites and derived allele-specific motifs. With the increasing availability of experimental MHC peptide-binding data, more tools have been developed using various statistical and computational techniques (see Figure 1 for a review). So-called matrix-based methods use site-specific score matrices to determine whether a peptide sequence matches the binding motif of a specific MHC allele. Another class of MHC binding prediction methods utilizes machine learning techniques, such as artificial neural networks or support vector machines (see Figure 1).The performance of these algorithms is highly dependent on the quantity and quality of the training datasets available for each genomic model (e.g., “HLA-A 02:01”, “H2-Db”, etc.) to “learn” potential patterns / features with predictive binding capabilities. Recently, structure-based methods have emerged that circumvent the bottleneck of large training sets by predicting peptide-MHC interactions through, for example, energy minimization, since they rely solely on the peptide-MHC crystal structure and scoring functions (e.g., different energy functions) to do so (see Figure 1). However, the accuracy of those methods remains significantly lower than that of sequence-based methods. Benchmark studies show that the artificial neural network-based tool NetMHC (C. Lundegaard et al., Nucleic Acids Research 36, W509 (2008)) and the matrix-based algorithm SMM (B. Peters, A. Sette, BMC bioinformatics 6, 132 (2005)) perform best on test evaluation data (B. Peters, A. Sette, BMC bioinformatics 6, 132 (2005); HH Lin, S. Ray, S. Tongchusak, EL Reinherz, V. Brusic, BMC immunology 9, 8 (2008)). Both methods have been integrated into the so-called IEDB consensus method, which is available in the immunoepitaxy database (Y. Kim et al., Nucleic Acids Research 40, W525 (2012)). Because MHC II molecules have binding grooves with open ends on either side, allowing them to bind peptides of varying lengths, the interaction models for peptide-MHC II binding are far more complex than those for MHC I. However, peptides binding to MHC I are primarily limited to 8–12 amino acids, a length that can differ significantly from MHC II peptides (9–30 amino acids). Recent benchmark studies have shown that available MHC II prediction methods offer limited accuracy compared to MHC I prediction (HH Lin, S. Ray, S. Tongchusak, EL Reinherz, V. Brusic, BMC immunology 9, 8 (2008)).

[0009] Moutaftsi et al. conducted the first large-scale systematic application of algorithms for identifying T-cell epitopes (M. Moutaftsi et al., Nature Biotechnology 24, 817 (2006)), combining different tools to predict potential candidate vaccines for vaccinia virus-infected C57BL76 mice, extracting spleen cells and measuring CD8+ T cell responses to the top 1% of predicted peptides. They identified 49 peptides (out of 2256 peptides) that induced T-cell responses. Since then, numerous studies have been published using different MHC-binding prediction tools to identify T-cell epitopes as candidate vaccines, primarily targeting pathogens such as Leishmania major (C. Herrera-Najera, R. Piña-Aguilar, F. Xacur-Garcia, M. J. Ramirez-Sierra, E. Dumonteil, Proteomics 9, 1293 (2009)). However, using MHC I binding prediction tools alone to predict immunogenicity is misleading, as these tools are trained to predict whether a given peptide has the potential to bind to a given MHC allele. The basic principle behind using MHC binding prediction to predict immunogenicity is the assumption that peptides binding with high affinity to individual MHC alleles are more likely to be immunogenic (A. Sette et al., The Journal of Immunology 153, 5586 (1994)). However, numerous studies have shown that low MHC binding affinity can also lead to high immunogenicity (MC Feltkamp, ​​MP Vierboom, WM Kast, CJ Melief, Molecular Immunology 31, 1391 (1994)), and that peptide-MHC stability may be a better predictor of immunogenicity than peptide affinity (M. Harndahl et al., European Journal of Immunology 42, 1405 (2012)). Therefore, immunogenicity prediction has not been very accurate to date, which is reflected in the low success rate of predicting immunogenicity. However, peptide binding is a necessary but not sufficient condition for T cell epitope recognition, and effective prediction would significantly reduce the number of peptides that can be tested experimentally.

[0010] Obviously, the development of models for predicting immunogenicity also needs to take into account the recognition of T cell receptors (TCRs) and central tolerance, i.e., the negative and positive selection of T cells during thymus development.

[0011] A predictive model is needed that can simulate all aspects mentioned above to accurately predict the immunogenicity of epitopes, not just binding.

[0012] Description of the Invention 2 / 48 pages 5 CN 121130065 A Summary of the Invention

[0014] In one aspect, the present invention relates to a method for predicting immunogenic amino acid modifications, the method comprising the steps of:

[0015] a) determining a score for the binding of a modified peptide to one or more MHC molecules, and

[0016] b) determining a score for the binding of an unmodified peptide to one or more MHC molecules, and / or

[0017] c) determining a score for the binding of a modified peptide to one or more T cell receptors when present in an MHC-peptide complex.

[0018] In one embodiment, the modified peptide comprises a fragment of a modified protein, the fragment containing the modification present in the protein. In one embodiment, the unmodified peptide or protein contains germline amino acids at a site corresponding to the modification site in the modified peptide or protein.

[0019] In one embodiment, the unmodified peptide or protein is identical to the modified peptide or protein except for the modification. Preferably, the unmodified peptide or protein has the same length and / or sequence (except for the modification) as the modified peptide or protein.

[0020] In one embodiment, the length of the unmodified peptide and the modified peptide is 8-15 amino acids, preferably 8-12 amino acids.

[0021] In one embodiment, one or more MHC molecules comprise different MHC molecule types, particularly different MHC alleles. In one embodiment, one or more MHC molecules are MHC class I molecules and / or MHC class II molecules. In one embodiment, one or more MHC molecules comprise a set of MHC alleles, such as a set of MHC alleles of an individual or a subset thereof.

[0022] In one embodiment, the binding score with one or more MHC molecules is determined by a method including sequence comparison with a database of MHC-binding motifs.

[0023] In one embodiment, step a) includes determining whether the score meets a predetermined threshold for binding to one or more MHC molecules and / or step b) includes determining whether the score meets a predetermined threshold for binding to one or more MHC molecules. In one embodiment, the threshold applied in step a) is different from the threshold applied in step b). In one embodiment, the predetermined threshold for binding to one or more MHC molecules reflects the likelihood of binding to one or more MHC molecules.

[0024] In one embodiment, one or more T cell receptors comprise a set of T cell receptors, such as an individual's set of T cell receptors or a subset thereof.In one embodiment, step c) includes assuming that the T cell receptor group does not contain T cell receptors that bind to unmodified peptides present in the MHC-peptide complex and / or does not contain T cell receptors that bind with high affinity to unmodified peptides present in the MHC-peptide complex.

[0025] In one embodiment, step c) includes determining a score for the chemical and physical similarity between unmodified and modified amino acids. In one embodiment, step c) includes determining whether the score meets a predetermined threshold for the chemical and physical similarity between amino acids. In one embodiment, the predetermined threshold for the chemical and physical similarity between amino acids reflects the likelihood that the amino acids are chemically and physically similar. In one embodiment, the chemical and physical similarity score is determined based on the likelihood of amino acids being interchanged in their natural state. In one embodiment, the higher the frequency of amino acid interchange in their natural state, the higher the similarity of the amino acids is considered, and vice versa. In one embodiment, chemical and physical similarity is determined using an evolution-based log-probability matrix.

[0026] In one embodiment, if the binding score of the unmodified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, and the binding score of the modified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, then the modified peptide is predicted to be immunogenic, provided that the chemical and physical similarity scores of the unmodified amino acid and the modified amino acid meet a threshold indicating chemical and physical dissimilarity.

[0027] In one embodiment, if the unmodified peptide binds to one or more MHC molecules or has the potential to bind to one or more MHC molecules, and the modified peptide binds to one or more MHC molecules or has the potential to bind to one or more MHC molecules, then the modified peptide is predicted to be immunogenic, provided that the unmodified amino acid and the modified amino acid are chemically and physically dissimilar or have the potential to be chemically and physically dissimilar.

[0028] In one embodiment, the modification is not located at an anchoring site for binding to one or more MHC molecules.

[0029] In one embodiment, if the binding score of an unmodified peptide to one or more MHC molecules meets a threshold indicating non-binding to one or more MHC molecules, and the binding score of a modified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, then the modification or modified peptide is predicted to be immunogenic.

[0030] In one embodiment, if an unmodified peptide does not bind to one or more MHC molecules or has the potential not to bind to one or more MHC molecules, and a modified peptide binds to one or more MHC molecules or has the potential to bind to one or more MHC molecules, then the modification or modified peptide is predicted to be immunogenic.

[0031] In one embodiment, the modification is located at an anchoring site for binding to one or more MHC molecules.

[0032] In one embodiment, the method of the present invention includes performing step a) on two or more differently modified peptides, the two or more differently modified peptides containing the same modification. In one embodiment, the two or more differently modified peptides containing the same modification contain different fragments of a modified protein, the different fragments containing the same modification present in the protein. In one embodiment, the two or more differently modified peptides containing the same modification contain all possible MHC-binding fragments of a modified protein, the fragments containing the same modification present in the protein. In one embodiment, the method of the present invention further includes selecting a modified peptide from two or more differently modified peptides containing the same modification, the two or more differently modified peptides having the potential or maximum potential to bind to one or more MHC molecules. In one embodiment, the two or more peptides with the same modification differ in modification length and / or site.

[0033] In one embodiment, the method of the present invention includes performing step a) on two or more peptides with different modifications, and optionally performing one or both of steps b) and c). In one embodiment, the two or more peptides with different modifications contain the same modification and / or contain different modifications. In one embodiment, the different modifications are present in the same protein and / or different proteins. The group of two or more peptides with different modifications used in step a) and optionally in one or both of steps b) and c) may be the same or different. In one embodiment, the group of two or more peptides with different modifications used in step b) and / or step c) is a subset of the group of two or more peptides with different modifications used in step a). Preferably, the subset includes the peptide with the highest score in step a).

[0034] In one embodiment, the method of the present invention includes comparing the scores of two or more peptides with different modifications. In one embodiment, the method of the present invention includes ranking the two or more peptides with different modifications.In one embodiment, the weight of the modified peptide binding to one or more MHC molecules is higher than the weight of the modified peptide binding to one or more T cell receptors when present in an MHC-peptide complex, preferably higher than the score of chemical and physical similarity between the unmodified amino acid and the modified amino acid, and the weight of the score of the modified peptide binding to one or more T cell receptors when present in an MHC-peptide complex, preferably the score of chemical and physical similarity between the unmodified amino acid and the modified amino acid, is higher than the score of the unmodified peptide binding to one or more MHC molecules.

[0035] In one embodiment, the method of the present invention further includes identifying non-similar mutations in one or more protein-coding regions.

[0036] In one embodiment, according to the present invention, the modification is identified by partially or completely sequencing the genome or transcriptome of one or more cells, such as one or more cancer cells and optionally one or more non-cancer cells, and identifying mutations in one or more protein-coding regions.

[0037] In one embodiment, the mutation is a somatic mutation. In one embodiment, the mutation is a cancer mutation.

[0038] In one embodiment, the method of the present invention is used for the preparation of a vaccine. In one embodiment, the vaccine is derived from one or more modified peptides or peptides that are predicted to be immunogenic by the method of the present invention.

[0039] On the other hand, the present invention provides a method for providing a vaccine, comprising the step of: determining one or more mutant or modified peptides that are predicted to be immunogenic by the method of the present invention.

[0040] In one embodiment, the method further comprises the step of: providing a vaccine comprising a peptide or polypeptide, or a vaccine comprising a nucleic acid encoding said peptide or polypeptide, said peptide or polypeptide containing a modified or modified peptide that is predicted to be immunogenic.

[0041] On the other hand, the present invention provides a vaccine obtained using the method according to the present invention. Preferred embodiments of such vaccines are described herein.

[0042] The vaccine provided according to the present invention may comprise a pharmaceutically acceptable carrier, and may optionally comprise one or more adjuvants, stabilizers, etc. The vaccine may be in the form of a therapeutic or prophylactic vaccine.

[0043] On the other hand, a method for inducing an immune response in a patient is involved, comprising administering to a patient a vaccine provided according to the present invention.

[0044] On the other hand, a method for treating a cancer patient is provided, comprising the steps of:

[0045] (a) providing a vaccine by means of the method according to the invention; and

[0046] (b) administering the vaccine to the patient.

[0047] On the other hand, a method for treating a cancer patient is provided, comprising administering a vaccine according to the invention to the patient.

[0048] In other aspects, the present invention provides the vaccines described herein for use in the treatments described herein, particularly for the treatment or prevention of cancer.

[0049] The treatment of cancer described herein can be combined with surgery and / or radiation and / or conventional chemotherapy.

[0050] Other features and advantages of the invention will be apparent from the following detailed description and claims. Detailed Description of the Invention

[0052] Although the invention is described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0053] Hereinafter, elements of the invention will be described. These elements are listed together with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The various descriptive examples and preferred embodiments should not be construed as limiting the invention to the explicitly described embodiments. This description should be understood to support and include embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any arrangement and combination of all the elements described in this application should be considered as disclosed in the description of this application, unless the context otherwise indicates.

[0054] Preferably, the terminology used herein is defined as follows: "A multilingual glossary of biotechnological terms: (IUPAC specification 5 / 48 pages 8 CN 121130065 A Recommendations)", HGW Leuenberger, B. Nagel, and H. Kölbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0055] Unless otherwise specified, the practice of this invention applies to conventional methods in biochemistry, molecular biology, immunology, and recombinant DNA technology as explained in the literature in this field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0056] Unless the context otherwise requires, throughout this specification and the following claims, the term "comprise" and variations such as "comprises" and "comprising" should be understood to mean including the stated member, integer, or step, or group of members, integers, or steps, but not excluding any other member, integer, or step, or group of members, integers, or steps, although in some embodiments such other members, integers, or steps, or group of members, integers, or steps may be excluded, i.e., the subject matter consists of including the stated member, integer, or step, or group of members, integers, or steps. The terms "a," "an," and "the," and similar references used in the text of this invention (particularly in the text of the claims) should be interpreted to cover both singular and plural forms unless otherwise specified herein or obviously contradicted by the context. The description of numerical ranges herein is intended only as a shorthand for each separate value that falls within the range. Unless otherwise specified herein, each individual value is incorporated into this specification as if it were described separately herein.

[0057] All methods described herein may be practiced in any suitable order unless otherwise indicated herein or obviously contradicted by the context. The application of any and all examples or exemplary language (e.g., “as”) provided herein is intended only to better illustrate the invention and does not limit the scope of the claimed invention. The language of this specification should not be construed as indicating any unclaimed element necessary for the implementation of the invention.

[0058] References are made throughout this specification to several documents. Each document referenced herein (including all patents, patent applications, specific disclosures, manufacturers’ specifications, descriptions, etc.), whether in the foregoing or hereinafter, is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to precede such disclosure by virtue of prior invention.

[0059] According to the invention, the term "peptide" refers to a substance containing two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty-one or more, and up to preferably eight, ten, twenty-one or more, particularly one hundred amino acids, covalently linked by peptide bonds. The terms "polypeptide" or "protein" refer to large peptides, preferably peptides containing more than one hundred amino acid residues, but generally the terms "peptide," "polypeptide," and "protein" are synonyms and are used interchangeably herein.

[0060] According to the invention, the term "modification" with respect to peptides, polypeptides, or proteins refers to a sequence change in a peptide, polypeptide, or protein compared to the sequence of a parental sequence such as a wild-type peptide, polypeptide, or protein.The term includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and amino acid substitution variants, preferably amino acid substitution variants. All these sequence changes according to the invention may produce new epitopes.

[0061] Amino acid insertion variants include the insertion of one or more amino acids in a specific amino acid sequence.

[0062] Amino acid addition variants include the fusion of one or more amino acids, such as 1, 2, 3, 4, or 5, or more amino acids, at the amino terminus and / or carboxyl terminus.

[0063] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 4, or 5, or more amino acids. Specification 6 / 48 pages 9 CN 121130065 A

[0064] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue at its site.

[0065] According to the invention, the modified or modified peptide used for testing in the method of the invention may be derived from a protein containing the modification.

[0066] The term "derived" means, according to the invention, that the sequence of the specific entity, in particular the specific peptide, exists in the object from which it originates. Regarding the amino acid sequence, particularly a specific sequence region, "derived" specifically means that the relevant amino acid sequence originates from the amino acid sequence in which it exists.

[0067] The protein containing the modification, which may be the source of the modification or modified peptide used for testing in the method of the present invention, can be a neoantigen.

[0068] According to the present invention, the term "neoantigen" refers to a peptide or protein containing one or more amino acid modifications compared to the parent peptide or protein. For example, a neoantigen can be a tumor-associated neoantigen, wherein the term "tumor-associated neoantigen" comprises a peptide or protein containing amino acid modifications resulting from a tumor-specific mutation.

[0069] According to the present invention, the term "tumor-specific mutation" or "cancer-specific mutation" refers to a somatic mutation present in the nucleic acids of tumor cells or cancer cells but not in the nucleic acids of the corresponding normal cells (i.e., non-tumor cells or non-cancer cells). The terms "tumor-specific mutation" and "tumor mutation" and "cancer-specific mutation" are used interchangeably herein.

[0070] The term “immune response” refers to a comprehensive bodily response to a target such as an antigen, and preferably to a cellular immune response or a cellular and humoral immune response. An immune response can be protective / preventive / protective and / or therapeutic.

[0071] “Inducing an immune response” can mean that no immune response exists prior to induction, but can also mean that a certain level of immune response exists prior to induction and that the immune response is enhanced after induction. Therefore, “inducing an immune response” also includes “enhancing an immune response.” Preferably, after inducing an immune response in a subject, the subject is protected from disease such as cancer, or the disease condition is improved by inducing an immune response.For example, an immune response against tumor-expressing antigens can be induced in patients with cancer or in individuals at risk of developing cancer. Inducing an immune response in this case may mean that the subject's disease condition has improved, that the subject has not metastasized, or that an individual at risk of developing cancer has not developed cancer.

[0072] The terms "cellular immune response" and "cellular response," or similar terms, refer to an immune response of cells characterized by antigen presentation by class I or II MHCs, including T cells or T-lymphocytes acting as "helper" or "killer" cells. Helper T cells (also known as CD4+ T cells) play a major role by modulating the immune response, while killer cells (also known as cytotoxic T cells, cytolytic T cells, CD8+ T cells, or CTLs) kill diseased cells such as cancer cells, thereby preventing the production of more diseased cells. In a preferred embodiment, the invention includes stimulating an antitumor CTL response against tumor cells expressing one or more tumor-expressing antigens, and preferably utilizing class I MHCs to present such tumor-expressing antigens.

[0073] The “antigen” according to the invention encompasses any substance, preferably a peptide or protein, that is a target of and / or induces an immune response, such as a specific reaction with an antibody or T-lymphocytes (T cells). Preferably, the antigen contains at least one epitope, such as a T-cell epitope. Preferably, the antigen in the context of the invention can be a molecule, optionally which, after processing, induces an immune response preferably specific to said antigen (including cells expressing said antigen). The antigen or its T-cell epitope is preferably presented by cells, preferably by antigen-presenting cells, and when it is an MHC molecule, said cells include diseased cells, particularly cancer cells, which lead to an immune response against the antigen (including cells expressing said antigen).

[0074] In one embodiment, the antigen is a tumor antigen (also referred to herein as a tumor-expressing antigen), i.e., a portion of a tumor cell, such as a protein or peptide expressed in a tumor cell, which may be derived from the cytoplasm, cell surface, or cell nucleus, particularly those mainly present within the cell, or surface antigens of tumor cells. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferrin and embryonal sulfoglycoprotein, α2-H-ferritin and γ-fetoprotein. According to the invention, tumor antigens preferably include any antigen expressed in tumors or cancers and tumor cells or cancer cells, and optionally characterized by its targeting of the type and / or expression level of tumors or cancers and tumor cells or cancer cells, i.e., tumor-associated antigens.In one embodiment, the term "tumor-associated antigen" refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or at a specific stage of development. For example, a tumor-associated antigen may be specifically expressed under normal conditions in gastric tissue, preferably in the gastric mucosa, in reproductive organs, such as in the testes, in trophoblastic tissue, such as in the placenta or germline cells, and expressed or aberrantly expressed in one or more tumor tissues or cancerous tissues. In this regard, "limited number" preferably means no more than three, more preferably no more than two. In the context of this invention, tumor antigens include, for example, differentiation antigens, preferably cell type-specific differentiation antigens, i.e., proteins specifically expressed under normal conditions at a specific stage of differentiation of a particular cell type, cancer / testis antigens, i.e., proteins specifically expressed under normal conditions in the testes and sometimes in the placenta, and germ cell-specific antigens. Preferably, cancer cells are identified based on tumor antigens or aberrant expression of tumor antigens. In the context of this invention, tumor antigens expressed by cancer cells of the object (e.g., a patient with cancer) are preferably the object's own proteins. In a preferred embodiment, the tumor antigen in the case of the invention is specifically expressed in non-essential tissues or organs under normal conditions, i.e., tissues or organs that do not cause death of the subject when damaged by the immune system, or in organs or structures of the body that cannot or are barely accessible to the immune system.

[0075] According to the invention, the terms “tumor antigen,” “tumor-expressing antigen,” “cancer antigen,” and “cancer-expressing antigen” are equivalent and are used interchangeably herein.

[0076] The term “immunogenicity” refers to the relative effectiveness of inducing an immune response preferably in relation to treatment, such as treatment for cancer. As used herein, the term “immunogenic” refers to having immunogenic properties. For example, the term “immunogenic modification” used for peptides, polypeptides, or proteins refers to the effectiveness of said peptide, polypeptide, or protein in inducing an immune response caused by and / or directly against said modification. Preferably, unmodified peptides, polypeptides, or proteins do not induce an immune response, induce different immune responses, or induce different levels, preferably lower levels, of immune responses.

[0077] The term “major histocompatibility complex” and the abbreviation “MHC” include MHC class I and MHC class II molecules and involve a range of genes present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen-presenting cells or diseased cells in immune responses, where MHC proteins or molecules bind to peptides and present them for recognition by T cell receptors. MHC-encoded proteins are expressed on the cell surface and present T cells with self-antigens (peptide fragments from the cell itself) as well as non-self antigens (e.g., fragments from invading microorganisms).

[0078] The MHC scope is divided into three subgroups: class I, class II, and class III.MHC class I proteins contain α-chains and β2-microglobulins (not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. On most immune system cells, particularly antigen-presenting cells, MHC class II proteins contain α-chains and β-chains, and they present antigen fragments to helper T cells. MHC class III regions encode other immune components such as complement components, and some encode cytokines.

[0079] The MHC is polygenic (there are several MHC class I and MHC class II genes) and polymorphic (each gene has multiple alleles).

[0080] As used herein, the term “haplotype” refers to an HLA allele found on a chromosome and the protein it encodes. Haplotype also refers to an allele present at any locus within the MHC. Each type of MHC is represented by several loci: for example, for class I, HLA-A (human leukocyte antigen-A), HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, HLA-L, HLA-P, and HLA-V; and for class II, HLA-DRA, HLA-DRB1-9, HLA-, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1, HLA-DMA, HLA-DMB, HLA-DOA, and HLA-DOB. In this document, the terms “HLA allele” and “MHC allele” are used interchangeably. Specification 8 / 48 pages 11 CN 121130065 A

[0081] MHC exhibits extreme polymorphism: in humans, a large number of haplotypes containing different alleles exist at each gene locus. Different polymorphisms of class I and class II MHC alleles have different peptide specificities: each allele encodes a protein that binds to a peptide exhibiting a specific sequence type.

[0082] In a preferred embodiment of all aspects of the invention, the MHC molecule is an HLA molecule.

[0083] In the context of the invention, the term "MHC-binding peptide" includes MHC class I and / or class II binding peptides or peptides that can be processed to produce MHC class I and / or class II binding peptides. In the case of class I MHC / peptide complexes, the length of the binding peptide is typically 8-12 amino acids, preferably 8-10 amino acids, although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, the length of the binding peptide is typically 9-30 amino acids, preferably 10-25 amino acids, and particularly 13-18 amino acids; however, longer or shorter peptides may be effective.

[0084] If the peptide is to be presented directly, i.e., without treatment, especially without cleavage, its length is suitable for binding to MHC molecules, particularly class I MHC molecules, and preferably 7-30 amino acids in length, such as 7-20 amino acids, more preferably 7-12 amino acids, even more preferably 8-11 amino acids, and especially 9 or 10 amino acids in length.

[0085] If the peptide is part of a larger entity containing other sequences, such as a vaccine sequence or a polypeptide, and will be presented after treatment, especially after cleavage, the length of the peptide produced by treatment is suitable for binding to MHC molecules, especially class I MHC molecules, and preferably 7-30 amino acids in length, such as 7-20 amino acids, more preferably 7-12 amino acids, even more preferably 8-11 amino acids, and especially 9 or 10 amino acids in length. Preferably, the sequence of the peptide to be presented after treatment is derived from the amino acid sequence of the antigen or polypeptide used for vaccination, i.e., its sequence substantially corresponds to, and preferably is identical to, a fragment of the antigen or polypeptide.

[0086] Therefore, in one embodiment, the MHC-binding peptide comprises a sequence substantially corresponding to and preferably identical to the antigen fragment.

[0087] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment of a molecule recognized by the immune system, for example, by T cells, particularly when presented by MHC molecules. Epitopes of proteins such as tumor antigens preferably comprise continuous or discontinuous portions of said protein, and are preferably 5-100 amino acids in length, more preferably 5-50, more preferably 8-30, and most preferably 10-25 amino acids. For example, the epitope length can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In the case of the present invention, the epitope is particularly preferably a T-cell epitope.

[0088] According to the present invention, the epitope may bind to MHC molecules, such as MHC molecules on the cell surface, and therefore may be an "MHC-binding peptide".

[0089] As used herein, the term “neo-epitope” refers to an epitope that is not present in a reference, such as normal non-cancerous or germ cells, but is present in cancer cells. Specifically, this includes cases where a corresponding epitope is present in normal non-cancerous or germ cells, but the epitope sequence is altered due to one or more mutations in the cancer cell, resulting in a new epitope.

[0090] As used herein, the term “T-cell epitope” refers to a peptide that binds to an MHC molecule of a structure recognized by a T-cell receptor. Typically, T-cell epitopes are present on the surface of antigen-presenting cells.

[0091] As used herein, the term “predictive T-cell epitope” refers to a peptide that predicts whether it will bind to an MHC molecule and be recognized by a T-cell receptor.The term “predicting T-cell epitopes” is essentially synonymous with the phrase “predicting whether a peptide is immunogenic.”

[0092] According to the invention, T-cell epitopes may be present in vaccines as part of larger entities such as vaccine sequences and / or polypeptides containing multiple T-cell epitopes. Presented peptides or T-cell epitopes are generated after appropriate treatment.

[0093] One or more residues of the T-cell epitope may be modified, said residues being non-essential for TCR recognition or binding to MHC. Such modified T-cell epitopes can be considered immunoequivalent. Specification 9 / 48 pages 12 CN 121130065 A

[0094] Preferably, when presented by MHC and recognized by a T-cell receptor, the T-cell epitope is capable of inducing clonal expansion of T cells carrying T-cell receptors that specifically recognize the peptide / MHC complex, in the presence of a suitable co-stimulatory signal.

[0095] Preferably, the T-cell epitope comprises an amino acid sequence substantially corresponding to the amino acid sequence of the antigen fragment. Preferably, the antigen fragment is a peptide presented by MHC class I and / or class II.

[0096] The T cell epitope according to the invention preferably relates to a portion or fragment of an antigen capable of stimulating an immune response, preferably a cellular immune response against an antigen or a cell characterized by the expression of said antigen, and preferably achieved by antigen presentation (such as diseased cells, particularly cancer cells). Preferably, the T cell epitope is capable of stimulating a cellular response against cells characterized by antigens presented by class I MHC, and preferably capable of stimulating antigen-responsive cytotoxic T lymphocytes (CTLs).

[0097] "Antigen treatment" or "treatment" refers to the degradation of a peptide, polypeptide, or protein into a treatment product, which is a fragment of said peptide, polypeptide, or protein (e.g., polypeptide degradation into peptide), and one or more of these fragments are linked (e.g., by binding) to an MHC molecule for presentation to a specific T cell, preferably by an antigen-presenting cell.

[0098] An "antigen-presenting cell" (APC) is a cell that presents a peptide fragment of a protein antigen linked to an MHC molecule on its cell surface. Some APCs can activate antigen-specific T cells.

[0099] Professional antigen-presenting cells are highly effective at internalizing antigens, either through phagocytosis or receptor-mediated endocytosis, and then display antigen fragments bound to class II MHC molecules on their cell membranes. T cells recognize and interact with the antigen-class II MHC molecule complex on the surface of antigen-presenting cells. The antigen-presenting cells then generate additional co-stimulatory signals, leading to T cell activation. The expression of co-stimulatory molecules is a defining characteristic of professional antigen-presenting cells.

[0100] The main types of professional antigen-presenting cells are dendritic cells, macrophages, B cells, and certain activated epithelial cells, among which dendritic cells have the broadest range of antigen presentation and are perhaps the most important antigen-presenting cells.Dendritic cells (DCs) are a population of leukocytes that present antigens captured in surrounding tissues to T cells via MHC class II and class I antigen presentation pathways. Dendritic cells are well-known potent inducers of immune responses, and their activation is a key step in inducing antitumor immunity. Conveniently classifying dendritic cells into “immature” and “mature” cells can serve as a simple method to distinguish between two well-studied phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as antigen-presenting cells with high antigen uptake and processing capabilities, associated with high expression of Fcγ receptors and mannose receptors. Mature phenotypes are typically characterized by low expression of these markers and high expression of cell surface molecules responsible for T cell activation, such as class I and II MHC, adhesion molecules (e.g., CD54 and CD11), and co-stimulatory molecules (e.g., CD40, CD80, CD86, and 4-1BB). Dendritic cell maturation refers to the state of dendritic cell activation, in which antigen-presenting dendritic cells induce T cell initiation, while the presentation of immature dendritic cells leads to tolerance. Dendritic cell maturation is mainly caused by the following factors: detection of microbial biomolecules (bacterial DNA, viral RNA, endotoxins, etc.) via genetic receptors, pro-inflammatory cytokines (TNF, IL-1, IFN), CD40 linked to CD40 on the surface of dendritic cells via CD40L, and substances released by cells undergoing stress-induced cell death. Dendritic cells can be obtained by culturing bone marrow cells in vitro with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-α.

[0101] Nonprofessional antigen-presenting cells do not continuously express MHC class II proteins required for interaction with naïve T cells; these proteins are only expressed when nonprofessional antigen-presenting cells are stimulated by certain cytokines such as IFNγ.

[0102] Antigen-presenting cells can be loaded with class I MHC-presented peptides by transducing cells with nucleic acids, wherein the nucleic acid is a nucleic acid encoding a peptide or polypeptide containing the presented peptide, preferably RNA, such as a nucleic acid encoding an antigen or polypeptide for vaccination.

[0103] In some embodiments, a pharmaceutical composition or vaccine containing a nucleic acid delivery medium targeting dendritic cells or other antigen-presenting cells can be administered to a patient, thereby resulting in in vivo transfection. In vivo transfection of dendritic cells can be carried out, for example, by any method known in the art, such as those described in WO 97 / 24447, or the gene gun method described in Mahvi et al. Immunology and cell Biology 75: 456-460, 1997.

[0104] According to the invention, the term "antigen-presenting cell" also includes target cells.

[0105] “Target cell” means a target cell of an immune response such as a cellular immune response. Target cells include cells that present antigens (i.e., peptide fragments derived from antigens) and include any undesirable cells such as cancer cells. In a preferred embodiment, the target cell is a cell that expresses the antigen described herein, and preferably the antigen is presented by a class I MHC.

[0106] The term “portion” refers to a fraction. With respect to a specific structure such as an amino acid sequence or a protein, the term “its portion” can refer to a continuous or discontinuous portion of the structure. Preferably, a portion of the amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the amino acids of the amino acid sequence. Preferably, if the portion is discontinuous, the discontinuous portion consists of 2, 3, 4, 5, 6, 7, 8 or more portions of the structure, each portion being a continuous element of the structure. For example, a discontinuous portion of an amino acid sequence may consist of 2, 3, 4, 5, 6, 7, 8 or more, preferably no more than 4, portions of the amino acid sequence, wherein each portion preferably contains at least 5, at least 10, preferably at least 20, and preferably at least 30 consecutive amino acids of the amino acid sequence.

[0107] In this document, the terms “part” and “fragment” are used interchangeably and refer to a continuous element. For example, a portion of a structure, such as an amino acid sequence or a protein, refers to a continuous element of that structure. A portion of, a part of, or a fragment of a structure preferably contains one or more functional properties of the structure. For example, a portion of an epitope, peptide, or protein is preferably immunoequivalent to the epitope, peptide, or protein from which it originates. In the context of this invention, a “part” of the structure (such as an amino acid sequence) preferably comprises, and preferably consists of, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the entire structure or amino acid sequence.

[0108] In the context of this invention, the term “immunoreactive cell” refers to a cell that functions as an effector during an immune response. An “immunoreactive cell” is preferably characterized by its ability to bind to an antigen or by presenting an antigen or a fragment thereof (e.g., a T-cell epitope) and mediating an immune response.For example, such cells secrete cytokines and / or chemokines, secrete antibodies, recognize cancer cells, and optionally eliminate such cells. Immune response cells include, for example, T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the case of the invention, the “immune response cell” is a T cell, preferably CD4+ and / or CD8+ T cells.

[0109] Preferably, the “immune response cell” recognizes an antigen or a peptide fragment thereof with a degree of specificity, particularly if presented by an MHC molecule, such as on the surface of antigen-presenting cells or diseased cells such as cancer cells. Preferably, the recognition makes the cell recognizing the antigen or its peptide fragment responsive or reactive. If the cell is a helper T cell (CD4+ T cell) containing a receptor that recognizes an antigen or its peptide fragment, in the case of MHC class II molecules, such a response or reaction may include the release of cytokines and / or the activation of CD8+ lymphocytes (CTLs) and / or B cells. If the cells are CTLs, such responses or reactions may include the clearance of cells presented by MHC class I molecules, i.e., cells characterized by antigens presented by class I MHCs, for example, through apoptosis or perforin-mediated cell lysis. According to the invention, CTL responses may include sustained calcium efflux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL responses may also be determined using artificial reporter genes that accurately indicate CTL responses. Such CTLs that recognize antigens or antigen fragments and are responsive or reactive are also referred to herein as "antigen-responsive CTLs." If the cells are B cells, such responses may include the release of immunoglobulins.

[0110] In this document, the terms “T cell” and “T lymphocyte” are used interchangeably and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) that include cytolytic T cells.

[0111] T cells belong to a population of white blood cells called lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells, by the presence of a special receptor called the T cell receptor (TCR) on their cell surface. The thymus is the main organ responsible for T cell maturation. Several different subsets of T cells have been identified, each with different functions.

[0112] In the immune process, helper T cells assist other white blood cells, among other functions, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages.These cells are also called CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when they encounter peptide antigens presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or aid in active immune responses.

[0113] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also associated with transplant rejection. Because these cells express the CD8 glycoprotein on their surface, they are also called CD8+ T cells. These cells recognize their targets by binding to MHC class I-associated antigens, which are present on the surface of almost every cell in the body.

[0114] Most T cells have a T cell receptor (TCR) that exists as a complex of some proteins. The true T cell receptor consists of two separate polypeptide chains produced by separate T cell receptor α and β (TCRα and TCRβ) genes and are called the α-TCR chain and the β-TCR chain. γδ T cells (γδ T cells) represent a small subset of T cells with different T cell receptors (TCRs) on their surface. However, in γδ T cells, the TCR consists of one γ-chain and one δ-chain. This T cell population is less common than the αβ T cell population (2% of total T cells).

[0115] The first signal in T cell activation is provided by the binding of the T cell receptor to a short peptide presented by MHC on another cell. This ensures that only T cells with TCRs specific to that peptide are activated. Companion cells are typically antigen-presenting cells such as professional antigen-presenting cells, and in the case of an initial response, dendritic cells are typically present, although B cells and macrophages are important APCs.

[0116] According to the invention, a molecule is capable of binding to a predetermined target if it has a significant affinity for the predetermined target and binds to the predetermined target in a standard analysis. “Affinity” or “binding affinity” is typically determined by the equilibrium dissociation constant (KD). If a molecule does not have a significant affinity for the target and does not bind significantly to the target in standard analysis, then the molecule cannot (substantially) bind to the target.

[0117] Cytotoxic T lymphocytes can be generated in vivo by incorporating an antigen or a peptide fragment thereof into antigen-presenting cells. The antigen or the peptide fragment thereof may be in the form of a protein, DNA (e.g., within a vector), or RNA. The antigen may be treated to produce peptide partners of an MHC molecule without further processing of the fragment for presentation. The latter is a special case if they are able to bind to an MHC molecule. Typically, administration to the patient is possible via intradermal injection. However, injection may also be performed via intranodal injection (Maloy et al. (2001), Proc Natl Acad Sci USA 98:3299-303).The generated cell presents a target complex and is recognized by autologous cytotoxic T lymphocytes, which then proliferate.

[0118] Specific activation of CD4+ or CD8+ T cells can be detected by various methods. Methods for detecting specific T cell activation include detecting T cell proliferation, the production of cytokines (e.g., lymphokines), or the production of cytolytic activity. Specification 12 / 48 pages 15 CN 121130065 A For CD4+ T cells, a preferred method for detecting specific T cell activation is to detect T cell proliferation. For CD8+ T cells, a preferred method for detecting specific T cell activation is to detect the production of cytolytic activity.

[0119] By the expression “cells characterized by antigen presentation” or “antigen-presenting cells” or similar expressions, it means that in the case of MHC molecules, especially MHC class I molecules, cells such as diseased cells (e.g., cancer cells) or antigen-presenting cells present antigens they express or fragments derived from said antigens, for example, by processing the antigen. Similarly, the term "disease characterized by antigen presentation" refers to a disease involving cells characterized by antigen presentation, particularly those presented by class I MHC. Antigen presentation by cells may be influenced by transfection of cells with nucleic acids, such as RNA encoding the antigen.

[0120] The term "fragment of presented antigen" or similar expressions mean that the fragment can be presented by MHC class I or II, preferably by MHC class I, for example, when added directly to antigen-presenting cells. In one embodiment, the fragment is a fragment naturally presented by the cell expressing the antigen.

[0121] The term "immunely equivalent" means that immunely equivalent molecules, such as immunely equivalent amino acid sequences, exhibit the same or substantially the same immune properties and / or exhibit the same or substantially the same immune effects, for example, regarding the type of immune effect such as induction of humoral and / or cellular immune responses, the strength and / or duration of the induced immune response, or the specificity of the induced immune response. In the context of the invention, the term "immunely equivalent" is preferably used with respect to the immune effects or properties of peptides used for immunization. For example, if an amino acid sequence induces an immune response specific to a reference amino acid sequence when the subject's immune system is exposed to it, then the amino acid sequence is immunely equivalent to the reference amino acid sequence.

[0122] In the context of this invention, the term "immune effector function" includes any function mediated by components of the immune system that results in, for example, the killing of tumor cells, or the inhibition of tumor growth and / or the inhibition of tumor development, including the inhibition of tumor dissemination and metastasis. Preferably, in the context of this invention, immune effector function is a T cell-mediated effector function.Such functions include, when the cells are helper T cells (CD4+ T cells), in the case of MHC class II molecules, recognition of antigens or antigen fragments by T cell receptors, release of cytokines and / or activation of CD8+ lymphocytes (CTLs) and / or B cells; and when the cells are CTLs, in the case of MHC class I molecules, recognition of antigens or antigen fragments by T cell receptors, clearance of cells presented by MHC class I molecules (i.e., cells characterized by antigens presented by class I MHC, for example, through apoptosis or perforin-mediated cell lysis), production of cytokines such as IFN-γ and TNF-α, and specific cytolytic killing of target cells expressing antigens.

[0123] According to the invention, the term “score” refers to the result of a test or examination, usually expressed numerically. Terms such as “better score” or “best score” refer to a better or best result of a test or examination.

[0124] Terms such as “predict,” “predicting,” or “prediction” refer to a determination of probability.

[0125] According to the present invention, determining the score for the binding of a peptide to one or more MHC molecules includes determining the likelihood of the peptide binding to one or more MHC molecules.

[0126] The score for the binding of a peptide to one or more MHC molecules can be determined by utilizing any peptide:MHC binding prediction tool. For example, the Immunotope Database Analysis Resource (IEDB-AR: http: / / tools.iedb.org) can be used.

[0127] Prediction is typically made for a set of MHC molecules, such as a different set of MHC alleles, such as all possible MHC alleles or a set of MHC alleles or subsets thereof present in a patient, preferably modified, and whose immunogenicity will be determined according to the present invention.

[0128] According to the present invention, determining the score for the binding of a modified peptide to one or more T cell receptors when present in an MHC-peptide complex includes determining the likelihood of the peptide binding to a T cell receptor when present in a complex formed with an MHC molecule. (See specification page 13 / 48, 16 CN 121130065 A)

[0129] Prediction can be made for a single T-cell receptor, such as a T-cell receptor present in the patient, or preferably for a group of T-cell receptors, such as a group of unknown different T-cell receptors or a group of T-cell receptors present in the patient, preferably modified, and whose immunogenicity will be determined according to the invention.

[0130] In addition, prediction is generally made for a group of MHC molecules, such as a group of different MHC alleles, such as all possible MHC alleles or a group of MHC alleles present in the patient, preferably modified, and whose immunogenicity will be determined according to the invention.

[0131] The binding score of a modified peptide present in the MHC-peptide complex to one or more T cell receptors can be determined by assessing the effect of the modification on the binding of the T cell receptor-peptide-MHC complex to a given (unknown) T cell receptor pool. The binding score of a modified peptide present in the MHC-peptide complex to one or more T cell receptors is generally defined as a representation of the recognition of a matching T cell receptor by a given peptide-MHC molecule.

[0132] The binding score of a modified peptide present in the MHC-peptide complex to one or more T cell receptors can be determined by the physicochemical differences between the modified and unmodified amino acids. For example, a substitution matrix can be used. Such a matrix describes the rate at which an amino acid in a sequence changes to another amino acid state over time.

[0133] For example, a log-odds matrix, such as an evolution-based log-odds matrix, can be used: a substitution with a low log-odds score has a greater chance of finding a matching T cell receptor from the (unknown) T cell receptor molecule pool than a substitution with a high log-odds score (due to the negative selection of T cell receptors that match the unmodified peptide). However, other methods exist for determining this score. For example, considering the site of mutation in the peptide (some sites may have a smaller impact on binding than others), taking into account the nearest neighbor of the substituted amino acid (which may affect the secondary structure of the substituted amino acid), taking into account the entire peptide sequence, taking into account the complete structural information of the peptide in the MHC molecule, etc. Determining the score also includes determining the T cell receptor repertoire (such as the patient's T cell receptor repertoire or a subset thereof), for example by NGS and performing docking simulations of the T cell receptor-peptide-MHC complex.

[0134] The invention may also include implementing the method of the invention on different peptides containing the same modification and / or different modifications.

[0135] In one embodiment, the term "different peptides containing the same modification" refers to different fragments of a modified protein or peptides composed thereof, said different fragments containing the same modification present in the protein, but different in length and / or modification site. If the protein is modified at site x, then two or more fragments of said protein that each contains a different sequence window of said protein covering said site x are considered to be different peptides containing the same modification.

[0136] In one embodiment, the term "different peptides containing different modifications" refers to peptides of the same and / or different lengths containing different modifications of the same and / or different proteins. If a protein is modified at sites x and y, then two segments of the protein containing a sequence window covering site x or site y are considered to be different peptides containing different modifications.

[0137] The present invention may also include cleaving a protein sequence containing a modification (whose immunogenicity is determined according to the invention) into peptide lengths suitable for MHC binding, and determining scores for the binding of peptides with the same and / or different modifications containing the same and / or different proteins to one or more MHC molecules. The outputs may be ranked, and the results may consist of a series of peptides and predicted scores showing their binding probabilities.

[0138] The steps of determining the binding scores of unmodified peptides to one or more MHC molecules and / or determining the binding scores of modified peptides present in the MHC-peptide complex to one or more T cell receptors may then be performed. This may be performed on all peptides with the same and / or different modifications, subsets thereof, such as those modified peptides containing the same and / or different modifications that have the highest binding scores to one or more MHC molecules, or only the modified peptide with the highest binding score to one or more MHC molecules.

[0139] Following the other steps, the results can be arranged, and the results may consist of a series of peptides and predictive scores indicating their likelihood of being immunogenic.

[0140] Preferably, in such an arrangement, the score for the binding of a modified peptide to one or more MHC molecules is given a higher weight than the score for the binding of a modified peptide present in the MHC-peptide complex to one or more T cell receptors, preferably with a higher weight than the score for the chemical and physical similarity between unmodified and modified amino acids, and the score for the binding of a modified peptide present in the MHC-peptide complex to one or more T cell receptors, preferably with a higher weight than the score for the chemical and physical similarity between unmodified and modified amino acids, is given a higher weight than the score for the binding of an unmodified peptide to one or more MHC molecules.

[0141] Amino acid modifications (whose immunogenicity is determined according to the present invention) may result from mutations in nucleic acids in the cell. Such mutations can be identified by known sequencing techniques.

[0142] In one embodiment, the mutation is a cancer-specific somatic mutation in a tumor sample from a cancer patient, which can be determined by identifying sequence differences between the genome, exome, and / or transcriptome of the tumor sample and the genome, exome, and / or transcriptome of a non-tumor sample.

[0143] According to the invention, the tumor sample refers to any sample, such as a body sample derived from a patient containing or potentially containing tumor cells or cancer cells. The body sample can be any tissue sample such as blood, a tissue sample obtained from a primary tumor or from a tumor metastasis, or any other sample containing tumor cells or cancer cells. Preferably, the body sample is blood, and the determination is of cancer-specific somatic mutations or sequence differences in one or more circulating tumor cells (CTCs) contained in the blood.In another embodiment, the tumor sample involves one or more isolated tumor cells or cancer cells such as circulating tumor cells (CTCs), or a sample containing one or more isolated tumor cells or cancer cells such as circulating tumor cells (CTCs).

[0144] The non-tumor sample involves any sample such as a body sample derived from a patient or other individual, preferably an individual of the same class as the patient, preferably a healthy individual that does not contain or is unlikely to contain tumor cells or cancer cells. The body sample can be any tissue sample such as blood or a sample derived from non-tumor tissue.

[0145] The invention may include the determination of cancer mutation markers in a patient. The term “cancer mutation marker” may refer to all cancer mutations present in one or more cancer cells in a patient or it may refer to only a portion of cancer mutations present in one or more cancer cells in a patient. Thus, the invention may include the identification of all cancer-specific mutations present in one or more cancer cells in a patient or may include the identification of only a portion of cancer-specific mutations present in one or more cancer cells in a patient. Typically, the method of the invention provides the identification of multiple mutations that provide a sufficient number of modifications or modified peptides included in the method of the invention.

[0146] Preferably, the mutation identified according to the invention is a non-synonymous mutation, preferably a non-synonymous mutation of a protein expressed in tumor cells or cancer cells.

[0147] In one embodiment, cancer-specific somatic mutations or sequence differences in the genome are determined, preferably the entire genome of a tumor sample. Therefore, the present invention may include identifying cancer mutation markers in the genome, preferably the entire genome of one or more cancer cells. In one embodiment, the step of identifying cancer-specific somatic mutations in a tumor sample from a cancer patient includes identifying a whole-genome cancer mutation profile.

[0148] In one embodiment, cancer-specific somatic mutations or sequence differences in the exome are determined, preferably the entire exome of a tumor sample. Therefore, the present invention may include identifying cancer mutation markers in the exome, preferably the entire exome of one or more cancer cells. In one embodiment, the step of identifying cancer-specific somatic mutations in a tumor sample from a cancer patient includes identifying a whole-exome cancer mutation profile. Specification 15 / 48 pages 18 CN 121130065 A

[0149] In one embodiment, cancer-specific somatic mutations or sequence differences in the transcriptome are determined, preferably the entire transcriptome of a tumor sample. Therefore, the present invention may include identifying cancer mutation markers in the transcriptome, preferably the entire transcriptome of one or more cancer cells. In one implementation, the step of identifying cancer-specific somatic mutations in tumor samples from cancer patients includes identifying a full transcriptome cancer mutation profile.

[0150] In one embodiment, the step of identifying cancer-specific somatic mutations or identifying sequence differences includes single-cell sequencing of one or more, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more cancer cells. Therefore, the present invention may include cancer mutation markers for identifying said one or more cancer cells. In one embodiment, the cancer cells are circulating tumor cells. Cancer cells, such as circulating tumor cells, may be isolated prior to single-cell sequencing.

[0151] In one embodiment, the step of identifying cancer-specific somatic mutations or identifying sequence differences includes using next-generation sequencing (NGS).

[0152] In one embodiment, the step of identifying cancer-specific somatic mutations or identifying sequence differences includes sequencing the genomic DNA and / or RNA of a tumor sample.

[0153] To reveal cancer-specific somatic mutations or sequence differences, preferably, the sequence information obtained from the tumor sample is compared with sequence information obtained from sequencing nucleic acids (such as DNA or RNA) from a reference, such as from normal non-cancer cells, such as germ cells, which may be obtained from a patient or different individuals. In one embodiment, normal genomic germline DNA is obtained from peripheral blood mononuclear cells (PBMCs).

[0154] The term “genome” refers to the total amount of genetic information in the chromosomes of an organism or cell.

[0155] The term “exome” refers to a portion of an organism’s genome formed by exons, which is the coding portion of expressed genes. The exome provides a genetic blueprint for the synthesis of proteins and other functional gene products. It is the most functionally relevant part of the genome and therefore most likely to influence the phenotype of an organism. It has been estimated that the exome of the human genome comprises 1.5% of the total genome (Ng, PC et al., PLoS Gen., 4(8): 1-15, 2008).

[0156] The term “transcriptome” refers to the entire RNA molecule set, including mRNA, rRNA, tRNA, and other non-coding RNA produced in a cell or cell population. In the context of this invention, the transcriptome means the entire RNA molecule set produced in a cell, cell population, preferably a cancer cell population, or all cells of a given individual at a given point in time.

[0157] According to the present invention, "nucleic acid" is preferably deoxyribonucleic acid (DNA or ribonucleic acid (RNA), more preferably RNA, and most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, nucleic acids include genomic DNA, cDNA, mRNA, recombinant and chemically synthesized molecules. According to the present invention, nucleic acids can exist in the form of single-stranded or double-stranded molecules and linear or covalently circular closed molecules. According to the present invention, nucleic acids can be isolated.According to the invention, the term "isolated nucleic acid" means that the nucleic acid (i) is amplified in vitro, for example by polymerase chain reaction (PCR); (ii) is produced by clonal recombination; (iii) is purified, for example by cleavage and separation by gel electrophoresis; or (iv) is synthesized, for example by chemical synthesis. Nucleic acids can be used for introduction into cells, i.e., transfection, particularly in the form of RNA prepared by in vitro transcription from a DNA template. Furthermore, prior to application, the RNA can be modified by stabilizing the sequence, capping, and polyadenylation.

[0158] The term "genetic material" refers to isolated nucleic acid, DNA or RNA, a segment of a double helix, a segment of a chromosome, or the entire genome of an organism or cell, particularly its exome or transcriptome.

[0159] The term "mutation" refers to an alteration or difference (nucleotide substitution, addition, or deletion) in the nucleic acid sequence compared to a reference. "Somatic mutations" can occur in any cell of the body except germ cells (sperm and egg) and are therefore not passed on to children. These alterations can (but are not always) cause cancer or other diseases. Preferably, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that results in an amino acid change (such as an amino acid substitution) in the translation product, preferably a nucleotide substitution.

[0160] According to the invention, the term "mutation" includes point mutations, insertions and deletions (Indel), fusions, chromosome fragmentation (chromothripsis), and RNA editing.

[0161] According to the invention, the term "insertions and deletions" describes a specific class of mutations defined as resulting in the coexistence of insertions and deletions of nucleotides, as well as net gains or losses. In the coding regions of the genome, frameshift mutations occur unless the length of the insertion or deletion is a multiple of 3. Insertions and deletions can be contrasted with point mutations; where insertions and deletions insert and delete nucleotides from the sequence, while point mutations are a form of substitution that replaces a nucleotide.

[0162] Fusions can produce hybrid genes formed from two previously separate genes. It can be caused by translocations, intermediate deletions, or chromosomal inversions. Typically, fusion genes are oncogenes. Oncogenes can produce gene products with novel functions or functions different from those of the two fusion genes from which they originate. Optionally, a proto-oncogene is fused with a strong promoter, thus initiating carcinogenesis through upregulation caused by the strong promoter of the upstream fusion portion. Oncogenes can also be caused by trans-splicing or readthrough events.

[0163] According to the invention, the term "chromosome fragmentation" refers to a genetic phenomenon in which specific regions of the genome are broken and then linked together via a single destructive event.

[0164] According to the present invention, the term “RNA edit” or “RNA editing” refers to a molecular process in which the information content of an RNA molecule is altered by chemical changes in its base composition. RNA editing includes nucleoside modifications such as deamination of cytosine (C) to uracil (U) and adenine (A) to hypoxanthine (I), as well as the addition and insertion of non-template nucleotides. RNA editing in mRNA effectively alters the amino acid sequence of the encoded protein, thereby making it different from that predicted by the genomic DNA sequence.

[0165] The term “cancer mutation marker” refers to a set of mutations present in cancer cells when compared with non-cancer reference cells.

[0166] According to the present invention, a “reference” can be used to correlate and compare results obtained from tumor samples using the methods of the present invention. Typically, a “reference” can be obtained based on one or more normal samples, particularly unaffected by cancer from patients or one or more different individuals, preferably healthy individuals, particularly individuals of the same species. A “reference” can be determined empirically by testing a sufficiently large number of normal samples.

[0167] According to the present invention, any suitable sequencing method can be used to determine mutations, with next-generation sequencing (NGS) technology being preferred. Future third-generation sequencing may replace NGS technology to accelerate the sequencing steps in the method. For clarification purposes: in the context of this invention, the terms “next-generation sequencing” or “NGS” mean all high-throughput sequencing technologies that, compared to “conventional” sequencing methodologies known as Sanger chemistry, read nucleic acid templates in parallel and randomly along the entire genome by dividing the entire genome into small fragments. Such NGS technologies (also known as massively parallel sequencing technologies) are capable of delivering nucleic acid sequence information of the entire genome, exome, transcriptome (the entire transcribed sequence of the genome), or methylome (the entire methylated sequence of the genome) in a very short time period, for example, within 1-2 weeks, preferably within 1-7 days, or most preferably within less than 24 hours, and in principle allow for single-cell sequencing methods.In the context of this invention, various commercially available NGS platforms or those mentioned in the literature may be used, such as those described in Zhang et al. 2011: The impact of next-generation sequencing on genomics. J. Genet Genomics 38 (3), 95-109; or Voelkerding et al. 2009: Next generation sequencing: From basic research to diagnostics. Clinical chemistry 55, 641-658. Non-limiting examples of such NGS technologies / platforms include:

[0168] 1) Synthetic sequencing technology called pyrophosphate sequencing, used in, for example, the GS-FLX 454 Genome Sequencer™ (Branford, Connecticut) of Roche Associates 454 Life Sciences, first described in Ronaghi et al. 1998: A sequencing method based on real-time pyrophosphate. Science 281 (5375), 363-365. This technology utilizes emulsion PCR (emulsion PCR amplification), in which single-stranded DNA-binding beads are encapsulated in aqueous micelles containing PCR reactants surrounded by oil by a powerful eddy current. During pyrophosphate sequencing, light emitted by phosphate molecules upon nucleotide incorporation is recorded as DNA strands synthesized by polymerase.

[0169] 2) By Solexa (now Illumina Inc., San Diego, A synthetic sequencing method developed by a company in California (part of the state) is based on reversible dye terminators and is used in instruments such as the Illumina / Solexa Genome Analyzer™ and Illumina HiSeq 2000 Genome Analyzer™. In this technique, all four nucleotides and DNA polymerase are simultaneously added to an oligonucleotide primer cluster fragment within a flow cell channel. Bridge amplification extends the cluster strand using all four fluorescently labeled nucleotides for sequencing.

[0170] 3) Ligation sequencing methods, such as the ligation sequencing method used in the SOLid™ platform of Applied Biosystems (now Life Technologies Corporation, Carlsbad, California). In this technique, a pool of all possible oligonucleotides of a fixed length is labeled according to the sequencing site. The oligonucleotides are annealed and ligated; the preferential ligation of matching sequences by DNA ligase generates signal information for the nucleotides at that site. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing only a copy of the same DNA molecule, are placed on a glass slide. As a second example, the Polonator™ G.007 platform of Dover Systems (Salem, New Hampshire) also employs ligation sequencing methods, amplifying DNA fragments for parallel sequencing by utilizing randomly arranged, bead-based emulsion PCR amplification.

[0171] 4) Single-molecule sequencing technologies, such as the single-molecule sequencing technology used in the PacBio RS system of Pacific Biosciences (Menlo Park, California) or the HeliScope™ platform of Helicos Biosciences (Cambridge, Massachusetts). A significant feature of this technology is its ability to sequence single DNA or RNA molecules without amplification, which is defined as single-molecule real-time (SMRT) DNA sequencing. For example, HeliScope uses a highly sensitive fluorescence detection system to directly detect each nucleotide during synthesis. Visigen Biotechnology (Houston, Texas) has developed a similar technology based on fluorescence resonance energy transfer (FRET). Other fluorescence-based single-molecule technologies come from US Genomics (GeneEngine™) and Genovox (AnyGene™).

[0172] 5) Nanotechnology for single-molecule sequencing, in which different nanostructures are used, for example, arranged on a chip to monitor the movement of polymerase molecules on a single strand during replication. Non-limiting examples of nanotechnology-based methods include Oxford Nanopore Technologies' (Oxford, UK) GridON™ platform, Nabsys' (Providence, Rhode Island) hybridization-assisted nanopore sequencing (HANS™) platform, and a proprietary ligase-based DNA sequencing platform with DNA nanosphere (DNB) technology called combinatorial probe anchoring ligation (cPAL™).

[0173] 6) Single-molecule sequencing technologies based on electron microscopy, such as those developed by LightSpeed ​​Genomics (Sunnyvale, California) and Halcyon Molecular (Redwood City, California).

[0174] 7) Ion semiconductor sequencing based on the detection of hydrogen ions released during DNA polymerization. For example, Ion Torrent Systems (San Francisco, California) implements this biochemical process in massively parallel using a high-density array of micromechanical pores. Each pore contains a different DNA template. Below the pore is an ion-sensitive layer, and below that is a proprietary ion sensor.

[0175] Preferably, DNA and RNA preparations are used as starting materials for NGS. Such nucleic acids can be readily obtained from samples such as biological materials, for example from fresh, rapidly frozen, or formalin-fixed paraffin-embedded tumor tissue (FFPE) or from freshly isolated cells or from CTCs present in the peripheral blood of a patient. Normal, non-mutated genomic DNA or RNA can be extracted from normal somatic tissues; however, germ cells are preferred in the case of this invention. Germline DNA or RNA can be extracted from peripheral blood mononuclear cells (PBMCs) of patients with non-hematologic malignancies. Although nucleic acids extracted from FFPE tissue or freshly isolated single cells are highly fragmented, they are suitable for NGS applications. Specification 18 / 48 pages 21 CN 121130065 A

[0176] Several targeted NGS methods for exome sequencing are described in the literature (for review, see, for example, Teer and Mullikin 2010: Human Mol Genet 19 (2), R145-51), all of which can be used in conjunction with the present invention. Many of these methods (described as, for example, genome capture, genome partitioning, genome enrichment, etc.) utilize hybridization techniques and include array-based hybridization methods (e.g., Hodges et al. 2007: Nat. Genet. 39, 1522–1527) and liquid-based hybridization methods (e.g., Choi et al. 2009: Proc. Natl. Acad. Sci. USA 106, 19096–19101). Commercial kits for DNA sample preparation and subsequent exome capture are also available: for example, Illumina Inc. (San Diego, California) offers the TruSeq™ DNA Sample Preparation Kit and the TruSeq™ Exome Enrichment Kit.

[0177] To reduce the number of false positives in the detection of cancer-specific somatic mutations or sequence differences, when comparing, for example, tumor sample sequences with reference sample sequences such as germ cell sample sequences, it is preferable to determine the replicate sequences of one or both of these sample types. Therefore, it is preferable to determine the sequence of the reference sample, such as the germline sample, two, three, or more times. Optionally or additionally, the sequence of the tumor sample is determined two, three, or more times. It is also possible to determine the sequence of the reference sample (e.g., the germline sample sequence) and / or the tumor sample multiple times by determining the genomic DNA sequence at least once and the RNA sequence in the reference sample and / or the tumor sample at least once. For example, by determining the changes between replicas of the reference sample, such as the germline sample, the expected rate of false positive (FDR) somatic mutations can be estimated as a statistic. Technical repetitions of the samples should produce identical results, and any changes in the detection during this "identical to identical comparison" are false positives. In particular, to determine the false detection rate of somatic mutation detection of the tumor sample relative to the reference sample, the technical repetition of the reference sample can be used as a reference to estimate the number of false positives. Furthermore, machine learning methods can be used to combine different quality-related metrics (e.g., coverage or SNP quality) with a single quality score. For a given somatic variant, all other variants exceeding the quality score can be calculated, making it possible to rank all variants in the dataset.

[0178] In the context of this invention, the term "RNA" refers to a molecule containing at least one ribonucleotide residue, and preferably consists entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide containing a hydroxyl group at the 2'-position of the β-D-furanose group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (such as partially or completely purified RNA), substantially purified RNA, synthetic RNA, and recombinant RNA such as modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances, such as addition to the ends or interior of the RNA, for example at one or more nucleotides of the RNA. Nucleotides in the RNA molecule may also contain non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0179] According to the invention, the term "RNA" includes and preferably refers to "mRNA". The term "mRNA" means "messenger RNA" and refers to a "transcript" generated using a DNA template and encoding a peptide or polypeptide. Typically, mRNA contains a 5'-UTR, a protein-coding region, and a 3'-UTR. mRNA has only a limited half-life in cells and in vitro.In the case of the present invention, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodology is known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0180] According to the present invention, the stability and translation efficiency of RNA can be improved as needed. For example, RNA can be stabilized and translation enhanced by one or more RNA modifications that have stabilizing effects and / or increase translation efficiency. Such modifications are described, for example, in PCT / EP2006 / 009448, which is incorporated herein by reference. In order to increase the expression of the RNA used according to the present invention, the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein, can be modified to increase GC- content, thereby enhancing the stability of the mRNA and implementing codon optimization, and thus enhancing translation in the cell.

[0181] In the case of the RNA used in the present invention, the term "modification" includes any RNA modification that is not naturally present in said RNA.

[0182] In one embodiment of the invention, the RNA used according to the invention does not contain uncapped 5'-triphosphate. Such uncapped 5'-triphosphate can be removed by treating the RNA with a phosphatase.

[0183] The RNA according to the invention may contain modified ribonucleotides to increase its stability and / or reduce cytotoxicity. For example, in one embodiment, the RNA used according to the invention is partially or completely substituted with 5-methylcytosine nucleoside, preferably completely substituted. Optionally or additionally, in one embodiment, the RNA used according to the invention is partially or completely substituted with pseudouridine, preferably completely substituted.

[0184] In one embodiment, the term "modification" refers to providing a 5'-cap or a 5'-cap analogue to the RNA. The term "5'-cap" refers to a cap structure found at the 5' end of an mRNA molecule and is typically composed of a guanine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate link. In one embodiment, the guanine nucleotide is methylated at the 7-position. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanine nucleoside cap (m7G). In the context of this invention, the term "5'-cap" includes 5'-cap analogs that are structurally similar to RNA caps and are modified to have the ability to stabilize RNA and / or enhance RNA translation (if attached thereto), preferably in vivo and / or in cells.

[0185] RNA having a 5'-cap or a 5'-cap analogue can be provided by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analogue, wherein said 5'-cap is co-transcribed into the resulting RNA chain, or said RNA can be produced, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using a capping enzyme, such as a capping enzyme of vaccinia virus.

[0186] The RNA may contain other modifications. For example, other modifications to the RNA used in this invention may be the extension or truncation of a naturally occurring poly(A) tail or alteration of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of said RNA, for example, alteration of an existing 3'-UTR or one or more, preferably derived from the insertion of two copies of the 3'-UTR of a globulin gene (such as α2-globulin, α1-globulin, β-globulin, preferably β-globulin, more preferably human β-globulin).

[0187] RNA with an unmasked poly-A sequence is transcribed more efficiently than RNA with a masked poly-A sequence. The terms “poly(A) tail” or “poly-A sequence” refer to an adenine (A) residue sequence that is normally located at the 3' end of an RNA molecule, and “unmasked poly-A sequence” means that the poly-A sequence at the 3' end of the poly-A sequence of the RNA molecule is terminated by an A in the poly-A sequence and that, except for the A at the 3' end, it is downstream of the poly-A sequence and contains no nucleotides. Furthermore, long poly-A sequences of about 120 base pairs produce optimal RNA transcript stability and translation efficiency.

[0188] Therefore, in order to enhance the stability and / or expression of the RNA used according to the invention, it can be modified to exist in a form linked to a poly-A sequence, preferably 10-500 residues long, more preferably 30-300 residues long, even more preferably 65-200 residues long, and particularly 100-150 adenosine residues long. In a particularly preferred embodiment, the poly-A sequence is approximately 120 adenosine residues in length. To further enhance the stability and / or expression of the RNA used according to the invention, the poly-A sequence may be unmasked.

[0189] Furthermore, incorporating a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can lead to increased translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3'-untranslated regions. The 3'-untranslated region and the RNA into which it is introduced may be autologous or heterologous. In one specific embodiment, the 3'-untranslated region is derived from the human β-globulin gene.Specification 20 / 48 pages 23 CN 121130065 A

[0190] The combination of modifications described above, namely the incorporation of poly-A sequences, the unmasking of poly-A sequences, and the incorporation of one or more 3'-untranslated regions, has a synergistic effect on enhancing RNA stability and translation efficiency.

[0191] The term "stability" of RNA refers to the half-life of RNA. "Half-life" refers to the time period required to eliminate half of the activity, amount, or number of molecules. In the case of the present invention, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the "sustainability of RNA expression". It is expected that RNA with a long half-life will express for an extended period.

[0192] Of course, if it is desired to reduce the stability and / or translation efficiency of RNA according to the present invention, RNA can be modified to interfere with the function of the elements that enhance the stability and / or translation efficiency of RNA as described above.

[0193] The term “expression” as used in this invention is its most common meaning and includes the production of RNA and / or peptides, polypeptides, or proteins, for example, through transcription and / or translation. Regarding RNA, the term “expression” or “translation” specifically relates to the production of peptides, polypeptides, or proteins. It also includes partial expression of nucleic acids. Furthermore, expression can be transient or stable.

[0194] According to this invention, the term expression also includes “aberrant expression” or “abnormal expression.” “Aberrant expression” or “abnormal expression” means that, according to this invention, expression is altered, preferably increased compared to a reference, for example, compared to the state of a subject not suffering from a disease associated with the aberrant or abnormal expression of a certain protein (e.g., a tumor antigen). Increased expression refers to an increase of at least 10%, particularly at least 20%, at least 50%, or at least 100% or more. In one embodiment, expression is present only in diseased tissue, while expression in healthy tissue is suppressed.

[0195] The term "specifically expressed" means that a protein is expressed essentially only in a specific tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that the protein is expressed primarily in the gastric mucosa and is not expressed in other tissues or not expressed to a significant extent in other tissue or organ types. Therefore, a protein that is expressed only in gastric mucosal cells and is expressed at a significantly lower level in any other tissue, such as the testes, is a protein specifically expressed in gastric mucosal cells. In some embodiments, under normal conditions, the tumor antigen may also be specifically expressed in multiple tissue types or organs, such as in two or three tissue types or organs, but preferably in no more than three different tissue or organ types.In this case, the tumor antigen is then specifically expressed in these organs. For example, if the tumor antigen is preferably expressed to approximately equivalent levels in the lungs and stomach under normal conditions, then the antigen is specifically expressed in the lungs and stomach.

[0196] In the case of the present invention, the term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA. Subsequently, the RNA can be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to the process of synthesizing RNA, particularly mRNA, in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are included in the term "vector" according to the present invention. According to the present invention, the RNA used is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter controlling transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription according to the present invention is controlled by the T7 or SP6 promoter. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0197] According to the invention, the term "translation" refers to the process by which a messenger RNA chain in a cellular ribosome directs the assembly of an amino acid sequence to produce a peptide, polypeptide, or protein.

[0198] According to the invention, an expression control sequence or regulatory sequence that can be functionally linked to a nucleic acid can be homologous or heterologous to the nucleic acid. If the coding sequence is controlled by or influenced by the regulatory sequence, the coding sequence and the regulatory sequence are "functionally" linked together. If the coding sequence is to be translated into a functional protein, the induction of the regulatory sequence with the functional linking of the regulatory sequence to the coding sequence results in the transcription of the coding sequence without causing a frame shift of the coding sequence or without causing the coding sequence to be untranslated into the desired protein or peptide.

[0199] According to the invention, the term "expression control sequence" includes promoters, ribosome-binding sequences, and other control elements that control the transcription of nucleic acids or the translation of the obtained RNA. In some embodiments of the invention, a regulatory sequence can be controlled. The precise structure of the regulatory sequence may vary depending on the species or cell type, but typically includes a 5'-untranscribed sequence involved in the initiation of transcription or translation, as well as 5'- and 3'-untranslated sequences, such as TATA-boxes, capped sequences, CAAT sequences, etc. In particular, the 5'-untranscribed regulatory sequence includes a promoter region containing a promoter sequence that functionally binds to the transcriptional control of a gene. The regulatory sequence may also include an enhancer sequence or an upstream activator sequence.

[0200] Preferably, according to the invention, RNA expressed in the cell is introduced into the cell. In one embodiment of the method according to the invention, the RNA to be introduced into the cell is obtained by in vitro transcription of a suitable DNA template.

[0201] According to the invention, terms such as “RNA capable of expressing…” and “RNA encoding…” are used interchangeably herein, and with respect to a specific peptide or polypeptide, it means that, if present in a suitable environment, preferably in a cell, the RNA is capable of expression to produce the peptide or polypeptide. Preferably, according to the invention, the RNA is capable of interacting with cellular translation mechanisms to provide the peptide or polypeptide it is capable of expressing.

[0202] Terms such as “transferring,” “introducing,” or “transfecting” are used interchangeably herein, and refer to the introduction of nucleic acids, particularly exogenous or heterologous nucleic acids, particularly RNA, into cells. According to the invention, cells can form part of organs, tissues, and / or organisms. According to the invention, the application of nucleic acids is achieved by naked nucleic acids or in combination with an application reagent. Preferably, nucleic acids are applied in the form of naked nucleic acids. Preferably, RNA is applied in combination with a stabilizing substance such as an RNase inhibitor. The present invention also envisions the repeated introduction of nucleic acids into cells to allow for sustained expression over extended periods.

[0203] Cells can be transfected using any vector to which RNA can be linked, for example by forming a complex with RNA or forming a mediator, wherein the RNA is closed or encapsulated, resulting in increased RNA stability compared to naked RNA. Vectors available according to the invention include, for example, lipid-containing vectors, such as cationic lipids (cationic lipids), liposomes, cationic liposomes and micelles, and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used according to the invention.

[0204] Preferably, RNA encoding a peptide or polypeptide is introduced into cells, particularly into cells present in vivo, resulting in the expression of said peptide or polypeptide within the cells. In a particular embodiment, nucleic acids that target specific cells are preferred. In such embodiments, the vector used to administer nucleic acids to cells (e.g., retroviruses or liposomes) displays the target molecule. For example, molecules specific to surface membrane proteins on target cells (such as antibodies) or ligands of receptors on target cells can be incorporated into or bound to the nucleic acid vector. In the case of nucleic acid administration via liposomes, proteins that bind to surface membrane proteins associated with endocytosis can be incorporated into the liposome formulation, thereby enabling targeting and / or uptake. Such proteins include capsid protein fragments specific to specific cell types, antibodies against internalized proteins, proteins targeting intracellular sites, etc.

[0205] The term "cell" or "host cell" preferably refers to an intact cell, i.e., a cell with an intact membrane that has not yet released its normal intracellular components such as enzymes, organelles, or genetic material.Intact cells are preferably live cells, i.e., live cells capable of performing their normal metabolic functions. According to the invention, the term preferably refers to any cell that can be transformed or transfected with exogenous nucleic acids. According to the invention, the term "cell" includes prokaryotic cells (e.g., *Escherichia coli*) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). Exogenous nucleic acids may be present within the cell in the following forms: (i) freely dispersed, (ii) incorporated into a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. Mammalian cells, such as those from humans, mice, hamsters, pigs, sheep, and primates, are particularly preferred. Cells can be derived from a variety of tissue types and include primary cells and cell lines. Specific examples include cytoplasmic cells, peripheral blood lymphocytes, bone marrow stem cells, and embryonic stem cells. (See page 22 / 48 of the specification, CN 121130065 A). In other embodiments, the cells are antigen-presenting cells, particularly dendritic cells, monocytes, or macrophages.

[0206] Cells containing nucleic acid molecules preferably express peptides or polypeptides encoded by said nucleic acids.

[0207] The term "clonal expansion" refers to the process of increasing the number of specific entities. In the context of the invention, this term is preferably used for the immune response in which lymphocytes are stimulated by an antigen, proliferate, and recognize said antigen through the expansion of specific lymphocytes. Preferably, clonal expansion leads to lymphocyte differentiation.

[0208] Terms such as "reduction" or "inhibition" relate to the ability to cause an overall reduction in levels, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more. The term "inhibition" or similar phrases include complete or substantially complete inhibition, i.e., reduction to zero or substantially reduced to zero.

[0209] Terms such as “increase,” “enhance,” “promote,” or “prolong” preferably refer to an increase, enhancement, promotion, or prolongation of about 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 80%, preferably at least 100%, preferably at least 200%, and particularly at least 300%. These terms may also refer to an increase, enhancement, promotion, or prolongation of more than zero or to a measurable or detectable level from zero or an unmeasurable or undetectable level.

[0210] The present invention provides vaccines, such as cancer vaccines, designed based on amino acid modifications or modified peptides predicted to be immunogenic by the methods of the present invention.

[0211] According to the present invention, the term “vaccine” refers to a pharmaceutical preparation (pharmaceutical composition) or product that, upon administration, induces an immune response, particularly a cellular immune response, to recognize and attack antigens or diseased cells such as cancer cells. Vaccines can be used for the prevention or treatment of diseases.The term "personalized cancer vaccine" or "individualized cancer vaccine" refers to a specific cancer patient and means a cancer vaccine tailored to the needs or special circumstances of an individual cancer patient.

[0212] In one embodiment, the vaccine provided according to the invention may comprise a peptide or polypeptide or a nucleic acid, preferably RNA, encoding said peptide or polypeptide, wherein said modification or modified peptide contains one or more amino acid modifications or one or more modified peptides predicted to be immunogenic by the methods of the invention.

[0213] The cancer vaccine provided according to the invention, when administered to a patient, provides one or more T cell epitopes suitable for stimulating, initiating, and / or amplifying patient tumor-specific T cells. The T cells are preferably targeting cells expressing antigens from which the T cell epitopes are derived. Therefore, the vaccine described herein is preferably capable of inducing or promoting a cellular response against cancer characterized by presentation of one or more tumor-associated neoantigens by class I MHC, preferably cytotoxic T cell activity. Since the vaccine provided according to the invention targets cancer-specific mutations, it is specific to the patient's tumor.

[0214] The vaccine provided according to the invention relates to a vaccine, which, when administered to a patient, preferably provides one or more T-cell epitopes, such as 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, and preferably up to 60, 55, 45, 40, 35, or 30 T-cell epitopes, which are combined with amino acid modifications or modified peptides predicted to be immunogenic by the method of the invention. Such cell epitopes are also referred to herein as “novel epitopes.” These epitopes, presented by the patient’s cells, particularly antigen-presenting cells, preferably result in T cells targeting the epitopes when bound to MHC, thus the patient’s tumor, preferably a primary tumor and tumor metastases, expresses antigens derived from the T-cell epitopes and presents the same epitopes on the surface of tumor cells.

[0215] The method of the invention may include further steps of determining the availability of the identified amino acid modifications or modified peptides in a cancer vaccine.Therefore, other steps may include one or more of the following: (i) assessing whether the modification is located within a known or predicted MHC-presenting epitope; (ii) in vitro and / or computer simulation testing to determine whether the modification is located within an MHC-presenting epitope, for example, testing whether the modification is part of a peptide sequence presented as and / or as an MHC-presenting epitope, as per specification page 23 / 48, page 26, CN 121130065 A; and (iii) in vitro testing whether the epitope of the intended modification, particularly when present in its native sequence, such as when flanked by an amino acid sequence of an epitope also flanked in a naturally occurring protein, and when expressed on antigen-presenting cells, can stimulate T cells such as patient T cells with the desired specificity. Each such flanking sequence may contain 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, 45, 40, 35 or 30 amino acids, and may have epitope sequences flanking the N-terminus and / or C-terminus.

[0216] Modified peptides determined according to the invention may be arranged according to their availability as epitopes in cancer vaccines. Thus, in one aspect, the method of the invention includes a manual or computer-based analytical process in which identified modified peptides are analyzed and their availability in their respective provided vaccines is selected. In a preferred embodiment, the analytical process is a computational algorithm-based process. Preferably, the analytical process includes determining and / or arranging epitopes based on predictions of their immunogenicity.

[0217] The novel epitopes identified according to the invention and provided by the vaccines of the invention are preferably in the form of polypeptides containing said novel epitopes, such as multi-epitope polypeptides or nucleic acids, particularly RNA, encoding said polypeptides. Furthermore, the novel epitope can exist in the polypeptide in the form of a vaccine sequence, i.e., in its native sequence environment, for example, flanked by an amino acid sequence flanked by the epitope in a naturally occurring protein. Each such flanking sequence may contain 5 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, 45, 40, 35 or 30 amino acids, and epitope sequences may be flanked at the N-terminus and / or C-terminus. Thus, the vaccine sequence may contain 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, and preferably up to 50, 45, 40, 35 or 30 amino acids. In one embodiment, the novel epitope and / or vaccine sequence is arranged head-to-tail in the polypeptide.

[0218] In one embodiment, the novel epitope and / or vaccine sequence is separated by linkers, particularly neutral linkers.According to the present invention, the term "linker" refers to a peptide added between two peptide domains, such as epitopes or vaccine sequences, for linking said peptide domains. There are no particular limitations on the linker sequence. However, it is preferred that the linker sequence reduces steric hindrance between the two peptide domains, is well translated, and supports or allows processing of said epitopes. Furthermore, the linker should be free of immunogenic sequence elements or contain only low-immunogenic sequence elements. The linker preferably should not generate non-endogenous novel epitopes that may produce undesirable immune responses (such as those generated by sutures between adjacent novel epitopes). Therefore, multi-epitope vaccines should preferably contain linker sequences capable of reducing the number of undesirable NHC-binding linker epitopes. Hoyt et al. (EMBO J. 25(8), 1720–9, 2006) and Zhang et al. (J. Biol. Chem., 279(10), 8635–41, 2004) have shown that glycine-rich sequences impair proteasome processing, and therefore the application of glycine-rich linker sequences minimizes the number of linker-containing peptides that can be processed by the proteasome. Furthermore, glycine has been observed to inhibit strong binding within MHC binding groove sites (Abastado et al., J. Immunol. 151(7), 3569–75, 1993). Schlessinger et al. (Proteins, 61(1), 115–26, 2005) have found that the presence of glycine and serine in amino acid sequences leads to more efficient translation and proteasome processing of more flexible proteins, enabling better access to the encoded epitopes. Each linker may contain 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, 45, 40, 35, or 30 amino acids. Preferably, the linker is rich in glycine and / or serine. Preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the amino acids in the linker are glycine and / or serine. In a preferred embodiment, the linker is essentially composed of glycine and serine.In one embodiment, the linker comprises the amino acid sequence (GGS)a(GSS)b(GGG)c(SSG)d(GSG)e, wherein a, b, c, d, and e are numbers independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and wherein a + b + c + d + e is not 0 and is preferably 2 or more, 3 or more, 4 or more, or 5 or more. In one embodiment, the linker comprises a sequence as described herein, such as the linker sequence GGSGGGGSG.

[0219] In a particularly preferred embodiment, according to the invention, a polypeptide, such as a multi-epitope polypeptide, incorporating one or more novel epitopes, is administered to a patient in the form of a nucleic acid, preferably RNA, such as in vitro transcribed or synthesized RNA, which can be expressed in patient cells, such as antigen-presenting cells, to produce the polypeptide. The present invention also contemplates the administration of one or more multi-epitope polypeptides, which, for the purposes of the invention, include the term "multi-epitope polypeptide," preferably administered in the form of nucleic acids, preferably RNA such as in vitro transcribed or synthesized RNA, which can be expressed in patient cells, such as antigen-presenting cells, to produce one or more polypeptides. In the case of administration of multiple multi-epitope polypeptides, the new epitopes provided by the different multi-epitope polypeptides may be different or partially overlapping. Once present in patient cells, such as antigen-presenting cells, the polypeptides are processed according to the invention to produce new epitopes identified according to the invention. Administration of the vaccine provided according to the invention can provide MHC class II presenting epitopes capable of eliciting a CD4+ helper T cell response against cells expressing the source antigen of the MHC presenting epitope. Optionally or additionally, administration of the vaccine provided according to the invention can provide MHC class I presenting epitopes capable of eliciting a CD8+ T cell response against cells expressing the source antigen of the MHC presenting epitope. Furthermore, administration of the vaccine provided according to the invention can provide one or more novel epitopes (including known novel epitopes and novel epitopes identified according to the invention) and one or more epitopes that do not contain cancer-specific somatic mutations but are expressed by cancer cells and preferably induce an immune response against cancer cells (preferably a cancer-specific immune response). In one embodiment, administration of the vaccine provided according to the invention provides a novel epitope that is an MHC class II presented epitope and / or capable of eliciting a CD4+ helper T cell response against a source antigen expressing an MHC presented epitope, and provides an epitope that does not contain cancer-specific somatic mutations that is an MHC class I presented epitope and / or capable of eliciting a CD8+ T cell response against a source antigen expressing an MHC presented epitope.In one embodiment, the epitope that does not contain cancer-specific somatic mutations is derived from a tumor antigen. In one embodiment, the novel epitope and the epitope that does not contain cancer-specific somatic mutations have a synergistic effect in the treatment of cancer. Preferably, the vaccine provided according to the invention is suitable for multi-epitope stimulation of cytotoxic T cells and / or helper T cell responses.

[0220] The vaccine provided according to the invention may be a recombinant vaccine.

[0221] In the context of the invention, the term “recombinant” means “generated by genetic engineering.” Preferably, in the context of the invention, “recombinant entities” such as recombinant polypeptides are not naturally occurring and are preferably the result of combining entities such as amino acids or nucleic acids that are not naturally combined. For example, in the context of the invention, a recombinant polypeptide may comprise several amino acid sequences, such as novel epitopes or vaccine sequences derived from different proteins fused together (e.g., by peptide bonds or suitable linkers) or different portions of the same protein.

[0222] As used herein, the term “naturally occurring” refers to the fact that the object can exist naturally. For example, peptides or nucleic acids present in organisms (including viruses), which can be isolated from natural sources and have not yet been intentionally modified by researchers are naturally occurring.

[0223] The reagents, compositions, and methods described herein can be used to treat subjects suffering from diseases, such as diseases characterized by the presence of diseased cells expressing antigens and the presentation of antigen fragments. Particularly preferred diseases are cancer. The reagents, compositions, and methods described herein can also be used for immunization or vaccination to prevent diseases described herein.

[0224] According to the invention, the term "disease" refers to any pathological state, including cancer, particularly those forms of cancer described herein.

[0225] The term "normal" refers to the healthy state or condition of a healthy subject or tissue, i.e., a non-pathological condition, wherein "healthy" preferably means non-cancerous. Specification 25 / 48 pages 28 CN 121130065 A

[0226] According to the invention, "disease involving cells expressing antigens" means that the expression of antigens is detected in the cells of diseased tissues or organs. The expression in the cells of diseased tissues or organs may be increased compared to the state in healthy tissues or organs. An increase refers to an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed. According to the invention, diseases involving cells expressing antigens or associated with cells expressing antigens include cancer.

[0227] According to the invention, the term "tumor" or "tumor disease" refers to the abnormal growth of cells (called neoplastic cells, tumor-producing cells, or tumor cells), preferably forming swelling or lesions."Tumor cells" means abnormal cells that grow through rapid, uncontrolled cell proliferation and continue to grow after a stimulus that stops the initiation of new growth. Tumors exhibit partial or complete loss of the structural organization and functional coordination of normal tissue and typically form dissected clumps of tissue, which can be benign, pre-malignant, or malignant.

[0228] Cancer (medical term: malignant tumor) is a group of diseases in which cells exhibit uncontrolled growth (dividing beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body via the lymphatic or bloodstream). These three malignant characteristics of cancer distinguish them from benign tumors that are self-limiting and do not invade or metastasize. Most cancers form tumors, but some, such as leukemia, do not. Malignancy, malignant neoplasm, and malignant tumor are essentially synonymous with cancer.

[0229] A neoplasm is an abnormal clump of tissue resulting from neoplasm formation. Tumor formation (new growth in Greek) is the abnormal proliferation of cells. The cells grow beyond the surrounding normal tissue and are out of sync with it. Even after the stimulation ceases, the growth continues in the same excessive manner. It typically causes a mass or tumor. Tumors can be benign, pre-malignant, or malignant.

[0230] According to the invention, “tumor growth” or “tumor development” refers to the tendency of a tumor to increase its size and / or the tendency of tumor cells to proliferate.

[0231] For the purposes of this invention, the terms “cancer” and “cancer disease” are used interchangeably with the terms “tumor” and “tumor disease”.

[0232] Cancers are classified according to the type of tumor-like cells and the tissue of presumed tumor origin. These are histological and site-specific.

[0233] According to the present invention, the term "cancer" includes carcinomas, adenocarcinomas, germ cell tumors, leukemia, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, leukemia, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, and lung cancer, as well as their metastases. Examples of these include lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or metastases of the cancer types or tumors described above. According to the present invention, the term cancer also includes cancer metastasis and cancer recurrence.

[0234] The term “metastasis” refers to the spread of cancer cells from their original site to other parts of the body. The formation of metastasis is a highly complex process and depends on the separation of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membrane into body cavities and blood vessels, and then, after being transported by the blood, infiltration into the target organ. Finally, the growth of the new tumor, i.e., the secondary tumor or metastatic tumor, at the target site depends on angiogenesis. Tumor metastasis often occurs, even after the removal of the primary tumor, because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention refers to “distant metastasis,” which involves metastasis away from the primary tumor and the local lymph node system. Specification 26 / 48 pages 29 CN 121130065 A

[0235] The cells of a secondary tumor or metastatic tumor are the same as those in the original tumor. This means, for example, if ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells, rather than abnormal liver cells. The tumor in the liver is then referred to as metastatic ovarian cancer, rather than liver cancer.

[0236] The term “circulating tumor cells” or “CTCs” refers to cells isolated from a primary tumor or tumor metastasis and circulating in the blood. CTCs can serve as seed cells for the subsequent growth of other tumors (metastases) in different tissues. Circulating tumor cells are found at a frequency of approximately 1–10 CTCs per mL of whole blood in patients with metastatic disease. Research methods for isolating CTCs have been developed. Several research methods for isolating CTCs have been described in the art, such as techniques that utilize the fact that epithelial cells typically express the cell adhesion protein EpCAM, which is not present in normal blood cells. Capture based on immunomagnetic beads involves treating a blood sample with an EpCAM antibody linked to magnetic particles, followed by separation of the labeled cells in a magnetic field. The separated cells are then stained with antibodies against another epithelial marker, cytokeratin, and a common leukocyte marker, CD45, thereby distinguishing the small number of CTCs from contaminated leukocytes. This robust and semi-automated method identifies CTCs with an average yield of approximately 1 CTC / mL and a purity of 0.1% (Allard et al., 2004: Clin Cancer Res 10, 6897–6904). A second method for isolating CTCs utilizes a microfluidic-based CTC capture device, which involves passing whole blood through a chamber embedded with 80,000 microspheres, functionalizing the microspheres by coating them with an EpCAM antibody. The CTCs are then stained with a second antibody targeting cytokeratin or tissue-specific markers (such as PSA in prostate cancer or HER2 in breast cancer), and visualized by automated scanning of the microspheres along three-dimensional coordinates in multiple planes.CTC-arrays can identify cytokeratin-positive circulating tumor cells in patients with an average yield of 50 cells / ml and a purity range of 1–80% (Nagrath et al., 2007: Nature 450, 1235–1239). Another possibility for isolating CTCs is using Veridex, LLC (Raritan, NJ)’s CellSearch™ circulating tumor cell (CTC) detection, which captures, identifies, and counts CTCs in in-tube blood. The CellSearch™ system is an FDA-approved method for counting CTCs in whole blood, based on a combination of immunomagnetic labeling and automated digital microscopy. Other methods for isolating CTCs are described in the literature, all of which can be used in conjunction with this invention.

[0237] Relapse or recurrence occurs when a person is again affected by a past condition that affected them. For example, if a patient has suffered from a tumor, received successful treatment for the tumor, and then suffers from the tumor again, the newly acquired disease can be considered a relapse or recurrence. However, according to the present invention, a relapse or recurrence of a tumor may, but does not necessarily, occur at the site of the original tumor. Thus, for example, if a patient suffers from an ovarian tumor and has received successful treatment, a relapse or recurrence could be the occurrence of the ovarian tumor or the tumor in a location different from the ovary. A tumor relapse or recurrence also includes the occurrence of the tumor in a location different from the original tumor site, as well as the occurrence at the original tumor site. Preferably, the original tumor that the patient has already treated is a primary tumor, and the tumor located in a location different from the original tumor site is a secondary tumor or a metastatic tumor.

[0238] By “treatment” means administering to a subject a compound or composition as described herein to prevent or eliminate a disease, including reducing the size or number of tumors in the subject; preventing or slowing the development of a disease in the subject; inhibiting or slowing the development of a new disease in the subject; reducing the frequency and severity of symptoms and / or recurrences in a subject currently suffering from or having previously suffered from the disease; and / or prolonging, i.e., increasing the lifespan of the subject. In particular, the term “disease treatment” includes curing, shortening the duration, improving, preventing, slowing or inhibiting the progression or deterioration of a disease, or preventing or delaying the onset of a disease or its symptoms.

[0239] By “at risk of” means a subject, i.e. a patient, who is identified as having a higher probability of having a disease, particularly cancer, than the general population. Furthermore, subjects who have had or currently have a disease, particularly cancer, are subjects with an increased probability of having the disease because such subjects may continue to have the disease.Subjects currently suffering from or having previously suffered from cancer (pages 27 / 48, 30 CN 121130065 A) also have an increased likelihood of cancer metastasis.

[0240] The term "immunotherapy" refers to treatment involving activation of a specific immune response. In the context of this invention, terms such as "protective," "preventive," "prophylactic," "protective," or "protective" refer to both the prevention or treatment of a disease in a subject, or the occurrence and / or spread of the disease, particularly to minimize the likelihood of the patient developing the disease or to delay the development of the disease. For example, individuals at risk of developing cancer, as described above, are candidates for cancer prevention treatments.

[0241] Prophylactic administration of immunotherapy, such as prophylactic administration of the vaccine of this invention, preferably protects the recipient from the disease. Therapeutic administration of immunotherapy, such as therapeutic administration of the vaccine of this invention, can result in inhibition of disease development / growth. This includes slowing the development / growth of the disease, particularly disrupting the development of the disease, which preferably leads to the clearance of the disease.

[0242] Immunotherapy can be implemented using any of a variety of techniques, wherein the function of the agents provided herein is to remove diseased cells from the patient. Such removal can occur due to enhanced or induced specific immune responses in the patient to antigens or cells expressing antigens.

[0243] In some embodiments, immunotherapy can be active immunotherapy, wherein treatment depends on the in vivo stimulation of the endogenous host immune system by the administration of immune response modifiers (such as peptides and nucleic acids described herein), thereby acting against diseased cells.

[0244] The reagents and compositions provided herein can be used alone or in combination with conventional treatment regimens such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0245] The terms “immune” or “vaccination” describe a process of treating a subject with the aim of inducing an immune response for a cause of treatment or prevention.

[0246] The term “in vivo” refers to a location within the subject.

[0247] The terms “subject,” “individual,” “organism,” or “patient” are used interchangeably and refer to vertebrates, preferably mammals. For example, in the context of this invention, mammals are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals such as animals in zoos. The term "animal" as used herein also includes humans. The term "object" may also include patients, i.e., animals, preferably humans suffering from diseases as described herein.

[0248] The term "autologous" is used to describe anything derived from the same object. For example, "autologous transplantation" refers to the transplantation of tissues or organs derived from the same object. Such arrangements are advantageous because they overcome immune barriers that would otherwise lead to rejection.

[0249] The term "heterogeneous" is used to describe something composed of a variety of different elements.As an example, transplanting bone marrow from one individual into another constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the recipient.

[0250] As part of a composition for immunization or vaccination, it is preferred to administer one or more of the agents described herein in conjunction with one or more adjuvants to induce or enhance an immune response. The term "adjuvant" refers to a compound that prolongs, enhances, or promotes an immune response. It is preferred that the compositions of the invention function without the addition of an adjuvant. However, the compositions of the invention may contain any known adjuvant. Adjuvants include a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), liposomes, and immunostimulatory complexes. An example of an adjuvant is monophospholipid A (MPL SmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96 / 33739), QS7, QS17, QS18, and QS-L1 (So et al., 1997, Mol.Cells 7: 178-186), Freund's incomplete adjuvant, Freund's complete adjuvant, vitamin E, montanid, alum, CpG oligonucleotides (Krieg et al., 1995, Nature 374: 28 / 48 pages, 31 CN 121130065 A 546-549), and various water-in-oil emulsions prepared from biodegradable oils such as squalene and / or tocopherol.

[0251] Other substances that stimulate the patient's immune response may also be administered. It is possible, for example, that cytokines are used in vaccination due to their regulatory properties on lymphocytes. Such cytokines include, for example, interleukin-12 (IL-12), GM-CSF, and IL-18, with interleukin-12 showing increased protective effects against vaccines (cf. Science 268:1432-1434, 1995).

[0252] Many compounds exist that enhance immune responses and can therefore be used in vaccination. These compounds include co-stimulatory molecules such as B7-1 and B7-2 (CD80 and CD86, respectively), provided in protein or nucleic acid form.

[0253] According to the invention, a body sample can be a tissue sample, including body fluids and / or cell samples. Such body samples can be obtained by conventional means, such as by biopsy, including drill biopsy, and by obtaining blood, bronchial aspirate, sputum, urine, feces, or other body fluids. According to the invention, the term "sample" also includes fragments or isolates of processed samples such as biological samples, for example, nucleic acid or cell isolates.

[0254] The reagents, vaccines, and compositions described herein can be administered via any conventional route, including by injection or infusion. Administration can be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, or percutaneously. In one embodiment, administration can be intranodal, such as by intralymphatic injection. Other forms of administration envision in vitro transfection of antigen-presenting cells, such as dendritic cells, with the nucleic acids described herein, followed by administration of the antigen-presenting cells.

[0255] The reagents described herein are administered in an effective amount. An "effective amount" refers to an amount, alone or in combination with other doses, that achieves the desired response or desired effect. In the case of treatment for a specific disease or condition, the desired response preferably involves the inhibition of disease progression. This includes slowing the progression of the disease, and in particular, blocking or reversing the progression of the disease. The desired response in the treatment of a disease or condition can also be the delay or prevention of the onset of said disease or condition.

[0256] The effective amount of the reagents described herein depends on the condition to be treated, the severity of the disease, individual patient parameters including age, physical condition, size and weight, duration of treatment, type of concomitant treatment (if present), specific route of administration, and similar factors. Therefore, the dosage of the reagents described herein may depend on different such parameters. Higher doses may be used (or more effectively, through different, more localized routes of administration) where the initial dose does not elicit a sufficient response in the patient.

[0257] The pharmaceutical compositions described herein are preferably sterile and contain an effective amount of the therapeutically active substance to produce the desired response or desired effect.

[0258] The pharmaceutical compositions described herein are generally administered in pharmaceutically compatible amounts and pharmaceutically compatible formulations. The term "pharmaceutically compatible" refers to a non-toxic substance that does not interact functionally with the active component in the pharmaceutical composition. Such formulations may typically contain salts, buffers, preservatives, carriers, complementary immune enhancers such as adjuvants (e.g., CpG oligonucleotides, cytokines, chemokines, saponins, GM-CSF, and / or RNA), and other suitable therapeutically active compounds. When used in pharmaceuticals, the salt should be pharmaceutically compatible. However, non-pharmaceutically compatible salts may be used to prepare pharmaceutically compatible salts, and these are included in this invention. Such pharmacologically and pharmaceutically compatible salts include, in a non-limiting manner, those prepared from acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically compatible salts may also be prepared as basic metal salts or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0259] The pharmaceutical compositions described herein may comprise pharmaceutically compatible carriers. The term "carrier" refers to a natural or synthetic organic or inorganic component in which the active component is combined to facilitate application.According to the present invention, the term "pharmaceutically compatible carrier" includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a patient. The components of the pharmaceutical compositions of the present invention generally do not undergo interactions that significantly impair the desired pharmaceutical efficacy.

[0260] The pharmaceutical compositions described herein may contain suitable buffering substances such as acetate, citric acid, borate, and phosphate salts.

[0261] The pharmaceutical compositions may also suitably contain suitable preservatives such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0262] The pharmaceutical compositions are generally provided in a uniform dosage form and can be prepared in ways known per se. The pharmaceutical compositions of the present invention may be in the form of, for example, capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs, or emulsions.

[0263] Compositions suitable for parenteral administration generally comprise sterile aqueous or non-aqueous formulations of the active compound, preferably isotonic with the recipient's blood. Examples of compatible carriers and solvents are Ringer's solution and isotonic sodium chloride solution. Additionally, sterile fixative oils are typically used as the solution or suspension medium.

[0264] The invention is described in detail below with reference to the figures and examples, which are for illustrative purposes only and are not intended to be limiting. Other embodiments of the invention will be available to those skilled in the art due to the description and examples.

[0265] This application also relates to the following embodiments:

[0266] Embodiment 1. A method for predicting the immunogenicity of amino acid modifications, the method comprising the steps of:

[0267] a) determining the binding score of a modified peptide to one or more MHC molecules, and

[0268] b) determining the binding score of an unmodified peptide to one or more MHC molecules, and / or

[0269] c) determining the binding score of a modified peptide to one or more T cell receptors when present in an MHC-peptide complex.

[0270] Embodiment 2. The method of Embodiment 1, wherein the modified peptide comprises a fragment of a modified protein, the fragment comprising modifications present in the protein.

[0271] Embodiment 3. The method of Embodiment 1 or 2, wherein the unmodified peptide contains germline amino acids at a site corresponding to the modification site in the modified peptide.

[0272] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the unmodified peptide is identical to the modified peptide except for the modification.

[0273] Embodiment 5. The method of any one of Embodiments 1 to 4, wherein the length of the unmodified peptide and the modified peptide is 8 to 15 amino acids, preferably 8 to 12 amino acids.

[0274] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the one or more MHC molecules comprise different MHC molecule types, particularly different MHC alleles.

[0275] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the one or more MHC molecules are MHC class I molecules and / or MHC class II molecules.

[0276] Embodiment 8. The method of any one of Embodiments 1 to 7, wherein the binding score with the one or more MHC molecules is determined by a method including sequence comparison with an MHC binding motif database.

[0277] Embodiment 9. The method of any one of Embodiments 1 to 8, wherein step a) includes determining whether the score meets a predetermined threshold for binding with the one or more MHC molecules.

[0278] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein step b) includes determining whether the score meets a predetermined threshold for binding with the one or more MHC molecules.

[0279] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein the threshold applied in step a) is different from the threshold applied in step b).

[0280] Embodiment 12. The method of any one of Embodiments 1 to 11, wherein the predetermined threshold for binding to one or more MHC molecules reflects the likelihood of binding to one or more MHC molecules. Specification 30 / 48 pages 33 CN 121130065 A

[0281] Embodiment 13. The method of any one of Embodiments 1 to 12, wherein step c) includes determining a chemical and physical similarity score between the unmodified amino acid and the modified amino acid.

[0282] Embodiment 14. The method of any one of Embodiments 1 to 13, wherein step c) includes determining whether the score satisfies a predetermined threshold for chemical and physical similarity between amino acids.

[0283] Embodiment 15. The method of any one of Embodiments 1 to 14, wherein the chemical and physical similarity score is determined based on the likelihood of amino acid interchange in the natural state.

[0284] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the higher the frequency of amino acid interchange in the natural state, the higher the amino acid similarity is considered.

[0285] Embodiment 17. The method of any one of Embodiments 1 to 16, wherein the chemical and physical similarity is determined using an evolution-based log-probability matrix.

[0286] Embodiment 18. The method of any one of Embodiments 1 to 17, wherein if the binding score of the unmodified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, and the binding score of the modified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, then the modified or modified peptide is predicted to be immunogenic, provided that the chemical and physical similarity score of the unmodified amino acid and the modified amino acid meets a threshold indicating chemical and physical dissimilarity.

[0287] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein if the unmodified peptide binds to one or more MHC molecules or has the potential to bind to one or more MHC molecules, and the modified peptide binds to one or more MHC molecules or has the potential to bind to one or more MHC molecules, then the modified or modified peptide is predicted to be immunogenic, provided that the unmodified amino acid and the modified amino acid are chemically and physically dissimilar or have the potential to be chemically and physically dissimilar.

[0288] Embodiment 20. The method of Embodiment 18 or 19, wherein the modification is not located at an anchor site for binding to one or more MHC molecules.

[0289] Embodiment 21. The method of any one of Embodiments 1 to 17, wherein if the binding score of the unmodified peptide to one or more MHC molecules meets a threshold indicating non-binding to one or more MHC molecules, and the binding score of the modified peptide to one or more MHC molecules meets a threshold indicating binding to one or more MHC molecules, then the modification or modified peptide is predicted to be immunogenic.

[0290] Embodiment 22. The method of any one of Embodiments 1 to 17 and 21, wherein if the unmodified peptide does not bind to one or more MHC molecules or has the possibility of not binding to one or more MHC molecules, and the modified peptide binds to one or more MHC molecules or has the possibility of binding to one or more MHC molecules, then the modification or modified peptide is predicted to be immunogenic.

[0291] Embodiment 23. The method of Embodiment 21 or 22, wherein the modification is located at an anchor site for binding to one or more MHC molecules.

[0292] Embodiment 24. The method of any one of Embodiments 1 to 23, comprising performing step a) on two or more differently modified peptides, the two or more differently modified peptides comprising the same modification.

[0293] Embodiment 25. The method of Embodiment 24, wherein the two or more differently modified peptides comprising the same modification comprise different fragments of a modified protein, the different fragments comprising the same modification present in the protein.

[0294] Embodiment 26. The method of Embodiment 24 or 25, wherein two or more differently modified peptides comprising the same modification comprise all possible MHC-binding fragments of the modified protein, said fragments comprising the same modification present in said protein as described on page 34 of specification 31 / 48, CN 121130065 A.

[0295] Embodiment 27. The method of any one of Embodiments 24 to 26, further comprising selecting (said) the modified peptide from two or more differently modified peptides comprising the same modification having the greatest probability of binding to one or more MHC molecules.

[0296] Embodiment 28. The method of any one of Embodiments 24 to 27, wherein the two or more differently modified peptides comprising the same modification have different lengths and / or modification sites.

[0297] Embodiment 29. The method of any one of Embodiments 1 to 28, comprising performing step a) on two or more differently modified peptides, and optionally performing one or both of steps b) and c).

[0298] Embodiment 30. The method of Embodiment 29, wherein the two or more differently modified peptides contain the same modification and / or contain different modifications.

[0299] Embodiment 31. The method of Embodiment 30, wherein the different modifications are present in the same and / or different proteins.

[0300] Embodiment 32. The method of any one of Embodiments 24 to 31, comprising comparing scores of two or more of the differently modified peptides.

[0301] Embodiment 33. The method of Embodiment 32, wherein the binding score of the modified peptide to one or more MHC molecules is weighted higher than the binding score of the modified peptide to one or more T cell receptors when present in an MHC-peptide complex, preferably higher than the chemical and physical similarity score between the unmodified amino acid and the modified amino acid, and the binding score of the modified peptide to one or more T cell receptors when present in an MHC-peptide complex, preferably the chemical and physical similarity score between the unmodified amino acid and the modified amino acid, is weighted higher than the binding score of the unmodified peptide to one or more MHC molecules.

[0302] Embodiment 34. The method of any one of embodiments 1 to 33, further comprising identifying non-synonymous mutations in one or more protein-coding regions.

[0303] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the modification is identified by partially or completely sequencing the genome or transcriptome of one or more cells, such as one or more cancer cells and optionally one or more non-cancer cells, and by identifying mutations in one or more protein-coding regions.

[0304] Embodiment 36. The method of embodiment 34 or 35, wherein the mutation is a somatic mutation.

[0305] Embodiment 37. The method of embodiments 34 to 36, wherein the mutation is a cancer mutation.

[0306] Embodiment 38. The method of any one of embodiments 1 to 37, for preparing a vaccine.

[0307] Embodiment 39. The method of embodiment 38, wherein the vaccine is derived from one or more modified peptides or peptides predicted to be immunogenic by the method.

[0308] Embodiment 40. A method of providing a vaccine, comprising the steps of:

[0309] identifying one or more modified peptides or peptides predicted to be immunogenic by the method of any one of embodiments 1 to 37.

[0310] Embodiment 41. The method of embodiment 40, further comprising the step of:

[0311] providing a vaccine comprising a peptide or polypeptide or a nucleic acid encoding said peptide or polypeptide, said peptide or polypeptide containing said modified peptide or peptide predicted to be immunogenic.

[0312] Embodiment 42. A vaccine produced according to the method of any one of Embodiments 38 to 41.

[0313] Figures

[0314] Figure 1. Overview of MHC binding prediction. Specification 32 / 48 pages 35 CN 121130065 A

[0315] Figure 2. Immunogenicity analysis as a function of Mmut score for 50 optimized B16F10 mutations and 82 optimized CT26.WT mutations (a total of 132 mutations), of which 30 were immunogenic. All vaccinations were administered using RNA. For B16F10, immunogenicity was analyzed by stimulating BMDCs with RNA and by measuring the immune response of spleen cells with ELISPOT and FACS. For CT26.WT, immunogenicity was analyzed by stimulating BMDCs with RNA and peptides respectively and by measuring the immune response of spleen cells with ELISPOT; if either the peptide or RNA elicited an immune response, the mutation was considered immunogenic. The cumulative distribution of immunogenic mutations was a function of the Mmut score. The graphs show the total number of mutations below a given Mmut score (red), the number of immunogenic mutations among these mutations (blue), and the percentage of immunogenic mutations in the total mutations (black). Histograms of the percentage of immunogenic mutations for each Mmut library within the following ranges: ≤ 0.3, (0.3, 1], >1. The errors shown are standard errors.

[0316] Figure 3. Immunogenicity analysis of B16F10 and CT26.WT as a function of Mmut. The cumulative distribution of immunogenic mutations is a function of the Mmut scores for B16 (A) and CT26 (C).Histograms of the percentage of immunogenic mutations in each Mmut library for the following ranges: [0.1, 0.3], (0.3, 1], (1, ∞) for B16 (B) and CT26 (D). Figures A and B are based on an analysis of 50 B16F10 preferential mutations, of which 12 were immunogenic. Figures C and D are based on an analysis of 82 B16F10 preferential mutations, of which 30 were immunogenic. See the description of Figure 2 for more details. Errors are standard errors.

[0317] Figure 4. Immunogenicity and Control Hypothesis Models. Class I immunogenicity, represented by HA, assumes that both WT and MUT epitopes are presented by cells, and that mutations are sufficient to alter the physicochemical properties of amino acids so that the immune system records the change and generates an immune response (represented by lightning balls). The Hn hypothesis, as a control of HA, is simply the opposite of the HA hypothesis, i.e., the mutation does not significantly alter the physicochemical properties of amino acids, and therefore is less likely to be "detected" by the immune system and generate an immune response. In Class II immunogenicity (HB U HC), the WT epitope is not presented, but the MUT epitope is presented. HB and HC are distinguished by high (T>τ) vs. low (T≤τ) T scores, respectively. Note that for =α, the HBC1 model of immunogenicity (Mmut<β) is a composite of all four groups: HBC1 = U [HA, HB, HC, Hn].

[0318] Figure 5. Relationship between T score and immunogenicity hypothesis. According to the Class I immunogenicity model, during T cell development, TCRs that strongly bind to wild-type epitopes are eliminated. Existing TCRs should only show weak or no binding affinity to wild-type epitopes (A). Epitopes containing amino acid substitutions with high T scores have similar physicochemical properties to wild-type amino acids and therefore may have little effect on binding to existing TCRs (B). Epitopes containing amino acid substitutions with T scores have a greater likelihood of increasing binding affinity to existing TCRs and therefore a greater likelihood of becoming immunogenic (C). In this schematic diagram, color coding is used to pair T cells with matching peptides. Orange / yellow mutations represent mutations with high T scores (similar to WT), while blue / purple mutations represent mutations with low T scores (significant physicochemical differences compared to WT).

[0319] Figure 6. Cumulative distribution of immunogenic mutations as a function of Mmut. A. Percentage of immunogenic mutations satisfying the background control hypothesis HBC1: and satisfying partial hypotheses HA':, partial hypotheses HBC2:, and complete hypotheses HA: Comparison of the percentage of immunogenic mutations, α=1, τ=1. Comparison of the percentage of immunogenic mutations of HBC1 satisfying the control hypothesis: and satisfying the opposite partial hypothesis: and.The analyses in A and B are based on the merged B16F10 and CT26.WT datasets containing 132 mutations, of which 30 are immunogenic. Each data point in the graph is based on ≥4 mutations.

[0320] Figure 7. Cumulative distribution of immunogenic mutations as a function of Mmut score. Comparison of the percentage of immunogenic mutations satisfying the background control hypothesis HBC1: (Page 33 / 48, CN 121130065 A) with the percentage of immunogenic mutations satisfying the partial hypothesis HA':, the partial hypothesis HBC2:, and the complete hypothesis HA:, for B16 (A) and CT26 (C), given α=1, τ=1. Comparison of the percentage of immunogenic mutations satisfying the background control hypothesis HBC1: in B16 (B) and CT26 (D) with the percentage of immunogenic mutations satisfying the opposite partial hypothesis: and.

[0321] Figures A and B are based on the analysis of 50 B16F10-prioritized mutations, of which 12 are immunogenic. Figures C and D are based on 82 B16F10-prioritized mutations, of which 30 are immunogenic. Each data point in the figures is based on ≥4 mutations.

[0322] Figure 8. WT immunogenicity control. To examine whether omitting the MUT+ / WT+ protocol affected these results, we excluded 9 MUT+ / WT+ mutations and 2 mutations for which WT was not measured from the dataset, leaving a total of 121 mutations (43 B16 and 78 CT26), of which 19 were MUT+ / WT- (5 B16 and 14 CT26). We again found the same trend as in the complete dataset, namely, a highly nonlinear response as a function of Mmut score, the advantage of the HA hypothesis over the partial hypothesis, and the disadvantage of the opposite hypothesis compared to the background control HBC1. A. Accumulated distribution of immunogenicity as a function of Mmut score. B. Histogram of the percentage of immunogenic mutations in each Mmut library. C. Comparison of the percentage of immunogenic mutations satisfying the background control hypothesis HBC1 with the percentages satisfying HA', HBC2, and HA. D. Comparison of the percentage of immunogenic mutations satisfying the background control hypothesis HBC1 with the percentages satisfying the opposite hypothesis. See Figure 5 for further details.

[0323] Figure 9. Percentage of immunogenic mutations as a function of RPKM. Red: All 50 unfiltered B16 mutations and 82 CT26 mutations (132 mutations in total). Blue: Mutations according to the HA hypothesis with α=1, β=0.5, and τ=1. B. Percentage of immunogenic mutations in different unfiltered RPKM ranges. The RPKM library is: 1=(0,1], 2=(1,5], 3=(5,50], 4=(50, ∞). C. Percentage of immunogenic mutations in different RPKM ranges satisfying the HA hypothesis of α=1, β=0.5, τ=1.The RPKM library is: 1 = (0, 1], 2 = (1, ∞). The error is SE.

[0324] Figure 10. Type II immunogenic epitopes with anchored and non-anchored site mutations. Anchored site motifs were analyzed using SYFPEITHI.

[0325] Figure 11. Proposed model of immunogenic tumor-associated epitopes.

[0326] Figure 12. Example of a method for weighting mutation ranking. For each mutation, its ranking in the mutation ranking list can be further weighted by the number of solutions resulting from the patient's HLA type, the possible window length of the HLA type, and the combination of solutions within the epitope that results in low Mmut or HA and / or HBUHC classification. Since all solutions for each mutation can potentially be proposed in parallel, this weighting factor may be an important contributor to mutation ranking.

[0327] Figure 13. For Mmut and ΔM = Mmut – An example of a scatter plot of all epitope schemes of the CT26 mutation chr14_52837882 in Mwt

[0328] Examples

[0329] The techniques and methods used herein are described herein, or in ways known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. All methods, including the use of kits and reagents, are performed according to the manufacturer’s information unless specifically indicated.

[0330] Example 1: Establishing a model for predicting the immunogenicity of T cell epitopes

[0331] We previously investigated the immunogenicity of 50 somatic mutations identified in the B16F10 mouse melanoma cell line. (Instructions for use 34 / 48 pages 37 CN 121130065 A (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081) (2012)). These 50 mutations were selected from a pool of 563 nonsynonymous somatic mutations that primarily maximized the expression of MHC class I (JC Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)) (see also Example 2).When searching all possible MHC class I allele spaces, potential epitope lengths, and sequence windows (locating mutation sites), we predicted the minimum epitope for each mutation, i.e., the epitope with the lowest MHC class I concordance score (Y. Kim et al., Nucleic Acids Research 40, W525 (2012)) (defined here as Mmut) (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)). The immunogenicity of these mutations was measured using RNA vaccination, followed by peptide vaccination to confirm previously identified peptide readouts (see Example 2) (JC Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)), showing that only 12 (24%) of the 50 mutations were immunogenic (Table 1), with MUT+ / WT- sequences comprising only 10% of all tested mutations.

[0332] Table 1. Number of immunogenic mutations after RNA vaccination in B16F10 and CT26.WT mouse strains

[0333]

[0334] This table excludes two CT26 MUT+ mutations because their WT reactivity has not yet been measured. To date, 18 MUT+ mutations out of 82 CT26 mutations have been measured, yielding a success rate of 22%.

[0335] Results from the B16F10 mouse test showed that initial selection of non-synonymous mutations with low Mmut scores (≤3.9) yielded a low success rate in immunogenicity prediction. Therefore, a better understanding of the mechanisms driving immunogenicity is needed if personalized vaccines targeting tumor-specific neoantigens are to become effective therapies. To attempt to discover additional variables contributing to immunogenicity, we investigated the immunogenicity of non-synonymous somatic mutations identified in the mouse colon cancer cell line CT26.WT. In total, 96 mutations were selected based on mutation Mmut score (low vs. high), mean RPKM (low vs. high), and cellular localization (intracellular vs. extracellular), and immunogenicity was tested using peptide and RNA readouts via RNA vaccination (see Example 2 for further details). Together with the B16F10 cell line, our dataset includes 132 epitopes, whose immunogenicity against mouse spleen cells was measured in vitro.

[0336] MHC Conformity Score. To investigate the dependence of immunogenicity on Mmut, we defined the cumulative percentage of immunogenic mutations as a function of Mmut, i.e., the percentage of mutations with an Mmut score less than a given threshold (denoted as β) for immunogenicity. Analysis of the combined B16 and CT26 datasets, spanning a total of 132 mutations, showed a highly nonlinear dependence of immunogenicity success on Mmut (Figure 2A). Figure 2A shows that immunogenic mutations are rich in very low Mmut scores (≤~0.2). For Mmut≤0.1, the percentage of immunogenic mutations peaks at ~60% and rapidly decays with increasing Mmut, falling to below ~25% for Mmut≥2. The percentage of immunogenic mutations with Mmut≤0.3 versus >0.3 was 44.4% vs. 17.1%, a statistically significant difference (P = 0.004, Fisher exact test, one-tailed). Histograms of the percentage of immunogenic mutations in three Mmut libraries: ≤0.3, (0.3, 1], and >1 showed that the percentage of immunogenic mutations decreased with increasing Mmut (Figure 2B). The success rate of 44.4% for the lowest library (Mmut ≤0.3) in Figure 2B was statistically significant compared to 20.7% for the intermediate library and 15.8% for the highest library (Mmut>1) (P = 0.05 and 0.004, respectively, Fisher's exact test, one-tailed), showing that for Mmut >~0.3, the success rate decreased in a statistically significant manner. A similar trend in success rate was also observed when analyzing the B16 and CT26 mutation groups separately (Figure 3).

[0337] So far, our selection criteria for mutations have focused on presentation, and we have observed that MHC binding scores restricting mutant epitopes allow for immunogenic epitope prediction with up to 60% accuracy. However, presentation is a necessary but not sufficient condition for inducing immunogenicity. By identifying other criteria for TCR recognition, we may be able to further improve the accuracy of our predictions. We hypothesize two mutually exclusive mechanisms driving immunogenicity, which we call the Type I and Type II immunogenicity models.

[0338] Type I Immunogenicity. In order for the TCR library to recognize mutant epitopes and generate an immune response, we hypothesize that three conditions must be met (HA Figure 4): (i) wild-type epitopes are presented to the immune system at some point in the organism’s development, clearing matching TCRs through strong TCR / pMHC binding; (ii) mutant epitopes are presented; and (iii) the physicochemical properties of the mutant amino acids are sufficiently “different” from those of the wild-type amino acids (by some of the metrics we define below) so that the TCR library can “detect” or “record” the substitution. Conditions (i) and (ii) ensure that the immune system is actually exposed to the change, i.e., the mutation.Condition (iii) requires a mutation that significantly alters the physicochemical characteristics of the wild-type amino acid so that the binding affinity of the mutant epitope to the existing (unremoved) TCR may increase, thereby initiating a signaling cascade leading to an immune response (Figure 5).

[0339] TCT recognition score. Class I immunogenicity models require a metric to estimate the physicochemical differences between two amino acids. It is known in molecular evolution that amino acids that are frequently interchanged may have chemical and physical similarities, while amino acids that are rarely interchanged may have different physicochemical properties. The likelihood of a substitution occurring naturally is measured by a log-probability matrix compared to the likelihood of a given substitution occurring by chance. The patterns observed in the log-probability matrix imposed by natural selection “reflect the similarity of amino acid residue functions in the weak interactions between amino acid residues and other amino acid residues in the three-dimensional conformation of the protein” (M. O. Dayhoff, R. M. Schwartz, B. C. Orcutt, A model for evolutionary change. MO Dayhoff, ed. Atlas of protein sequence and structure Vol.5, 345 (1978)). Therefore, we utilize an evolution-based log-odds matrix, termed the “T-score,” to reflect TCR recognition as an effective scoring matrix for cancer-related amino acid substitutions. Substitutions with positive T-scores (i.e., log-odds) are likely to occur spontaneously and therefore correspond to two amino acids with similar physicochemical properties. Class I models predict that substitutions with positive T-scores are less likely to be immunogenic. Conversely, substitutions with negative T-scores reflect the likelihood of spontaneous occurrence and therefore correspond to two amino acids with significantly different physicochemical properties. According to our model, such substitutions are more likely to be immunogenic. We compared different methods for estimating the log-odds matrix and found the results to be quite stable relative to the chosen method. The maximum probability (ML)-based estimation method, termed WAG (S. Whelan, N. Goldman, Molecular biology and evolution 18, 691 (2001)), using a PAM (acceptable point mutation) distance of 250, appears to best separate predicted immunogenicity from non-immunogenic mutations; therefore, we use this matrix to represent the results (see Example 2 for further details).

[0340] Class II immunogenicity. In the Class II model of immunogenicity, we hypothesize that if the immune system has never seen the wild-type epitope before, the mutation may be immunogenic and thus stimulated by the mutated epitope.Therefore, in this model, for a mutation to be immunogenic, we hypothesize that two conditions must be met: (i) the wild-type epitope has never been presented to the immune system, and (ii) the mutated peptide has been presented. These conditions can be met if, for example, the mutation is located at an anchoring site, thus changing a "non-binding" epitope into a "binding" epitope. Formally, class II immunogenicity can be divided into two subhypotheses: high T score (HB in Figure 4) and low T score (HC in Figure 4). However, since it is assumed that the wild-type epitope is not presented, the nature of amino acid substitutions is not expected to affect TCR recognition; therefore, we consider class II immunogenicity equivalent to the consensus hypothesis: HB U HC.

[0341] Testing for Class I Immunogenicity. The Class I immunogenicity (HA in Figure 4) hypothesis can be mathematically restated as follows: Specification 36 / 48 Page 39 CN 121130065 A We need wild-type epitopes to be presented ( ), mutant epitopes to be presented ( ), and amino acid substitutions to be insignificant ( ), where Mwt is defined as the MHC consistency score (same HLA alleles and window length) of the mutant epitope with wild-type amino acid substitution for mutant amino acid, and T represents the T score. Since all three conditions are required, we expect the HA classifier to be more accurate than the classifier based solely on M mut (HBC 1 in Figure 4) or the partial hypothesis: and compared to. Therefore, we calculate the percentage of immunogenic mutations (the number of true positives divided by the sum of true positives and false positives) as a function of β for HBC1, partial hypothesis HA': and partial hypothesis HBC2:. We found that the conservative threshold in the range of ≈0.5 to 1 performed best (the WAG250 matrix ranges from -5.1 (FG substitution) to +5.4 (FY substitution)). We also found that α can be strictly conserved compared to β, setting α≈1. Figure 6A indeed shows that when considering the combined mutant group of B16 and CT26, the classifier based on HBC2 and HA' is more accurate than the background control hypothesis HBC1. In addition, the classifier based on the complete hypothesis HA is more accurate than the partial hypotheses HBC1 and HBC2, thus showing an additive effect. The same conclusion is held when analyzing the B16 and CT26 datasets separately (Figure 7).

[0342] Due to the condition and the presumed necessary condition for immunogenicity, it is expected that the analyzer based on the condition or condition (i.e., the rejection of the second condition) will perform worse than HBC1. Indeed, we found this to be the case for B16 and CT26 when analyzed together (Figure 6B) or separately (Figure 7). Therefore, we infer that the B16 and CT26 datasets together or individually support the HA hypothesis. Omitting WT RNA also shows that reactive mutations do not affect these conclusions (Figure 8).

[0343] HA Hypothesis Comparison. Although mutations with high T scores may still be immunogenic, the hypothesis rich in such mutations should be statistically rich in non-immunogenic mutations. Therefore, if we compare the HA hypothesis ( ) with its opposite (in Figure 4), we should observe a statistically significant lack of immunogenic mutations. Table 2 does indeed show that for Mmut≤β=0.5, Mwt≤α=1, and T≤τ=1, HA outperforms Hn, with a success rate of 52.5% (n=21) vs. 21.4% (n=14; P=0.068, one-tailed Fisher exact test).

[0344] Table 2. Percentage of immunogenic mutations under different hypotheses based on the merged B16 and CT26 dataset containing 133 mutations. Instructions for Use, pages 37 / 48, 40 CN 121130065 A

[0345]

[0346] HA also performed better than the background control HBC2, reaching 41.2% (n=35). As the condition for β becomes more stringent, more false positives are removed from the HA group, and thus the difference in success rates between HA and Hn becomes larger as we lower β. For example, for β = 0.25, the success rate of the HA group was 67% (n=14) compared to 17% (n=6) in the Hn group (P=0.066, one-tailed Fisher exact test) – see Table 3.

[0347] Table 3. Ranking list of 133 measured B16F10 / CT26. WT mutations satisfying the basic control hypothesis HBC1 (Mmut ≤ 0.25) were divided into three disjoint hypothesis categories: the HA hypothesis for immunogenic mutations (Mwt ≤ 0.8, T ≤ 0.5), the Hn / opposite HA hypothesis rich in non-immunogenic mutations (Mwt ≤ 0.8, T > 0.5), and the HBUHC hypothesis for immunogenic mutations (Mwt > 0.8). It is proposed to rank candidate HA and HBUHC based on the relative importance of the discriminant variables. For HA, the proposed order is: Mmut (decreased) → T score (decreased) → MWT decrease. For HBUHC, the proposed order is: Mmut (decreased) → MWT (increased). Error is standard error. Specification 38 / 48 pages 41 CN 121130065 A

[0348]

[0349] Examples of other weighting factors that may further improve the immunogenicity ranking are given in Example 3.

[0350] More generally, the list of mutations satisfying HBC1 (Mmut≤β) can be divided into three categories: HA, Hn and HBUHC (Table 3), where HA is rich in immunogenic mutations and Hn is rich in non-immunogenic mutations. In the cases of B16 and CT26, all three candidates in the HBUHC group are non-immunogenic, contrary to our expectations.However, if a more appropriate threshold α is chosen for Mwt so that α >> α, then there will be no predictions for the HBUHC test.

[0351] Highest accuracy of the immunogenicity classifier. According to Table 1, the average success rate of predicting immunogenicity in the combined B16 and CT26 dataset is 22.7% (=30 / 132). By applying the most stringent threshold ( ) to the Mmut score, the accuracy of the immunogenicity classifier is increased to 60% (=6 / 10; HBC1 in Table 2). By combining HBC1 with condition or with condition ( ), the accuracy is increased to 66.7% (=6 / 9). The HA-based classifier that combines both criteria results in an additive response, which increases the accuracy to 75% (=6 / 8) (Table 2).

[0352] B16 epitope MUT33. In the combined B16 / CT26 dataset, the highest-ranking HA class epitope by all evolutionary models (except the PAM matrix) is B16's MUT33 (see Table 3). Further analysis showed that MUT33 did indeed elicit an MHC class I restricted CD8+ response and exhibited in vitro immunogenicity against the minimal predicted epitope (data not shown).

[0353] Role of gene expression. The proportion of immunogenic mutations (the ratio of the number of immunogenic mutations with RPKM values ​​below a given threshold to the total number of immunogenic mutations) was formulated as a function of the RPKM values ​​of B16 and CT26 candidates, showing that the ratio plateaued somewhat at very low RPKM values ​​(Fig. 9). This effect was observed regardless of whether the HA criteria were applied. Plotting the percentage of immunogenic mutations in different RPKM libraries (Fig. 9) showed that RPKM values ​​≤ ~1 had a slightly lower success rate (with or without the HA filtering hypothesis), and although heuristic, these results should be noted to be within the margin of error.

[0354] Study of disclosed CD8+ epitopes. Next, we wanted to understand whether disclosed T-cell-restricted tumor antigens with single amino acid substitutions elicited CD8+-restricted responses that satisfied our immunogenicity model. Of the 17 published epitopes (P. Van der Bruggen, V. Stroobant, N. Vigneron, B. Van den Eynde. (Cancer Immun, http: / / www.cancerimmunity.org / peptide / , 2013)) (Table 4), 5 met the HA criteria (α = 0.7, β = 0.2, τ = 0.5), four met the HCUHB criteria (α = 2.2, β = 0.4), and two met the Hn criteria (α = 0.6, β = 0.3, τ = 1.7).

[0355] Table 4. Published epitopes with a single amino acid substitution generate a CD8+ response. See Example 2 for a list of references. Anchor site mutations in the HBUHC group are highlighted in red.

[0356]

[0357] Thus, the HA and HCUHB hypotheses roughly together account for 50% of the published epitopes. Interestingly, due to anchor site mutations, three of the four published epitopes that meet the HCUHB criteria (red boxes in Table 4) have Mwt scores greater than 10 (Fig. 10). Since the HCUHB hypothesis requires that the likelihood of any cell presenting a wild-type epitope remain very low during organismal development, it is expected that the Mwt threshold should remain high, i.e. Indeed, when α increases from 0.8 to >3, the B16 / CT26 false positives in Table 3 disappear. Therefore, a more suitable threshold for Mwt in the HCUHB hypothesis might be 3–10.

[0358] MZ7-MEL cell line. To test the ability of our immunogenicity model to predict immunogenic epitopes in a human tumor model setting, we investigated the MZ7-MEL cell line established in 1988 from spleen metastases from patients with malignant melanoma (V. Lennerz et al., Proceedings of the National Academy of Sciences of the United States of America 102, 16013 (2005)). Screening of cDNA libraries from MZ7-MEL cells containing autologous tumor-reactive T cells showed that at least five neoantigens could elicit a CD8+ response (V. Lennerz et al., Proceedings of the National Academy of Sciences of the United States of America 102, 16013 (2005)). This constitutes the largest set of CD8+ neoantigens from patient-derived sources to date. Applying our immunogenicity model to these epitopes, we found that three neoantigens were classified as HA epitopes, and one neoantigen with an anchor site mutation was classified as an HBUHC epitope (arrows in Table 4 and Figure 10). Therefore, four of these five epitopes can be explained by our immunogenicity model.

[0359] To test our ability to re-predict these epitopes in the MZ7-MEL cell line, we sequenced the exome of the MZ7-MEL cell line (see Methods). A total of 743 expressed nonsynonymous mutations were identified.All five mutations previously identified by Lennerz et al. (V. Lennerz et al., Proceedings of the National Academy of Sciences of the United States of America 102, 16013 (2005)) were found. We then calculated the T score, Mmut, and Mwt for each mutation, and also reported the HLA alleles and epitopes leading to the minimum MHC concordance score for a given mutation. Mutations were grouped into one of three groups—HA, HBUHC, and Hn—using thresholds α=0.8, β=0.2, and τ=0.5 (plus the condition RPKM >0.2), and then ranked based on their immunogenic potential as described in Table 3. We found that of the 743 mutations, 32 met the HA criteria (Table 5), 12 met the HBUHC criteria (Table 6), and 15 met the Hn criteria. Instructions 40 / 48 pages 43 CN 121130065 A

[0360] Table 5. MZ7-MEL cell mutations of the HA class. Using thresholds α=0.8, β=0.2 and τ=0.5, 32 of the 743 expressed nonsynonymous mutations of MZ7-MEL were classified as HA-immunogenic. The order was based on the Mmut (decreasing) → T score (decreasing) ranking scheme. Neoantigens identified by Lennerz et al. as immunogenic are highlighted in yellow. Furthermore, RPKM needs to be greater than 0.2.

[0361]

[0362] Table 6. MZ7-MEL cell mutations of the HBUHC class. Using thresholds = 0.8, β=0.2 and RPKM > 2, 12 of the 743 expressed nonsynonymous mutations of MZ7-MEL were classified as HBUHC-immunogenic. The order was based on the Mmut (decreasing) → Mwt (increasing) ranking scheme. Neoantigens with immunogenicity identified by Lennerz et al. are highlighted in yellow. Specification 41 / 48 pages 44 CN 121130065 A

[0363]

[0364] Of the 32 mutations classified as HA, using the Mmut→T scoring scheme, the three HA mutations identified by Lennerz et al. (SIRT2, SNRPD1 and RBAF600) ranked 2nd, 4th and 13th in 18 ranked classes (see Table 3). Of the 12 mutations classified as HBUHC, the previous mutation by Lennerz et al. (SNRP116) ranked 3rd. Furthermore, if a higher (more practical) Mwt threshold (e.g., α ~ 5) is applied, the previous mutation by Lennerz et al. ranked 1st (along with another anchor site mutation – Table 7).Finally, as reported by Lennerz et al., the four predicted mutations had the correct HLA alleles, epitope lengths, and mutation sites.

[0365] Table 7. MZ7-MEL cell mutations of the HBUHC class. Using a threshold of 5, β = 0.2, and RPKM > 2, two of the 743 expressed nonsynonymous mutations of MZ7-MEL were classified as HBUHC-immunogenic. The order was based on the Mmut (decreasing) → Mwt (increasing) sorting scheme. Neoantigens identified by Lennerz et al. as immunogenic are highlighted in yellow.

[0366]

[0367] Conclusion

[0368] The analysis of the B16 and CT26 datasets supports the following model: immunogenicity is conferred if three conditions are met: wild-type peptides are presented, mutant peptides are presented, and amino acid substitutions have sufficiently low log-odds scores (Figure 11A). This immunogenicity model, which we call Class I immunogenicity, is further supported in the human melanoma cell line model MZ7-MEL. The MZ7-MEL model and the published CD8+-restricted neoantigens support a second model, which we call Class II immunogenicity, in which wild-type epitopes are not presented, but substitution (e.g. at anchor sites) leads to a significant increase in the MHC concordance score (>5 to 10), generating new, previously unseen epitopes (Figure 11B). This framework for limiting immunogenicity is derived from a three-variable classification scheme (Mmut, Mwt, T score). Using this classification scheme, we were able to reduce the 743 mutations in MZ7-MEL to a single column of 34 mutations, with 3 of the 5 epitopes from Lennerz et al. ranking in the top 5. Specification 42 / 48 pages 45 CN 121130065 A

[0369] Table 7 shows that Class II immunogenic mutations are rare. Of the 743 mutations, only 2 were classified as class II immunogenic (using an appropriate threshold of Mwt) compared to approximately 30 class I immunogenic mutations. A small number of HBUHC class mutations were also observed in a mouse melanoma model (Table 8). This observation highlights the importance of class I immunogenic mutations in personalized vaccines, which are expected to be the main mutation types present in patient samples that can be used for vaccination. Meanwhile, the fact that one of the five epitopes found by Lennerz et al. is class II immunogenic may suggest that class II immunogenic mutations are more effective or are selected by the immune system in some way.

[0370] Table 8. Number of candidate HA and HBUHC mutations in different tumor models. (α=0.8, α=0.5, β=0.2, τ=0.5)

[0371]

[0372] Example 2: Materials and Methods

[0373] The materials and methods used in Example 1 are described below.

[0374] Animals

[0375] C57BL / 6J and Balb / cJ mice (CRL) were housed at the University of Mainz in accordance with federal and state policies for animal research.

[0376] Cells for melanoma and colorectal tumor models in mice

[0377] The B16F10 melanoma cell line (product: ATCC CRL-6475, lot number: 58078645) and the CT26.WT colon cancer cell line (product: ATCC CRL-2638, lot number: 58494154) were purchased from the American Type Culture Collection in 2010. Sequencing experiments were performed using early passaged cells (generations 3 and 4). Routine mycoplasma testing was performed on the cells. The cells have not been reconfirmed since receipt. The MZ7-MEL cell line (established in January 1988) and the autologous Epstein-Barr virus-transformed B cell line were obtained from Dr. Thomas Wölfel (Department of Medicine, Hematologic Oncology, Johannes Gutenberg University).

[0378] Synthetic Peptides

[0379] Peptides were purchased from Jerini Peptide Technologies (Berlin, Germany) or synthesized using TRON peptide equipment. The synthesized peptides were 27 amino acids in length, with the mutant (MUT) or wild-type (WT) amino acid located at position 14.

[0380] Immunization of Mice

[0381] Age-matched female C57BL / 6 or Bal b / c mice were intravenously injected with 20 µg of in vitro transcribed mRNA in PBS containing 20 µl of Lipofectamine™ RNAiMAX (Invitrogen), for a total injection volume of 200 µl (3 mice per group). Mice were immunized on days 0, 3, 7, 14, and 18. Twenty-three days after the initial injection, mice were killed and spleen cells were isolated for immunological assays (see ELISPOT analysis). DNA sequences representing one (single epitope) or two (double epitope) mutations were constructed using a 27-amino acid (aa) sequence containing the mutation at site 14 and cloned into the pST1-2BgUTR-A120 backbone (S. Holtkamp et al., Blood 108, 4009 (2006)). In vitro transcription and purification from this template have been previously described (S. Kreiter et al., Cancer Immunology, Immunotherapy 56, 1577 (2007)).

[0382] Enzyme-Linked Immunospot Assay Manual 43 / 48 pages 46 CN 121130065 A

[0383] Enzyme-linked immunospot (ELISPOT) assay (S. Kreiter et al., Cancer Research 70, 9031 (2010)) and the generation of syngeneic bone marrow-derived dendritic cells (BMDCs) as stimulants have been previously described (L. MB et al., J. Immunol. Methods 223, 77 (1999)). For the B16F10 model, BMDCs are pulsed with a peptide containing the indicated mutant peptide, the corresponding wild-type peptide, or the control peptide (VSV-NP) (6 µg / ml). For the CT26 model, in addition to peptide restimulation, BMDCs are transfected with the corresponding in vitro transcribed mRNA, and the mRNA is also used for restimulation. For analysis, 5 × 10⁴ BMDCs were co-incubated with 5 × 10⁵ freshly isolated spleen cells in a microtiter plate coated with anti-IFN-γ antibody (10 μg / mL, clone AN18; Mabtech). After incubation at 37°C for 18 hours, cytokine secretion was detected using anti-IFN-γ antibody (clone R4-6A2; Mabtech). The number of spots was counted and analyzed using an ImmunoSpot® S5 Versa ELISPOT analyzer, ImmunoCapture™ Image Acquisition software, and ImmunoSpot® analysis software version 5. Statistical analysis was performed using Student's t-test and Man-Whitney test (non-parametric test). A p-value < 0.05 was considered a significant response.

[0384] Intracellular Cytokine Analysis

[0385] The prepared aliquots of spleen cells for ELISPOT analysis were analyzed for cytokine production using intracellular flow cytometry. For this purpose, 2 × 10⁶ spleen cells from each sample were placed in 96-well plates in medium (RPMI + 10% FCS) supplemented with the Golgi inhibitor brefidobacterium A (10 µg / mL). Cells from each animal were restimulated at 37°C with BMDCs transfected with 2 × 10⁵ peptide pulses or RNA for 5 h. After incubation, cells were washed with PBS, resuspended in 50 µl PBS, and stained extracellularly at 4°C for 20 min with the following anti-mouse antibodies: anti-CD4 FITC and anti-CD8 APC-Cy7 (BD Pharmingen). After incubation, cells were washed with PBS and then resuspended at 4°C in 100 µL Cytofix / Cytoperm (BD Bioscience) solution for 20 min to allow for outer membrane permeabilization.After permeabilization, cells were washed with Perm / Wash-Buffer (BD Bioscience), resuspended in 50 µL of Perm / Wash-Buffer per sample, and stained intracellularly at 4°C for 30 min with the following anti-mouse antibodies: anti-IFN-γ PE, anti-TNF-α PE-Cy7, and anti-IL2 APC (BD Pharmingen). After washing with Perm / Wash-Buffer, cells were resuspended in PBS containing 1% paraformaldehyde for flow cytometry analysis. Samples were analyzed using a BD FACSCanto™ II hematology counter and FlowJo (version 7.6.3).

[0386] Next-Generation Sequencing

[0387] Nucleic Acid Extraction: DNA and RNA were extracted from mixed cells and from mouse tissue using the Qiagen DNeasy Blood and Tissue Kit (for DNA) and the Qiagen RNeasy Micro Kit (for RNA).

[0388] DNA exome sequencing: As previously described, three exon captures of B16F10, C57BL / 6J, and CT26.WT, and DNA resequencing of Balb / cJ were performed (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)). Two exome captures for MZ7-MEL / EBV-B DNA resequencing were performed using a capture analysis based on the Agilent XT Human all Exon 50 Mb protocol to capture all protein-coding regions. 3 µg of purified genomic DNA (gDNA) was split into 150–200 bp fragments using a Covaris S2 sonication device. The fragments were end-repaired and 5' phosphorylated and 3' adenosineted according to the manufacturer's instructions. A 10:1 aptamer-to-gDNA molar ratio was used to ligate Agilent-indexed end-paired aptamers to gDNA fragments. Four cycles of pre-capture amplification were performed using Agilent's InPE 1.0 and SureSelect indexing pre-capture PCR primers and Herculase II polymerase. At 65°C, 500 ng of the aptamer-ligated, PCR-enriched gDNA fragment was hybridized with Agilent exome capture bait for 24 hours. The hybridized gDNA / RNA bait complex was removed using streptavidin-coated magnetic beads, washed, and the RNA bait was cleaved during elution with SureSelect elution buffer.The eluted gDNA fragments were captured and amplified using SureSelect Indexing Post-Capture PCR (SPCR) with primers and Herculase II polymerase (pages 44 / 48, CN 121130065 A, manual specification). Ten cycles of amplification were performed. All cleanup was carried out using 1.8 volumes of Agencourt AMPure XP magnetic beads. All quality control was performed using Invitrogen's Qubit HS assay, and fragment size was determined using Agilent's 2100 Bioanalyzer HS DNA assay. A 7 pM library was used to aggregate exon-rich gDNA on cBots using the Truseq SR cluster kit v2.5, and sequencing was performed at 1 x 100 bps using the Truseq SBS kit on an Illumina HiSeq 2000.

[0389] RNA gene expression profiling (RNA-Seq): Two barcoded mRNA-seq cDNA libraries were prepared. mRNA was isolated from 5 µg of total RNA using Seramag Oligo(dT) magnetic beads (Thermo Scientific) (modified Illumina mRNA-seq protocol using NEB reagents), and fragmented using divalent cations and heat. The resulting fragments (160–220 bp) were converted into cDNA using random primers and SuperScript II (Invitrogen), and then the second strand was synthesized using DNA polymerase I and RNase H. The cDNA was end-repaired, and 5' phosphorylation and 3' adenylation were performed according to the NEB RNA library kit instructions. Using an aptamer to cDNA insertion molar ratio of 10:1, aptamers specific to the Illumina polyploid identifier with a single 3' protrusion were ligated using T4 DNA ligase. The cDNA library was purified and selected to a size of 300 bp (E-Gel 2% SizeSelect gel, Invitrogen). Enrichment was performed by PCR using Phusion DNA polymerase and Illumina-specific PCR primers, followed by the addition of Illumina hexabase indexing and flow cell-specific sequences. All prior to this step was cleaned using 1.8 volumes of Agencourt AMPure XP magnetic beads.All quality control was performed using Invitrogen's Qubit HS analysis, and fragment size was determined using Agilent's 2100 Bioanalyzer HS DNA analysis. Barcoded RNA-Seq libraries were aggregated, and 50 bps were sequenced as described above.

[0390] NGS data analysis, gene expression: Output sequence reads of RNA samples were preprocessed according to Illumina's standard protocol, including filtering out low-quality reads. Sequence reads were aligned with mm9 (A. T. Chinwalla et al., Nature 420, 520 (2002)) or hg18 (F. Collins, E. Lander, J. Rogers, R. Waterston, I. Conso, Nature 431, 931 (2004)) reference genome sequences using bowtie (version 0.12.5) (B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Genome Biol 10, R25 (2009)). For genome alignment, two mismatches were allowed, and only the best alignment (“-v2-best”) was reported; for transcript alignment, the default parameters were used. Reads that did not match the genome sequence were compared with a database of all possible exon-exon linker sequences of known genes from the UCSC (F. Hsu et al., Bioinformatics 22, 1036 (2006)). Expression values ​​were determined by crossing the read coordinates with the coordinates of the RefSeq transcripts, counting overlapping exons and linker reads, and normalizing to RPKM expression units (the number of reads located at each 1K base of an exon per million located reads) (A. Mortazavi, B. A. Williams, K. McCue, L. Schaeffer, B. Wold, Nature methods 5, 621 (2008)).

[0391] NGS data analysis to explore somatic mutations: somatic mutations were identified as previously described (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)).Sequence reads were aligned to mm9 or hg18 reference genomes using bwa (default option, version 0.5.8c) (H. Li, R. Durbin, Bioinformatics 25, 1754 (2009)). Indeterminate reads located at multiple sites in the genome were removed. Mutations were identified using a combination of two software programs: samtools (version 0.1.8) (H. Li, Bioinformatics 27, 1157 (2011)) and SomaticSniper (A. McKenna et al., Genome Research 20, 1297 (2010)). For B16F10 and C57BL / 6J, GATK (A. McKenna et al., Genome Research 20, 1297 (2010)) was also included. Assign a “false discovery rate” (FDR) confidence value to potential somatic variations identified in all their respective replications (M. Löwer et al., PLoS computational biology 8, e1002714 (2012)) (CT26 and MZ7-MEL only). Specification 45 / 48 pages 48 CN 121130065 A

[0392] Mutation selection and confirmation

[0393] The selection criteria for 50 B16F10 mutations for immunogenicity testing have been previously described (JC Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)). These mutation criteria include: (i) present in all three B16F10 replications and not in all three C57BL / 6 replications; (ii) occurring in RefSeq transcripts; (iii) causing nonsynonymous alterations; (iv) occurring in genes expressing B16F10 (median RPKM > 10 in replications, exon expression > 0); and (v) for each mutation, an Mmut score (see below) < 5.Of the remaining 59 mutations, MHC class I scores, MHC class II scores, and transcript expression quantile rankings were generated, and the top 50 mutations (0.1 ≤ Mmut ≤ 3.9) were selected for confirmation by PCR (see (J. C. Castle et al., Exploiting the mutanome for tumor vaccination. Cancer Research 72, 1081 (2012)) for further details). The selection criteria for the 96 CT26.WT mutations used for immunogenicity testing were further refined to include the following criteria: (i) present in all three CT26.WT replications and not in all three Balb / cJ replications; (ii) FDR ≤ 0.05; (iii) occurring in UCSC-known gene transcripts; (iv) causing nonsynonymous alterations; (v) not present in the dbSNP database; and (vi) not located in genomic repetitive regions. From the remaining 493 mutations, eight 12-member groups were identified based on three characteristics: Mmut score (lowest - [0.1, 1.9] vs. highest - [3.9-20.3]), protein compartment (extracellular, intracellular), and gene expression (median 7.1 RPKM or lower vs. higher). Mutations were selected using a greedy algorithm, and thresholds were adjusted accordingly. 94 of the resulting 96 mutations were confirmed by PCR and then subjected to Sanger sequencing.

[0394] The selection criteria for the MZ7-ML mutations used for analysis included: (i) presence in two MZ7-MEL replications and absence in two autologous EBV-B replications, followed by steps (ii) to (vi) of CT26.WT described above. Steps (i) to (vi) reduced the initial list of ~8000 mutations to 743.

[0395] MHC binding prediction and Mmut score calculation

[0396] MHC binding prediction was performed using the IEDB analysis resource consistency tool (http: / / tools.immuneepitope.org / analyze / html / mhc_binding.html) (Y. Kim et al., Nucleic Acids Research 40, W525 (2012)), which combines the best-performing prediction methods based on benchmark studies from ANN (C. Lundegaard et al., Nucleic Acids Research 36, W509 (2008); M. Nielsen et al., Protein Science 12, 1007 (2009)), SMM (B. Peters, A. Sette, BMC bioinformatics 6, 132 (2005)) and also some allele models (J. Sidney et al., Immunome Research 4, 2 (2008)) (HH Lin, S. Ray, S. Tongchusak, EL Reinherz, V. Brusic, BMC immunology 9, 8 (2008); B. Peters et al., PLoS computational biology 2, e65 (2006). The consistency method combines the predicted scores of all tools by generating a percentage ranking that reflects the binding prediction score of a given peptide against 5 million random peptides from SWISSPROT.

[0397] For each mutation, we calculate the predicted MHC consistency score for all possible (i) sequence windows (locating the mutation), (ii) epitope determination, and (iii) possible mouse MHC class I alleles. The minimum score of all MHC consistency scores is defined as the Mmut score.

[0398] The log-odds matrix and T score are calculated.

[0399] The log-odds matrix can be estimated from sequence alignment comparisons from large protein databases.Early log-odds matrices were based on pairwise comparisons of sequences (BLOSUM62 (S. Kreiter et al., Cancer Immunology, Immunotherapy 56, 1577 (2007))) and maximum parsimony (MP) estimation methods (e.g., PAM250 (M. O. Dayhoff, R. M. Schwartz, BC Orcutt, A model for evolutionary change. MO Dayhoff, ed. Atlas of protein sequence and structure Vol. 5, 345 (1978)), JTT250 (S. Q. Le, O. Gascuel, Molecular biology and evolution 25, 1307 (2008)) and Gonnet matrices (CC Dang, V. Lefort, VS Le, QS Le, O. Gascuel, ...). Bioinformatics 27, 2758 (2011)). Recently, methods based on maximum probability (ML) have been developed (e.g., VT160 (P. G. Higgs, T. K. Attwood, Bioinformatics and molecular evolution. (Wiley-Blackwell, 2009)), WAG (S. Whelan, N. Goldman, Molecular biology and evolution 18, 691 (2001)), and LG (V. Lennerz et al., Proceedings of the National Academy of Sciences of the United States of America 102, 16013 (2005)). Since ML is not limited to comparing only closely related sequences, and neither are MP-based methods, this estimation method is expected to be the most accurate.

[0400] The calculation of the log-probability matrix has been described in detail elsewhere (CC Dang, V. Lefort, VS Le, QS Le, O. Gascuel, Bioinformatics 27, 2758 (2011)). In short, the standard model of amino acid substitution is assumed to be a Markovian, time-continuous, and time-reversible model represented by a 20×20 rate matrix, where is the number of substitutions from amino acid i to j per unit time, and the diagonal elements are chosen to satisfy . Q can be decomposable such that for i ≠ j, , where is a symmetric commutative matrix, and is the probability of observing amino acid i (CC Dang, V. Lefort, VS Le, QS Le, O. Gascuel, Bioinformatics 27, 2758 (2011)). Finally, Q is normalized so that the time unit t = 1.0 corresponds to 1.0 expected substitutions per site or one “acceptable point mutation” per site, expressed as a PAM distance of 100 (MO Dayhoff, RM Schwartz, BC Orcutt, A model for evolutionary change. MO Dayhoff, ed. Atlas of protein sequence and structure Vol. 5, 345 (1978); S. Q. Le, O. Gascuel, Molecular biology and evolution 25, 1307 (2008); C. C. Dang, V. Lefort, V. S. Le, Q. S. Le, O. Gascuel, Bioinformatics 27, 2758 (2011)). After time t, the probability that amino acid i is substituted by amino acid j is given by a 20×20 probability matrix (using symbolic representation of matrix exponentiation). The log-probability matrix for time t is given by a log-probability 20×20 matrix (MO Dayhoff, RM Schwartz, BC Orcutt, A model for evolutionary change. MO Dayhoff, ed. Atlas of protein sequence and structure Vol.5, 345 (1978, 1978)).Time reversibility means that it is therefore symmetric (P. G. Higgs, T. K. Attwood, Bioinformatics and molecular evolution. (Wiley-Blackwell, 2009)).

[0401] Here, the T-score is defined as , and depends on the evolutionary model and time t. We investigated various models and PAM distances for the T-score, including PAM, BLOSUM62, JTT, VT160, Gonnet, WAG, WAG, and LG (see references below). The figures in this report are generated using the T-score based on the WAG model and PAM distance 250. This large PAM distance implies a high probability of amino acid changes (P. G. Higgs, T. K. Attwood, Bioinformatics and molecular evolution. (Wiley-Blackwell, 2009)), and can be used to detect distant relationships between sequences whose residues may differ but retain the physicochemical properties of amino acids (MO Dayhoff, R. M. Schwartz, BC Orcutt, A model for evolutionary change. MO Dayhoff, ed. Atlas of protein sequence and structure Vol.5, 345 (1978); PG Higgs, T. K. Attwood, Bioinformatics and molecular evolution. (Wiley-Blackwell, 2009)).

[0402] Using t-distribution test statistics, we compared the average T-scores of the WAG matrix for immunogenic and non-immunogenic epitopes in Table 3 using various PAM scores (1, 10, 25, 50, 100, 150, 200, and 250). As expected, the analysis of the test statistics showed that the P-value monotonically decreased with PAM distance, suggesting that a PAM distance of 250 was the optimal solution (data not shown). For all matrices except the PAM matrix, which had the lowest accuracy among all evolutionary models, the classification to HA and Hn was the same.Among all evolutionary models, the WAG250 model results in the largest separation between the HA and Hn epitopes in Table 3, measured by test statistics: [maximum T-score (HA) - minimum T-score (Hn)] / σ(T-score (HA), T-score (Hn)) (data not shown). The same test statistics also result in the largest separation for the PAM distance 250 compared to smaller PAM distances.

[0403] Published CD8+ Epitopes

[0404] CD8+ epitopes with single mutated amino acids were collected from the list of tumor antigens resulting from mutations published in the Cancer Immunity Journal (P. Van der Bruggen, V. Stroobant, N. Vigneron, B. Van den Eynde. (Cancer Immunity, http: / / www.cancerimmunity.org / peptide / , 2013) (http: / / cancerimmunity.org / peptide / mutations / ). HLA alleles were obtained from the published table or from the original paper (if the latter is more precise).The references listed in Table 4 are as follows: (1) Lennerz et al. PNAS 102 (44), pp. 16013–16018 (2005); (2) Karanikas et al. Cancer Res 61 (9), pp. 3718–3724 (2001); (3) Sensi et al. Cancer Res 65 (2), pp. 632–640 (2005); (4) Linard et al. J. Immunol 168 (9), pp. 4802–4808 (2002); (5) Zorn et al. Eur. J. Immunol 29 (2), pp. 592–601 (1999); (6) Graf et al. Blood 109 (7), pp. 2985–2988 (2007); (7) Robbins et al. J. Exp. Med 183 (3) , pp. 1185– 1192 (1996); (8) Vigneron et al. Cancer Immun 2, pp. 9 (2002); (9) Echchakir et al. Cancer Res 61 (10) , pp. 4078–4083 (2001); (10) Hogan et al. Cancer Res 58 (22), pp. 5144–5150 (1998); (11) Ito et al. Int. J. Cancer 120 (12), pp. 2618–2624 (2007); (12) Wölfel et al. Science 269 (5228), pp. 1281–1284 (1995); (13) Gjertsen et al. Int. J. Cancer 72 (5), pp. 784–790 (1997).

[0405] Example 3: Example of a scheme to improve the prioritization of immunogenic mutations by weighting mutation scores

[0406] RNA injected into cells, once translated and cleaved into short peptides, can be presented by different HLA types within the cell. Therefore, it is obvious that the more HLA types predicted to have low MHC concordance (or similarity) scores, the more likely a given mutation is to be immunogenic, as it may be presented in parallel on multiple HLA types.Therefore, weighting mutations by the number of HLA types classified as HA and / or HBUHC, or even simply by the number of HLA types with low Mmut scores, may improve immunogenicity ranking. In the most common protocols, when we inject cells with 27-polymer RNA or peptides, we have the freedom not only to choose the HLA type but also the freedom to choose the peptide length and the mutation site of that peptide. Thus, we can look at all possible HLA types, all possible window lengths, and all possible mutation sites within the window, and calculate the number of protocols classified as HA and / or HBUHC (for each given mutation) (Figure 12). This can be an important weighting factor for mutation prioritization, thereby selecting the most effective epitopes for vaccination. Figure 13 shows an example of a scatter plot of all these protocols as a function of Mmut and ΔM = Mmut – Mwt.Instruction manual page 48 / 48 51 CN 121130065 A Figure 1 Instruction manual figure 1 / 13 page 52 CN 121130065 A Figure 2 Instruction manual figure 2 / 13 page 53 CN 121130065 A Figure 3 Instruction manual figure 3 / 13 page 54 CN 121130065 A Figure 4 Instruction manual figure 4 / 13 page 55 CN 121130065 A Figure 5 Instruction manual figure 5 / 13 page 56 CN 121130065 A Figure 6 Instruction manual figure 6 / 13 page 57 CN 121130065 A Figure 7 Instruction manual figure 7 / 13 page 58 CN 121130065 A Figure 8 Instruction manual figure 8 / 13 page 59 CN 121130065 A Figure 9 Instruction manual figure 9 / 13 page 60 CN 121130065 A Figure 10 Figure 11 (Page 10 / 13, CN 121130065 A) Figure 12 (Page 12 / 13, CN 121130065 A) Figure 13 (Page 13 / 13, CN 121130065 A) Abstract: The present invention relates to methods for predicting T cell epitopes. In particular, the present invention relates to methods for predicting whether modifications in peptides or polypeptides such as tumor-associated neoantigens are immunogenic or not. The methods of the invention are useful, in particular, for the provision of vaccines which are specific for a patient's tumor and, thus, in the context of personalized cancer vaccines.

Claims

1. A pharmaceutical composition comprising one or more modified peptides, Each of these modified peptides is immunogenic. Each modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a specified position relative to the corresponding unmodified peptide. Each modified peptide and its corresponding unmodified peptide are identical except for the one or more amino acid modifications described, which are impossible to occur in nature, and each modified amino acid and its corresponding unmodified amino acid are chemically and physically distinct. Each modified peptide and its corresponding unmodified peptide is 8 to 15 amino acids in length. Each modified peptide and its corresponding unmodified peptide bind to one or more MHC molecules.

2. Immunogenic modified peptides, The immunogenic modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a specified position relative to the corresponding unmodified peptide. The immunogenic modified peptides and their corresponding unmodified peptides are identical except for one or more amino acid modifications, wherein the amino acid modifications are impossible to occur in nature, and each modified amino acid and its corresponding unmodified amino acid are chemically and physically distinct. Each type of immunogenic modified peptide and its corresponding unmodified peptide has a length of 8 to 15 amino acids. Each immunogenic modified peptide and its corresponding unmodified peptide bind to one or more MHC molecules.

3. Use of the immunogenic peptide of claim 2 in the preparation of a medicament for treating cancer.

4. Nucleic acid sequences encoding modified peptides that promote immunogenicity. The immunogenic modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a specified position relative to the corresponding unmodified peptide. The immunogenic modified peptides and their corresponding unmodified peptides are identical except for one or more amino acid modifications, wherein the amino acid modifications are impossible to occur in nature, and each modified amino acid and its corresponding unmodified amino acid are chemically and physically distinct. Each type of immunogenic modified peptide and its corresponding unmodified peptide has a length of 8 to 15 amino acids. Each immunogenic modified peptide and its corresponding unmodified peptide bind to one or more MHC molecules.

5. Use of the nucleic acid sequence of claim 4 in the preparation of a medicament for treating cancer.

6. Vaccines, which include: One or more modified peptides; One or more nucleic acids encoding the one or more modified peptides; or Multiple cells expressing one or more of the modified peptides, Each of these modified peptides is immunogenic. Each modified peptide comprises one or more amino acid modifications, wherein each amino acid modification is located at a specified position relative to the corresponding unmodified peptide. Each modified peptide and its corresponding unmodified peptide are identical except for the one or more amino acid modifications described, which are impossible to occur in nature, and each modified amino acid and its corresponding unmodified amino acid are chemically and physically distinct. Each modified peptide and its corresponding unmodified peptide is 8 to 15 amino acids in length. Each modified peptide and its corresponding unmodified peptide bind to one or more MHC molecules.

7. The vaccine of claim 6, wherein the one or more modified peptides comprise one or more non-synonymous mutations corresponding to tumor-specific cancer mutations.

8. The vaccine of claim 7, wherein the vaccine is a personalized cancer vaccine designed to target tumor-specific cancer mutations in a patient.

9. A method for predicting the immunogenicity of modified peptides containing amino acid modifications, wherein the modification is a substitution, the method comprising the following steps: a) Determine the binding score of the modified peptide to one or more MHC molecules. b) Determine the binding score of the unmodified peptide to one or more MHC molecules, and c) Determine the chemical and physical similarity score between the unmodified amino acid and the modified amino acid using an evolution-based log-probability matrix for determining chemical and physical similarity, wherein the chemical and physical similarity score is determined based on the probability of amino acid interchangeability in the natural state. The higher the frequency of amino acid exchange in the natural state, the higher the amino acid similarity is considered. Wherein, if (i) the binding score of the unmodified peptide to one or more MHC molecules meets the threshold indicating binding to one or more MHC molecules, (ii) the binding score of the modified peptide to one or more MHC molecules meets the threshold indicating binding to one or more MHC molecules, and (iii) the chemical and physical similarity score of the unmodified amino acid to the modified amino acid meets the threshold indicating chemical and physical dissimilarity, then the modified peptide is predicted to be immunogenic.

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