EA045699B1 - PERSONALIZED PLATFORM FOR IMMUNOGENIC PEPTIDE IDENTIFICATION - Google Patents

Description

Technical field

The present invention relates to methods for predicting whether a polypeptide is immunogenic for a particular human subject, methods for identifying polypeptide fragments that are immunogenic for a particular human subject, methods for producing personalized or precision pharmaceutical compositions or kits containing such polypeptide fragments specific to the subject. -human pharmaceutical compositions containing such polypeptide fragments, and methods of treatment using such compositions.

State of the art

For decades, scientists have assumed that chronic diseases cannot be cured through natural human defenses. Recently, however, significant tumor regressions have been observed in individuals treated with antibodies that block immunoinhibitory molecules, accelerating the development of the field of cancer immunotherapy. These clinical data demonstrate that reactivation of existing T-lymphocyte responses results in significant clinical benefit for individuals. These advances have renewed enthusiasm for the development of cancer vaccines that induce tumor-specific T lymphocyte responses. Contrary to promise, existing immunotherapy is only effective for a subset of individuals. In addition, most cancer vaccine trials have failed to demonstrate statistically significant efficacy due to low rates of tumor regression and antitumor T cell responses in individuals. Similar failures have been reported with therapeutic and prophylactic vaccines that attempt to engage T-lymphocyte responses in the areas of human immunodeficiency virus (HIV) and allergy. There is a need to overcome clinical failures of immunotherapies and vaccines.

The essence of the invention

Antigen presenting cells (APCs) convert protein antigens into peptides. These peptides bind to human leukocyte antigen (HLA) molecules and are presented on the cell surface as peptide-HLA complexes with T lymphocytes. Different individuals express different HLA molecules, and different HLA molecules present different peptides. Thus, in accordance with the current art, a peptide or fragment of a larger polypeptide is identified as immunogenic for a particular human subject if it is presented by an HLA molecule that is expressed by the subject. In other words, the current state of the art describes immunogenic peptides as HLA-restricted epitopes. However, HLA-restricted epitopes induce T-lymphocyte responses in only a subset of individuals who express the HLA molecule) Peptides that activate T-lymphocyte responses in one individual are inactive in others, despite HLA allele compatibility) Thus, it was unknown why the molecules An individual's HLA presents antigen-derived epitopes that positively activate T-lymphocyte responses.

As stated herein, it is necessary for multiple HLAs expressed by an individual to present the same peptide to initiate a T lymphocyte response. Thus, fragments of a polypeptide antigen that are immunogenic for a particular individual are fragments that can bind to multiple HLA class I (activate cytotoxic T lymphocytes, killer T cells) or class II (activate helper T lymphocytes, helper T cells) expressed by this individual. Accordingly, in a first aspect, the present invention provides methods for predicting whether a polypeptide or polypeptide fragment is immunogenic for a particular human subject, the methods comprising the steps of (i) determining whether the polypeptide contains:

(a) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (b) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least two molecules the subject's HLA class II, and (ii) predicting that:

A) the polypeptide is immunogenic in a subject if the polypeptide contains at least one sequence that meets the requirements of step (i), or

B) the polypeptide is not immunogenic for the subject if the polypeptide does not contain at least one sequence that meets the requirements of step (i).

The invention also provides methods for identifying a polypeptide fragment as immunogenic for a particular human subject, the methods comprising the steps of:

(i) determining that the polypeptide contains:

(a) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (b) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least two molecules HLA class II of the subject, and (ii) the specified sequence as a fragment of the polypeptide that is for the subject.

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In some embodiments, the methods of the invention include the step of determining or obtaining the HLA class I genotype and/or the HLA class II genotype of a particular human subject.

A particular polypeptide antigen may contain more than one fragment, which is a T-lymphocyte epitope capable of binding to multiple HLAs of a particular individual. The combined group of all such fragments characterizes the set of antigen-specific T-lymphocyte responses of an individual, while the amino acid sequence of each fragment characterizes the specificity of each activated T-lymphocyte clone.

Accordingly, in some cases, the method is repeated until all polypeptide fragments that represent a T lymphocyte epitope capable of binding to at least two HLA class I and/or at least two HLA class II of the subject have been identified. This method characterizes the immune response of a subject to a polypeptide.

The present invention further provides methods of treating a human subject in need thereof, the method comprising administering to the subject a polypeptide, a pharmaceutical composition, or a set of polypeptides from a panel of polypeptides that has been identified or selected by any of the above methods, or contains a fragment of a polypeptide that has been identified or selected by any of the above methods; their use in a method of treating a respective human subject; and their use in the manufacture of a medicinal product for the treatment of the subject concerned.

Polypeptide fragments that are determined to be immunogenic for a particular human subject, in accordance with the methods described above, can be used to prepare human subject-specific immunogenic compositions.

Accordingly, in a further aspect, the invention provides methods for formulating or producing a human subject-specific pharmaceutical composition or kit or panel of polypeptides for use in a method of treating a particular human subject, the methods comprising:

(i) selecting a polypeptide fragment that has been identified as immunogenic for the subject by the method described above;

(ii) if the fragment selected in step (i) is an HLA class I binding epitope, optionally selecting a longer polypeptide fragment, the longer fragment being:

a) contains the fragment selected in step (i), and

b) is a T-lymphocyte epitope capable of binding to at least three or the most possible HLA class II molecules of the subject;

(iii) selecting a first sequence of up to 50 contiguous amino acids of the polypeptides, the contiguous amino acids comprising the amino acid sequence of the fragment selected in step (i) or the longer fragment selected in step (ii);

(iv) repeating steps (i)-(iii) to select a second amino acid sequence from up to 50 consecutive amino acids of the same or a different polypeptide relative to the first amino acid sequence;

(v) if necessary, further repeating steps (i)-(iii) to select one or more additional amino acid sequences from up to 50 consecutive amino acids of the same or different polypeptides relative to the first and second amino acid sequences, and (vi) compiling or obtaining a specific for the subject of a pharmaceutical composition, set or panel of polypeptides having as active ingredients one or more polypeptides that together contain all the amino acid sequences selected in the previous steps, optionally one or more, or each sequence flanked by N- and/or C -end with additional amino acids that are not part of the polypeptide sequence.

In some cases, each peptide either consists of one of selected amino acid sequences or consists of two or more amino acid sequences located end to end or overlapping in a single peptide.

The invention further provides a human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treating a particular human subject in need thereof, wherein the composition, kit or panel contains as active ingredients a first and a second peptide and optionally one of several additional peptides. peptides, wherein each peptide contains an amino acid sequence that is a T-lymphocyte epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T-lymphocyte epitope of the first , the second and optionally any additional peptides are different from one another, and the pharmaceutical composition or kit optionally contains at least one pharmaceutically acceptable diluent, carrier or preservative.

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The invention further provides a human subject-specific pharmaceutical composition, kit or panel of polypeptides for use in a method of treating a particular human subject in need thereof, wherein the composition or kit contains as an active ingredient a polypeptide comprising a first region and a second region, and optionally one of several additional regions, each region containing an amino acid sequence that is a T-lymphocyte epitope capable of binding to at least two HLA class I molecules and/or at least two HLA class II molecules of the subject, wherein the amino acid sequence of the T-lymphocyte the epitope of the first, second and optionally any additional regions are different from each other, and the pharmaceutical composition or kit optionally contains at least one pharmaceutically acceptable diluent, carrier or preservative.

The invention further provides a method of designing or producing a polypeptide for inducing an immune response in a particular human subject, comprising selecting an amino acid sequence that represents a T lymphocyte epitope capable of binding to at least three HLA class I molecules or at least three HLA class I molecules II subject, and composition or production of a polypeptide containing the selected amino acid sequence.

In additional aspects, the invention provides a method of inducing an immune response or a method of treatment comprising administering to a human subject in need thereof a human subject-specific pharmaceutical composition or kit or panel of polypeptides described above, wherein the composition, kit or panel of polypeptides is specific to the subject ;

a human subject-specific immunogenic composition, kit or panel described above for use in a method of inducing an immune response or method of treating a particular human subject, and use of a human subject-specific pharmaceutical composition or kit or panel of polypeptides described above in manufacturing a medicinal product, wherein the medicinal product is intended to induce an immune response or treat a specific subject.

In a further aspect, the invention provides a system comprising:

(a) a memory module configured to store data containing the HLA class I and/or class II genotype of the subject and the amino acid sequence of one or more test polypeptides, and (b) a computing module configured to identify and/or quantify amino acid sequences in one or more test polypeptides that are capable of binding to a plurality of HLA class I molecules of a subject and/or that are capable of binding to a plurality of HLA class II molecules of a subject.

The present invention provides a method of treating a human subject in need thereof, the method comprising administering to the subject a polypeptide, panel of polypeptides, pharmaceutical composition or active ingredient polypeptides from the set described above, wherein the subject is determined to express at least three HLA class I molecules and/or or at least three HLA class II molecules capable of binding to the polypeptide or one or more polypeptides of the active ingredient of the pharmaceutical composition or kit.

The invention will now be described in more detail by way of example and not by way of limitation, and with reference to the accompanying drawings. Upon review of the present invention, many equivalent modifications and variations will be apparent to those skilled in the art. Accordingly, exemplary embodiments of the present invention are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the scope of the invention. All documents cited, whether above or below, are incorporated herein by reference in their entirety.

The present invention includes a combination of the described aspects and preferred features, unless such combination is clearly prohibited or expressly stated to be avoided. As used in this specification and the accompanying claims, the singular number includes the plural unless the content clearly indicates otherwise. Thus, for example, a reference to “peptide” includes two or more such peptides.

Section headings are used herein for convenience only and should not be construed as limiting in any way.

Description of graphic materials

Fig. 1 - ROC curve (receiver operating characteristic, dependence of the number of correctly classified positive objects on the number of incorrectly classified negative objects) of HLA-restricted PEPI (personal epitope) biomarkers.

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Fig. 2 - ROC curve for test >1 PEPI3+ to determine diagnostic accuracy.

Fig. 3 - Distribution of PEPI3+ HLA class I versus CD8+ T cell responses, measured using a state-of-the-art assay, among peptide pools used in CD8+ T cell response assays. A: limited by HLA class IPEPI3+. The Overall Percent of Agreement (OPA) of 90% among T-lymphocyte responses and PEPI3+ peptides demonstrates the utility of the disclosed peptides for predicting the post-vaccination set of T-lymphocyte responses for individuals. B: HLA class I restricted epitopes (PEPI1+). The ORA between predicted epitopes and CD8+ T cell responses was 28% (not statistically significant). Darkest gray: true positive (TP), both peptides and T lymphocyte responses were detected; light gray: true negative (result) (FN, False negative), only T-lymphocyte responses were detected; lightest gray: false positive (result) (FP, False positive), only peptides were detected; dark gray: true negative (TN, True negative): neither peptides nor T-lymphocyte responses were detected.

Fig. 4 - Distribution of HLA class II PEPI versus CD4+ T cell responses measured using a state-of-the-art assay, among the peptide pools used in the assays. A: restricted by HLA class II PEPI4+. 67% ORA between PEPI4+ and CD4+ T cell responses (p=0.002). B: HLA class II restricted epitopes. The ORR between HLA class II-restricted epitopes and CD4+ T cell responses was 66% (not statistically significant). Darkest gray: true positive (TP), both peptides and T-lymphocyte responses were detected; light gray: true negative (result) (FN, False negative), only T-lymphocyte responses were detected; lightest gray: false positive (result) (FP, False positive), only peptides were detected; dark gray: true negative (TN, True negative): neither peptides nor T-lymphocyte responses were detected.

Fig. 5 - multi-HLA binding peptides that determine the HPV-16 LPV vaccine-specific set of T-lymphocyte responses in 18 patients with VIN-3 (Vulvar Intraepithelial Neoplasia, vulvar intraepithelial neoplasia) and 5 patients with cervical cancer. The number of HLA class I-restricted (A and B) PEPI3 and the number of HLA class II-restricted (C and D) PEPI3 obtained from LPV (Live Attenuated Poliovaccine) antigens of each patient. Light grey: patients with immune response measured after vaccination in a clinical trial; dark grey: patients without an immune response measured after vaccination in a clinical trial. Results show that peptides binding to >3 HLA class I predicted CD8+ T cell reactivity, and peptides binding to >4 HLA class II predicted CD4+ T cell reactivity.

Fig. 6 - multiple HLA class I binding peptides that determine the HPV (Human Papilloma Virus) vaccine-specific set of T-lymphocyte responses in 2 patients. A: Four HPV antigens in the HPV vaccine. The boxes represent the length of the amino acid sequences from the N-terminus to the C-terminus. B: Process for identifying multiple HLA binding peptides for two patients: patient HLA sequences annotated with the 4-digit HLA genotype to the right of the patient ID. The location of the 1st amino acid 54 and 91 epitopes that can bind to the HLA (PEPI1+) of patient 12-11 and patient 14-5, respectively, is depicted by lines. PEPI2 presents peptides selected from PEPI1+ that can bind to multiple patient HLAs (PEPI2+). PEPI3 represents peptides that can bind to the patient's HLA=3 (PEPI3+). PEPI4 represents peptides that can bind to the patient's HLA=4 (PEPI4+). PEPI5 represents peptides that can bind to the patient's HLA5 (PEPI5+). PEPI6 represents peptides that can bind to patient HLA 6 (PEPI6). C: A set of specific PEPI3+ DNA vaccines (DNA, deoxyribonucleic acid, deoxyribonucleic acid) from two patients characterizes their specific T-lymphocyte responses to the vaccine.

Fig. 7 - correlation between PEPI3+ score > 1 and cytotoxic T lymphocyte (CTL) response rate to peptide targets determined in clinical trials.

Fig. 8 - Correlation between PEPI3+ score > 1 and clinical immune response rate (IRR) of immunotherapeutic vaccines. Dashed lines: 95% confidence interval.

Fig. 9 - correlation between the indicator > 2 PEPI3+ and the disease control rate (DCR, Disease Control Rate) of immunotherapeutic vaccines. Dashed lines: 95% confidence interval.

Fig. 10 - IPI (International Prognostic Index) test of a patient with an HLA immune response) Overall survival (OS, Overall Survival) of patients with melanoma treated with ipilimumab. Data from 4 independent clinical trials: patients with HLA immune response (black line) and patients without HLA immune response (gray line). Statistical analysis: Cox regression for proportional hazards of survival. A: trial of 1:18 patients with HLA immune response and 30 patients without HLA immune response; B: trial 2: 24 patients with HLA immune response and 20 patients without HLA immune response; c: trial 3: 6 patients with immune response

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HLA and 11 patients without HLA immune response; D: trial 4:13 patients with immune response

HLA and 38 patients without HLA immune response.

Fig. 11 - multi-binding HLA peptides in mutational neoantigens. A: correlation of mutational load, neoantigen load (neoantigens are VanAllen neoepitopes) and B: correlation of PEPI3+ load and clinical effectiveness (min-Q1-mean-Q3-max).

Fig. 12 - HLA map for the Rindopepimut vaccine on the HLA alleles of subjects in the simulated population.

Fig. 13 - probability of expression of the antigen used in the preparation of the vaccine in the tumor cells of patient XYZ. There is a greater than 95% chance that 5 of the 12 target antigens in the vaccination regimen are expressed in the patient's tumor. Therefore, the 12 peptide vaccines together can induce immune responses against at least 5 ovarian cancer antigens with a probability of 95% (AGP95). Each peptide has an 84% chance of inducing immune responses in patient XYZ. AGP50 is the mean (expected value) = 7.9 (this is an indicator of the effectiveness of the vaccine on the tumor in patient XYZ).

Fig. 14 - CT scan results (MRI, Magnetic Renonance Imaging) of patient XYZ who received the personalized (PIT) vaccine. At this late stage, a patient with heavily pretreated ovarian cancer had an unexpected objective response after treatment with the PIT vaccine. These CT scan results suggest that the PIT vaccine in combination with chemotherapy significantly reduces the patient's tumor burden. The patient is now continuing treatment with the PIT vaccine.

Fig. 15 - probability of expression of the vaccine antigen in the patient's tumor cells ABC) There is a greater than 95% probability that 4 of the 13 vaccine target antigens are expressed in the patient's tumor. Therefore, the 12 peptide vaccines together can induce immune responses to at least 4 breast cancer antigens with a probability of 95% (AGP95). Each peptide has an 84% chance of inducing immune responses in patient ABC. AGP50 is the mean (expected value) of the discrete probability distribution = 6.45 (this is an indicator of the effectiveness of the vaccine on the tumor in patient ABC).

Fig. 16 is a schematic representation of examples of amino acid positions in overlapping HLA class I and HLA class II binding epitopes in a 30-mer peptide (a peptide having 30 amino acids).

Description of sequences

Sequence identification numbers (ID No.) 1-13 provide additional peptide sequences described in table. 17. Sequences with ID No.: 14-26 show personalized peptides of the vaccine compiled for patient XYZ, described in table. 26.

The sequences with ID No.: 27-38 show the personalized peptides of the vaccine compiled for patient ABC, described in table. 29.

Sequences with ID No.: 39-86 contain additional 9 mer T-lymphocyte epitopes described in table. 33.

Detailed description of the invention

HLA genotypes.

HLAs are encoded by the majority of polymorphic genes in the human genome. Each individual has maternal and paternal alleles for three HLA class I molecules (HLA-A*, HLA-B*, HLA-C*) and four HLA class II molecules (HLA-DP*, HLA-DQ*, HLA-DRB1 *, HLA-DRB3*/4*/5*). Almost every individual expresses a different combination of 6 HLA class I molecules and 8 HLA class II molecules, which represent different epitopes from the same protein antigen. The function of HLA molecules is to regulate T-lymphocyte responses. However, until now it was unknown how human HLA regulates T cell activation.

The nomenclature used to designate the amino acid sequence of an HLA molecule is as follows: gene name*allele: protein number, which for example might look like this: HLA-A*02:25. In this example, 02 refers to the allele. In most cases, alleles are defined by serotypes—meaning that the proteins of a given allele will not react with each other in serological assays. Protein numbers (25 in the example above) are assigned sequentially as the protein is discovered. A new protein number is assigned to some protein with a different amino acid sequence (for example, even a change in one amino acid sequence is considered a different protein number). Additional nucleic acid sequence information for a given locus may be applied to HLA nomenclature, but such information is not required for the methods described herein.

An individual's HLA class I genotype or HLA class II genotype may refer to the actual amino acid sequence of each HLA class I or class II individual, or may refer to the nomenclature described above, which designates, at a minimum, the allele and protein number of each HLA gene) In some In embodiments, an individual's HLA genotype is obtained or determined by

- 5 045699 analysis of a biological sample from an individual. A biological sample typically contains the subject's DNA. The biological sample may be, for example, blood, serum, plasma, saliva, urine, exhalation, cellular or tissue sample. In some embodiments, the biological sample is a saliva sample. In some embodiments, the biological sample is a buccal swab sample. The HLA genotype can be obtained or determined using any suitable method. For example, the sequence can be determined by sequencing the HLA gene loci using methods and protocols known in the art. In some embodiments, the HLA genotype is determined using site-specific primer (SSP, sequence specific primer) methods. In some embodiments, the HLA genotype is determined using sequence specific oligonucleotide (SSO) methods. In some embodiments, the HLA genotype is determined using sequence based typing (SBT) methods. In some embodiments, the HLA genotype is determined using next generation sequencing. Alternatively, an individual's HLA set may be stored in a database and accessed using methods known in the art.

HLA epitope binding.

A given HLA of a subject will present to T lymphocytes only a limited number of different peptides produced when protein antigens are processed into APC. As used herein, the term present or present when used in relation to HLA refers to the binding between a peptide (epitope) and HLA) In this regard, the term present or present peptide is synonymous with peptide binding.

As used herein, the term epitope or T-lymphocyte epitope refers to the sequence of contiguous amino acids contained in a protein antigen that has binding affinity (capable of binding) to one or more HLAs) The epitope is HLA- and antigen-specific (HLA-epitope pairs, predicted by known methods), but is not specific to the subject. An epitope, T-lymphocyte epitope, polypeptide, polypeptide fragment, or composition containing a polypeptide or fragment thereof is immunogenic in a particular human subject if it is capable of inducing a T-lymphocyte response (cytotoxic T-lymphocyte response or T-helper response) in that subject. In some cases, the T helper response is a Th1 type T helper response. In some cases, an epitope, T-lymphocyte epitope, polypeptide, fragment of a polypeptide, or a composition containing a polypeptide or fragment thereof is immunogenic in a particular human subject if it is more likely to induce a T-lymphocyte response or immune response in the subject than another T-lymphocyte an epitope (or in some cases each of two different T-lymphocyte epitopes) capable of binding to only one HLA molecule of a subject.

The terms T lymphocyte response and immune response are used interchangeably herein and refer to the activation of T lymphocytes and/or the induction of one or more effector functions upon recognition of one or more binding HLA-epitope pairs. In some cases, the immune response includes an antibody response, as HLA class II molecules stimulate helper responses that are involved in inducing both prolonged CTL and antibody responses. Effector functions include cytotoxicity, cytokine production, and proliferation. According to the present invention, an epitope, T-lymphocyte epitope, or polypeptide fragment is immunogenic for a particular subject if it is capable of binding to at least two, or in some cases at least three, HLA class I or at least two, or in some cases according to at least three, or at least four HLA class II of the subject.

For purposes of the present invention, the term personal epitope, or PEPI, is introduced to distinguish subject-specific epitopes from HLA-specific epitopes. PEPI is a polypeptide fragment composed of a sequence of contiguous amino acids of a polypeptide that is a T lymphocyte epitope capable of binding to one or more HLA class I molecules of a particular human subject. In other cases, PEPI is a polypeptide fragment composed of a sequence of contiguous amino acids of a polypeptide that is a T lymphocyte epitope capable of binding to one or more HLA class II molecules of a particular human subject. In other words, PEPI is a T-lymphocyte epitope that is recognized by the HLA set of a particular individual. Unlike an epitope, PEPIs are individual specific because different individuals have different HLA molecules, each of which binds to different T cell epitopes.

The term PEPP as used herein refers to a peptide or polypeptide fragment that can bind to one HLA class I molecule (or, in certain cases, an HLA class II molecule) of an individual. The term PEPI1+ refers to a peptide or polypeptide fragment that can bind to one or more HLA class I molecules of an individual.

The term PEPI2 refers to a peptide or polypeptide fragment that can bind to

- 6 045699 by two HLA class I (or II) molecules of an individual. The term PEPI2+ refers to a peptide or polypeptide fragment that can bind to two or more HLA class I (or II) molecules of an individual,

those. fragment identified in accordance with the method disclosed herein.

The term PEPI3+ refers to a peptide or polypeptide fragment that can bind to three HLA class I (or II) molecules of an individual. The term PEPI3+ refers to a peptide or polypeptide fragment that can bind to three or more HLA class I (or II) molecules of an individual.

The term PEPI4 refers to a peptide or polypeptide fragment that can bind to four HLA class I (or II) molecules of an individual. The term PEPI4+ refers to a peptide or polypeptide fragment that can bind to four or more HLA class I (or II) molecules of an individual.

The term PEPI5 refers to a peptide or polypeptide fragment that can bind to up to five HLA class I (or II) molecules of an individual. The term PEPI5+ refers to a peptide or polypeptide fragment that can bind to five or more HLA class I (or II) molecules of an individual.

The term PEPI6 refers to a peptide or polypeptide fragment that can bind to six HLA class I molecules (or six HLA class II molecules) of an individual.

Generally speaking, epitopes presented by HLA class I molecules are about nine amino acids long, and epitopes presented by HLA class II molecules are about fifteen amino acids long. However, for the purposes of the present invention, an epitope may be more or less nine (for HLA class I) or more or less fifteen (for HLA class II) amino acids in length within the range that the epitope is capable of binding to HLA) For example, an epitope that is capable of binding to HLA class I can be between 7 or 8 or 9 and 9 or 10 or 11 amino acids in length. The epitope that is capable of binding to HLA class II may be between 13 or 14 or 15 and 15 or 16 or 17 amino acids in length.

Thus, the invention described herein includes, for example, a method of predicting whether a polypeptide is immunogenic in a particular human subject, or identifying a fragment of a polypeptide as immunogenic in a particular human subject, the method comprising the steps of:

(i) determining whether the polypeptide contains:

a) a sequence of 7-11 contiguous amino acids that is capable of binding to at least two HLA class I of the subject, or

b) a sequence of 13-17 consecutive amino acids that is capable of binding to at least two HLA class II of the subject, and (ii) predicting that the polypeptide is immunogenic for the subject if the polypeptide contains at least one sequence that meets the requirements of the step (i); or predicting that the polypeptide is not immunogenic for the subject if the polypeptide does not contain at least one sequence that meets the requirements of step (i); or identifying said rigid amino acid sequence as a polypeptide fragment sequence that is immunogenic in a subject.

Using methods known in the art, epitopes that will bind to a known HLA can be determined. Any suitable method can be used, as long as the same method is used to determine multiple binding HLA-epitope pairs that are compared directly. For example, biochemical analysis may be used. You can also use lists of epitopes that are known to be associated with a given HLA) You can also use predictive or modeling software to determine which epitopes may be associated with a given HLA) Examples are given in Table. 1. In some cases, a T lymphocyte epitope is able to bind to a given HLA if it has a half-maximal inhibitory concentration or predicted IC50 of less than 5000 nM, less than 2000 nM, less than 1000 nM, or less than 500 nM.

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Table 1

Example software for epitope-HLA binding determination

TOOLS FORECASTING EPITOPE WEB ADDRESS BIMAS, NIH (National Institutes of Health, US National Institutes of Health) PPAPROC, University of Tübingen. Prediction of MHC (major histocompatibility complex, major histocompatibility complex), Vaccine Research Institute named after. Edward Jenner. www-bimas.cit.nih.gov/molbio/hla_bind/ EpiJen, Institute for Vaccine Research. Edward Jenner. http://www.ddg-pharmfac.net/epijen/EpiJen/EpiJen.htm NetMHC, Center for Biological Sequence Analysis http://www.cbs.dtu.dk/services/NetMHC/ SVMHC, University of Tübingen. http://abi.inf.uni-tuebingen.de/Services/SVMHC/ SYFPEITHI, Biomedical Informatics, Heidelberg http://www.svfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm ETC EPITOOLKIT, University of Jübingen. http://etk.informatik.uni-tuebingen.de/epipred/ PREDEP, Hebrew University, Jerusalem http://margalit.huii.ac.il/Teppred/mhc-bind/index.html RANCRER, MIF Bioinformatics http://bio.dfci.harvard.edu/RANKPEP/ IEDB, Immune Epitope Database http://tools.immuneepitope.org/main/html/tcell tools.html EPITOPES DATABASE WEB ADDRESS MHCBN, Institute of Microbial Technology, Chandigarh, INDIA http://www.imtech.res.in/raghava/mhcbn/ SYFPEITHI, Biomedical Informatics, Heidelberg http://www.syfpeithi.de/ AntiJen, Vaccine Research Institute Edward Jenner, EPIMHC MHC ligand database. http://www.ddg-pharmfac.net/antijen/AntiJen/antijenhomepage.htm EPIMHC MHC ligand database, MIF Bioinformatics http://immunax.dfci.harvard.edu/epimhc/ IEDB, Immune Epitope Database http://www.iedb.org/

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As stated herein, presentation of a T-lymphocyte epitope by multiple HLAs of an individual is generally required to initiate a T-lymphocyte response. Accordingly, the methods of the invention include determining whether the polypeptide has a sequence that is a T lymphocyte epitope capable of binding to at least two HLA class I molecules or at least two HLA class II molecules (PEPI2+) of a particular human subject.

The best predictor of cytotoxic T lymphocyte response to a given polypeptide is the presence of at least one T lymphocyte epitope that is present on three or more of an individual's HLA class I molecules (>1 PEPI3+). Accordingly, in some cases, the method includes determining whether the polypeptide has a sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of a particular human subject. In some cases, the method includes determining whether the polypeptide has a sequence that is a T lymphocyte epitope capable of binding to only three HLA class I molecules of a particular human subject. The T helper cell response can be predicted by the presentation of at least one T lymphocyte epitope that is presented by three or more (>1 PEPI3+) or 4 or more (>1 PEPI4+) HLA class II of an individual. Thus, in some cases, the method includes determining whether the polypeptide has a sequence that is a T lymphocyte epitope capable of binding to at least three HLA class II molecules of a particular human subject. In other cases, the method includes determining whether the polypeptide contains a sequence that is a T lymphocyte epitope capable of binding to at least four HLA class II molecules of a particular human subject. In other cases, the method includes determining whether the polypeptide contains a sequence that is a T lymphocyte epitope capable of binding to only three and/or only four HLA class II molecules of a particular human subject.

In some cases, the invention can be used to predict whether a polypeptide/fragment will induce both a cytotoxic T lymphocyte response and a helper T cell response in a particular human subject. The polypeptide/fragment contains both an amino acid sequence that is a T lymphocyte epitope capable of binding to a plurality of HLA class I molecules of a subject and an amino acid sequence that is a T lymphocyte epitope capable of binding to a plurality of HLA class II molecules of a subject. The epitopes that bind HLA class I and HLA class II may overlap completely or partially. In some cases, such polypeptide fragments may be identified by selecting an amino acid sequence that represents a T lymphocyte epitope capable of binding to multiple (e.g., at least two or at least three) HLA class I molecules of the subject, followed by screening one or more longer polypeptide fragments that are extended at the N- and/or C-terminus to bind to one or more HLA class II molecules of the subject.

Some subjects may have two HLA alleles that encode the same HLA molecule (for example, two copies for HLA-A*02:25 in the case of homozygosity). The HLA molecules encoded by these alleles bind all the same T-lymphocyte epitopes. For purposes of the present invention, the term binding to at least two HLA molecules of a subject as used herein includes binding to HLA molecules encoded by two identical HLA alleles in the same subject. In other words, the term binding to at least two HLA molecules of a subject and the like. could be expressed as binding to HLA molecules encoded by at least two HLA alleles of the subject.

Polypeptide antigens.

Described herein are methods for predicting whether a polypeptide is immunogenic in a particular human subject and identifying a fragment of the polypeptide as immunogenic in a particular human subject. As used herein, the term polypeptide refers to a full-length protein, a portion of a protein, or a peptide characterized as a chain of amino acids. As used herein, the term peptide refers to a short polypeptide containing between 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 , or 15 and 10, or 11, or 12, or 13, or 14, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50 amino acids.

As used herein, the terms fragment or fragment of a polypeptide refer to a chain of amino acids or amino acid sequence, generally of reduced length relative to the reference polypeptide, and containing, in general, an amino acid sequence identical to the reference polypeptide. Such a fragment according to the present invention may optionally be included in a larger polypeptide of which it is a part. In some cases, the fragment may contain the entire length of the polypeptide, for example, when the entire polypeptide,

- 9 045699 such as a 9 amino acid peptide, is a single T-lymphocyte epitope.

In some cases, the polypeptide is, or the polypeptide consists of all or part of an antigen expressed by a pathogenic organism (eg, a bacterium or parasite), a virus, or a cancer cell that is associated with an autoimmune disorder or response, or a pathogenic cell, or is an allergen, or an ingredient in a drug or pharmaceutical composition, such as a vaccine or immunotherapy composition. In some cases, the method of the invention includes the initial step of identifying or selecting a suitable polypeptide, for example a polypeptide, as further described below.

The polypeptide or antigen may be expressed in cells, or particularly in diseased cells, of a subject (eg, a tumor-associated antigen, a polypeptide expressed by a virus, intracellular bacteria or parasite, or the in vivo product of a vaccine or immunotherapy composition), or derived from environment (for example, food, allergen or medicine). The polypeptide or antigen may be present in a sample taken from a particular human subject. Both polypeptide antigens and HLA can be precisely defined by amino acid or nucleotide sequences and sequenced using methods known in the art.

The polypeptide or antigen may be a cancer-associated antigen (TAA, tumor-associated antigen). TAAs are proteins expressed in cancer or tumor cells. A cancer or tumor cell may be present in the sample obtained from the subject. Examples of TAAs include novel antigens (neoantigens) expressed during tumorigenesis, products of oncogenes and tumor suppressor genes, overexpressed or aberrantly expressed cellular proteins (e.g., HER2, MUC1), antigens produced by oncogenic viruses (e.g., EBV, HPV, HCV, HBV, HTLV), cancer testis antigens (CTAs) (e.g. MAGE family, NY-ESO) and cell type differentiation antigens (e.g. MART-1). TAA sequences can be found experimentally, or in published scientific papers, or in public databases such as the database of the Institute for Cancer Research. Ludwig (www.cta.lncc.br/). Cancer Immunity Database (cancerimmunity.org/peptide/) and TANTIGEN Tumor T-Cell Antigen Database (cvc. dfci) harvard. edu/tadb/).

In some cases, the polypeptide or antigen is not expressed or expressed to a minimal extent in normal healthy cells or tissues, but is expressed (in those cells or tissues) at a high rate (with a high frequency) by subjects having a particular disease or condition, such as a type of cancer or cancer that comes from a specific type of cell or tissue, such as breast cancer, ovarian cancer, or melanoma. Another example is colorectal cancer. Other, non-limiting examples of cancer include non-melanoma cancer of the skin, lung, prostate, kidney, bladder, stomach, liver, cervix, esophagus, non-Hodgkin's lymphoma, leukemia, pancreas, uterine body, lip, oral cavity, thyroid , brain, nervous system, gallbladder, larynx, pharynx, myeloma, nasopharynx, Hodgkin's lymphoma, testicles and Kaposi's sarcoma. Alternatively, the polypeptide may be expressed at low levels in normal healthy cells, but at high levels (overexpressed) in diseased (eg, cancer) cells or in subjects having a disease or condition. In some cases, the polypeptide is expressed or expressed at a high level compared to normal healthy cells or subjects in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such individuals, or a relevant subpopulation of people. For example, a subpopulation may be matched to a subject by ethnicity, geographic location, gender, age, disease, disease type or stage, genotype, or expression of one or more biomarkers.

In some cases, expression frequencies can be determined from published graphics and scientific publications. In some cases, the method of the invention includes the step of identifying or selecting such a polypeptide.

In some cases, the polypeptide is associated with, or is highly (over-)expressed in cancer cells or solid tumors. Typical types of cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors or blastomas. The cancer may or may not be a hormone-related or hormone-dependent cancer (eg, estrogen- or androgen-related cancer). The tumor can be malignant or benign. The cancer may or may not be metastatic.

In some cases, the polypeptide is cancer testis antigens (CTAs). CTAs are not typically expressed after embryonic development in healthy cells. In healthy adults, CTA expression is restricted to male germ cells, which do not express HLA and cannot present antigens to T lymphocytes. Therefore, CTAs are considered to express neoantigens when expressed in cancer cells. CTA expression is (i) tumor cell specific, (ii) more common in metastases than in primary tumors, and (iii) conserved among metastases from the same patient (Gajewski ed. Targeted Therapeutics in Melanoma) Springer New York. 2012).

The polypeptide may be a mutational neoantigen that is expressed by the cell

- 10 045699 by, for example, a cancer cell of an individual, but is changed relative to a similar protein in a normal or healthy cell. In some cases, the methods of the present invention include the step of identifying a polypeptide that is a mutational neoantigen or mutational neoantigen in a particular human subject, or identifying a neoepitope. For example, a neoantigen may be present in a sample obtained from a subject. Mutational neoantigens or neoepitopes can be used to target disease-associated cells, such as cancer cells, that express a neoantigen or a neoantigen containing a neoepitope. Mutations in a polypeptide expressed by a cell, such as a cell in a sample taken from a subject, can be detected, for example, by sequencing, but most do not induce an immune response against the neoantigen-expressing cells. Currently, the identification of mutational neoantigens that induce an immune response is based on the prediction of mutation-restricted HLA epitopes and further in vitro testing of the immunogenicity of the predicted epitopes in an individual's blood sample. This process is imprecise, time-consuming and expensive.

As discussed herein, the identification of mutational epitopes (neoepitopes) that bind to multiple HLA molecules reproducibly determines the immunogenicity of mutational neoantigens. Thus, in some cases in accordance with the present invention, the polypeptide is a mutational neoantigen, and the immunogenic fragment of the polypeptide contains a neoantigen-specific mutation (or consists of a neoepitope).

The polypeptide may be a viral protein that is expressed intracellularly. Examples include HPV16 E6, E7; HIV Tat, Rev, Gag, Pol, Env; HTLV-Tax, Rex, Gag, Env, human herpes virus proteins, Dengue virus proteins. The polypeptide may be a parasite protein that is expressed intracellularly, such as malaria proteins.

The polypeptide may be an active ingredient in a pharmaceutical composition, such as a vaccine or immunotherapeutic composition, optionally a potential active ingredient for a new pharmaceutical composition. As used herein, the term active ingredient refers to a polypeptide that is intended to induce an immune response, and may comprise a polypeptide product of a vaccine or immunotherapeutic composition that is produced in vivo after administration to a subject. For a DNA or RNA (ribonucleic acid, RNA, ribonucleic acid) immunotherapeutic composition, the polypeptide can be produced in vivo by cells of the subject to whom the composition is administered. For a cell-based composition, the polypeptide can be processed and/or presented by cells of the composition, for example, autologous dendritic cells or antigen presenting cells activated by the polypeptide or containing an expression construct encoding the polypeptide. The pharmaceutical composition may contain a polynucleoid or cell encoding one or more polypeptides of the active ingredient.

In other cases, the polypeptide may be a target polypeptide antigen of a pharmaceutical, vaccine or immunotherapeutic composition. A polypeptide is a target polypeptide antigen if the composition is intended or formulated to induce an immune response (eg, a cytotoxic T lymphocyte response) that is targeted or directed to the polypeptide. The target polypeptide antigen is typically a polypeptide that is expressed by a pathogenic organism, a virus, or a diseased cell, such as a cancer cell. The target polypeptide antigens may be TAA or CTA.

Currently, more than 200 clinical trials are investigating cancer vaccines with tumor antigens.

The polypeptide may be an allergen that enters the body of an individual, for example, through the skin, lungs or oral routes.

Non-limiting examples of suitable polypeptides include those listed in one or more tables. 2-7.

Genetic sequences can be obtained by sequencing biological materials. Sequencing can be performed by any suitable method that determines DNA and/or RNA and/or amino acid sequences. The invention uses both HLA genotypes and amino acid sequences. However, methods for identifying the HLA genotype from an individual's genetic sequences and methods for obtaining amino acid sequences derived from these DNA or RNA sequences are not the subject of the invention.

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table 2

List of named tumor antigens with corresponding accession numbers

5T4 Q13641.1 A1BG P04217.1 A3 3 Q99795.1 A4GALT Q9NPC4.1 AAST P01011.1 AAG Q9M6E9.1 ABI1 Q8TZP0.1 ABI2 Q9NYB9.1 ABL1 P00519.1 ABL-BCR Q8WUG5.1 ABLIM3 094929.1 ABLL R42684.1 AVTV1 Q969K4.1 ASASA Q13085.1 ACBD4 Q8NC06.1 ACO1 R21399.1 ACRBP Q8NEB7.1* ACTL6A 096019.1 ACTL8 Q9H568.1* ACTN4 043707.1 ACVR1 Q04771.1 ACVR1B P36896.1 ACVR2B Q13705.1 ACVRL1 R37023.1 ACS2B Q68CK6.1 ACSL5 Q9ULC5.1 ADAM-15 Q13444.1 ADAM17 R78536.1 ADAM2 Q99965.1* ADAM29 Q9UKF5.1* ADAM7 Q9H2U9.1 ADAP1 075689.1 ADFP Q99541.1 ADGRA3 Q8IWK6.1 ADGRF1 Q5T601.1 ADGRF2 Q8IZF7.1 ADGRL2 095490.1 ADHFE1 Q8IWW8.1 AEN Q8WTP8.1 AFF1 R51825.1 AFF4 Q9UHB7.1 AFP P02771.1 AGAP2 Q99490.1 AG01 Q9UL18.1 AG03 Q9H9G7.1 AGO4 Q9HCK5.1 AGR2 095994.1 ATFM2 Q9BRQ8.1 ATM2 014862.1 AKAR-13 Q12802.1 AKAP-3 075969.1* AKAR-4 Q5JQC9.1* AKIP1 Q9NQ31.1 ACT1 P31749.1 ACT 2 P31751.1 AKTZ Q9Y243.1 ALDH1A1 P00352.1 ALK Q9UM73.1 ALKBH1 Q13686.1 ALPK1 Q96QP1.1 AMIG02 Q86SJ2.1 ANG2 015123.1 ANKRD45 Q5TZF3.1* ANO1 Q5XXA6.1 ANP32A R39687.1 ANXA2 P07355.1 APC P25054.1 AREN R13798.1 AP0A2 P02652.1 APOD P05090.1 APOLl 014791.1 AE R10275.1 ARAF Р10398.1 ARF4L P49703.1 ARHGEF5 Q12774.1 ARID3A Q99856.1 ARID4A R29374.1 ARL6IP5 075915.1 ARMC3 B4DXS3.1* ARMC8 Q8IUR7.1 ARTC1 R52961.1 ARX Q96QS3.1* ATAD2 Q6PL18.1 ATTS R31939.1 AURKC Q9UQB9.1 AXIN1 015169.1 AXL P30530.1 VAAT Q14032.1 BAFF Q9Y275.1 BAGE-1 Q13072.1* BAGE-2 Q86Y30.1* BAGE-3 Q86Y29.1* BAGE-4 Q86Y28.1 BAGE-5 Q86Y27.1* BAI1 014514.1 BAL R19835.1 BALF2 P03227.1 BALF4 P03188.1 BALF5 P03198.1 BARF1 P03228.1 BBRF1 P03213.1 In CAN Q96GW7.1 BCAP31 P51572.1 BCL-2 R10415.1 BCL2L1 Q07817.1 BCL6 P41182.1 BCL9 000512.1 ALL R11274.1 BCRF1 P03180.1 BDLF3 P03224.1 BGLF4 P13288.1 BHLF1 P03181.1 BHRF1 P03182.1 BILF1 P03208.1 BILF2 P03218.1 BIN1 000499.1 BING-4 015213.1 BIRC7 Q96CA5.1 BLLF1 P03200.1 BLLF2 P03199.1 BMI1 R35226.1 BMLF1 Q04360.1 BMPR1B 000238.1 BMRF1 P03191.1 BNLF2a Р0С739.1 BNLF2b Q8AZJ3.1 BNRF1 P03179.1 BRAF1 R15056.1 BRD4 060885.1 BRDT Q58F21.1* BRI3BP Q8WY22.1 BRINP1 060477.1 BRLF1 P03209.1 BTBD2 Q9BX70.1 BUB1B 06 05 66.1 BVRF2 P03234.1 BXLF1 P03177.1 BZLF1 P03206.1 C15orf60 Q7Z4M0.1* SA 12-5 Q8WXI7.1 SA 19-9 Q969X2.1 CA195 Q5TG92.1 GA 9 Q16790.1 CABYR 075952.1* CADM4 Q8NFZ8.1 CAGE1 Q8CT20.1* CALCA P01258.1 CALR3 Q96L12.1 CAN R35658.1 CASC3 015234.1 CASC5 Q8NG31.1* CASP5 R51878.1 CASP8 Q14790.1 CBFA2T2 043439.1 CBFA2T3 075081.1 CBL R22681.1 CBLB Q13191.1 003 Q9BUP3.1 CCDC110 Q8TBZ0.1* CCDC33 Q8N5R6.1* CCDC36 Q8IYA8.1* CCDC6 Q16204.1 CCDC62 Q6P9F0.1* CCDC68 Q9H2F9.1 CCDC83 Q8IWF9.1* CCL13 Q99616.1 CCL2 P13500.1 CCL7 Р80098.1 CCNA1 Р78396.1* CCNA2 P20248.1 CCNB1 P14635.1 CCND1 R24385.1 CCNE2 096020.1 CCNI Q14094.1 CCNL1 Q9UK58.1 CCR2 R41597.1 CD105 R17813.1 CD123 P26951.1 CD13 P15144.1 CD133 043490.1 CD137 Q07011.1 CD138 P18827.1 CD157 Q10588.1 CD16A P08637.1 CD178 R48023.1 CD19 P15391.1 CD194 P51679.1 CD2 P06729.1 CD20 R11836.1 CD21 P20023.1 CD22 P20273.1 CD229 Q9HBG7.1 CD23 P06734.1 CD27 P26842.1 CD28 P10747.1 CD30 R28908.1 CD317 Q10589.1 CD33 P20138.1 CD350 Q9ULW2.1 CD36 R16671.1 CD37 R11049.1 CD4 P01730.1 CD40 P25942.1 CD40L R29965.1 CD45 P08575.1 CD47 Q08722.1 CD51 P06756.1 CD52 R31358.1 CD55 P08174.1 CD61 P05106.1 CD70 P32970.1 CD74 P08922.1 CD75 R15907.1 CD7 9B P40259.1 CD80 P33681.1 CD86 R42081.1 CD8a P01732.1 CD8b P10966.1 CD95 P25445.1 CD98 P08195.1 CDC123 075794.1 CDC2 P06493.1 CDC27 P30260.1 CDC73 Q6P1J9.1 CDCA1 Q9BZD4.1* CDCP1 Q9H5V8.1 CDH3 P22223.1 CDK2AP1014519.1 CDK4 R11802.1 CDK7 P50613.1 CDKN1A P38936.1 CDKN2A R42771.1 CEA P06731.1 CEACAM1 Q86UE4.1 CENPK Q9BS16.1 CEP162 Q5TB80.1 CEP290 015078.1* CEP55 Q53EZ4.1* CFLl P23528.1 CH3L2 Q15782.1 SNACK1 014757.1 CK2 P19784.1 CLCA2 Q9UQC9.1 CLOCK 015516.1 CLPP Q16740.1 CMC 4 P56277.1 CML66 Q96RS6.1 SO-029 R19075.1 C0TL1 Q14019.1 00X2 P35354.1 COX6B2 Q6YFQ2.1* CPSF1 Q10570.1 CPXCR1 Q8N123.1* CREBL2 060519.1 CREG1 075629.1 Cripto R13385.1 CRISP2 Р16562.1* *CRK P46108.1 CRKL P46109.1 CRLF2 Q9HC73.1 CSAGE Q6PB30.1 CT45 Q5HYN5.1* CT45A2 Q5DJT8.1* ST45AZ Q8NHU0.1* ST45A4 Q8N7B7.1* CT45A5 Q6NSH3.1* CT45A6 P0DMU7.1* ST46 Q86X24.1* ST47 Q5JQC4.1* CT47B1 P0C2P7.1* CTAGE2 Q96RT6.1* CTAGE5 015320.1* CTCFL Q8NI51.1* CTDSP2 014595.1 CTGF P29279.1 CTLA4 R16410.1 CTNNA2 Р26232.1* CTNNB1 P35222.1 CTNNDl 060716.1 CTSH P09668.1 CTSP1 A0RZH4.1* CTTN Q14247.1 CXCR4 P61073.1 CXorf48 Q8WUE5.1* CXorf61 Q5H943.1* Cyclin- -E P24864.1 CYP1B1 Q16678.1 SurV R23284.1 CYR61 000622.1 CS1 P28290.1 CSAG1 Q6PB30.1*

- 12 045699

CSDE1 075534.1 CSFl P09603.1 CSF1R P07333.1 CSF3R Q99062. 1 CSK P41240.1 CSK23 Q8NEV1.1 DAPK3 043293.1 DAZ1 Q9NQZ3.1 DBPC Q9Y2T7.1 DCAF12 Q5T6F0.1* DCT P40126.1 DCUN1D1 Q96GG9.1 DCUN1D3 Q8IWE4.1 DDR1 Q08345.1 DDX3X 000571.1 DDX6 P26196.1 DEDD 075618.1 DEK P35659.1 DENR 043583.1 DEPDC1 Q5TB30.1 DFNA5 060443.1 DGAT2 Q96PD7.1 DHFR P00374.1 DKK1 094907.1 DKK3 Q9UBP4.1 DKKL1 Q9UK85.1* DLEU1 043261.1 DMBT1 Q9UGM3.1 DMRT1 Q9Y5R6.1* DNAJB8 Q8NHS0.1* DNAJC8 075937.1 DNMT3A Q9Y6K1.1 DPPA2 Q7Z7J5.1* DR4 000220.1 DR5 014763.1 DRGl Q9Y295.1* DSCR8 Q96T75.1 E2F3 000716.1 E2F6 075461.1 E2F8 A0AVK6.1 EBNA1 P03211.1 EBNA2 P12978.1 EBNA3 P12977.1 EBNA4 P03203.1 EBNA6 P03204.1 EBNA-LP Q8AZK7.1 E-cadhe rin P12830.1 ECT2 Q9H8V3.1 ECTL2 Q008S8.1 EDAG Q9BXL5.1* EEF2 P13639.1 EFNA1 P20827.1 EFS 043281.1 EFTUD2 Q15029.1 EGFL7 Q9UHF1.1 EGFR p00533.1 EI24 014681.1 EIF4EBP1 Q13541.1 ELF3 P78545.1 ELF4 Q99607.1 ELOVL4 Q9GZR5.1* EMP1 P54849.1 ENAH Q8N8S7.1 Endosialin Q9HCU0.1 ENO1 P06733.1 ENO2 P09104.1 ENO3 P13929.1 ENTPD5 075356.1 EpCAM P16422.1 EPHA2 P29317.1 EPHA3 P29320.1 EPHB2 P29323.1 EPHB4 P54760.1 EPHB6 015197.1 EPSS Q12929.1 ERBB3 P21860.1 ERBB4 Q15303.1 EREG 014944.1 ERG P11308.1 ERVK-18 042043.1 ERVK-19 071037.1 ESR1 P03372.1 ETAA1 Q9NY74.1 ETS1 P14921.1 ETS2 P15036.1 ETV1 P50549.1 ETV5 P41161.1 ETV6 P41212.1 EVI5 060447.1 EWSRl Q01844.1 EYA2 000167.1 EZH2 Q15910.1 FABP7 015540.1 FAM133A Q8N9E0.1* FAM13A 094988.1 FAM46D Q8NEK8.1* FAM58BP P0C7Q3.1 FANCG 015287.1 FATE1 Q969F0.1* FBXO39 Q8N4B4.1* FBXW11 Q9UKB1.1 FCHSD2 094868.1 FER P16591.1 FES P07332.1 FEV Q99581.1 FGF10 015520.1 FGF23 Q9GZV9.1 FGF3 P11487.1 FGF4 P08620.1 FGF5 P12034.1 FGFR1 P11362.1 FGFR2 P21802. 1 FGFR3 P22607.1 FGFR4 P22455.1 FGR P09769.1 F.L.U. Q01543.1 FLT3 P36888.1 FMNL1 095 4 6 6.1 FMOD Q06828.1 FMR1NB Q8N0W7.1* FNl P02751.1 Fnl4 Q9NP84.1 FNIP2 Q9P278.1 FOLRI P15328.1 F.O.S. P01100.1 FosB P53539.1 F0SL1 P15407.1 FOXM1 Q08050.1 FOXOL Q12778.1 FOXO3 043524.1 FRAT1 Q92837.1 FRMD3 A2A2Y4.1 FSIP1 Q8NA03.1 FSTP2 Q5CZC0. 1 FSTL3 095633.1 FTHL17 Q9BXU8.1* FUNDC2 Q9BWH2.1 FUS P35637.1 FUTl P19526.1 FUT3 P21217.1 FYN P06241.1 GAB 2 Q9UQC2.1 GADD45G 095257.1 GAGE-1 Q13065.1 GAGE12B/C/D/E A1L429.1 GAGE12F P0CL80.1 GAGE12G P0CL81.1 GAGE12H A6NDE8.1 GAGE12I P0CL82.1 GAGE12J A6NER3.1 GAGE-2 Q6NT46.1 GAGE-3 Q13067.1 GAGE-4 Q13068.1 GAGE-5 Q13069.1 GAGE-6 Q13070.1 GAGE-7 076087.1 GAGE-8 Q9UEU5.1 GALGT2 Q00973.1 GAS 7 060861.1 GASZ Q8WWH4.1 GATA-3 P23771.1 GBU4-5 Q587J7.1 GCDFP-15 P12273.1 GFAP P14136.1 GFT1 Q99684.1 Ghrelir Q9UBU3.1 GHSR Q92847.1 GTPC1 014908.1 GITR Q9Y5U5.1 GKAP1 Q5VSY0. 1 GLT1 P08151.1 Glypican-3 P51654.1 GML Q99445.1 GNA11 P29992.1 GNAQ P50148.1 GNB2L1 P63244.1 GOLGA5 Q8TBA6.1 gplOO P40967.1 gp7 5 P17643.1 Gp96 P14625.1 GPAT2 Q6NUI2.1* GPATCH2 Q9NW75.1* GPC-3 P51654.1 GPNMB Q14956.1 GPR143 P51810.1 GPR89A B7ZAQ6.1 GRB2 P62993.1 GRP7 8 P11021. 1 GUCY1A3 Q02108.1 H3F3A P84243.1 HAGE Q9NXZ2.1* hANP P01160.1 HBEGF Q99075.1 hCG-beta P01233.1 HDAC1 Q13547.1 HDAC2 Q92769.1 HDAC3 015379.1 HDAC4 P56524.1 HDAC5 Q9UQL6.1 HDAC6 Q9UBN7.1 HDAC7 Q8WUT4.1 HDAC8 Q9BY41.1 HDAC9 Q9UKV0.1 HEATR1 Q9H583.1 Hepsin P05981.1 Her2/neu P04626.1 HERC2 095714.1 HERV-K1 04 P61576.1 HEXB P07686.1 HEXIM1 094992.1 HGRG8 Q9Y5A9.1 HTPK2 Q9H2X6.1 HJURP Q8NCD3.1 HMGB1 P09429.1 HM0X1 P09601.1 HNRPL P14866.1 HOM-TES -85 Q9P127.1* HORMADl Q86X24.1* HORMAD2 Q8N7B1.1* HPSE Q9Y251.1 HPV16 E 6 P03126.1 HPV16 :7 P03129.1 HPV18 E6 P06463.1 HPV18 E7 P06788.1 HRAS P01112.1 HSD17B13 Q7Z5P4.1 HSP105 Q92598.1 HSP60 P10809.1 HSPA1A P08107.1 HSPB9 Q9BQS6.1* HST-2 P10767.1 HT001 Q2TB18.1 hTERT 014746.1 HUS1 060921.1 ICAM-1 P05362.1 IDH1 075874.1 I DOI P14902.1 IER3 P46 695.1 IGF1R P08069.1 IGFS11 Q5DX21.1*

- 13 045699

IL13RA2 Q14627.1* IMP-3 Q9NV31.1* ING3 Q9NXR8.1 INPPL1 015357.1 INTS6 Q9UL03.1 IRF4 Q15306.1 IRS4 014654.1 ITGA5 P08648.1 ITGB8 P26012.1 ITPA Q9BY32.1 ITPR2 Q14571.1 JAK2 060674.1 JAK3 P52333.1 JARID1B Q9UGL1.1* JAZF1 Q86VZ6.1 JNK1 P45983.1 JNK2 P45984.1 JNK3 P53779.1 JTB 076095.1 JUN P05412.1 JUP P14923.1 K19 P08727. 1 KAAG1 Q9UBP8.1 Kallikrein 14 Q9P0G3.1 Kallikrein 4 Q9Y5K2.1 KAT6A Q92794.1 To DM1A 060341.1 KDM5A P29375.1 KIAA0100 Q14667.1* KTAA033 6 Q8IWJ2.1 KIAA119 9 Q8WUJ3.1 KIAA1641 A6QL64. 1 KIF11 P52732.1 KIF1B 060333.1 KIF20A 095235.1 KIT P10721.1 KLF4 043474.1 KLHL41 060 6 62.1 KLK10 043240.1 KMT2D 014686.1 KOCl 000425.1 K-ras P01116.1 KRIT1 000522.1 KW-12 P62913.1 KW-2 Q96RS0.1 KW-5 (SEBD4) Q9H0Z9.1 KW-7 075475.1 LI CAM P32004.1 L53 Q96EL3.1 L6 Q9BTT4.1 LAG3 P18627.1 Lage-l 075638.1 LATS1 095835.1 LATS2 Q9NRM7.1 LCMT2 060294.1 LCP1 P13796.1 LDHC P07864.1* LDLR P01130.1 LEMDl Q68G75.1* Lengsi qQ5TDP6.1 LETMD1 Q6P1Q0.1 LGALS3BP Q08380.1 LGALS8 000214.1 LIN7A 014910.1 LIPI Q6XZB0.1* LIV-1 Q13433.1 LLGL1 Q15334.1 LM01 P25800.1 LMO2 P25791.1 LMPl P03230.1 LMP2 P13285.1 LOC647107 Q8TAI5 . 1* LOXL2 Q9Y4K0.1 LRP1 Q07954.1 LRRN2 075325.1 LTF P02788.1 LTK P29376.1 LZTS1 Q9Y250.1 LY6K Q17RY6.1* LYN P07948.1 LYPD6B Q8NI32.1* MAEA Q7L5Y9.1 MAEL Q96JY0.1* MAF 075444.1 NAFF Q9ULX9.1 MAFG 015525.1 MAFK 060675.1 MAGE-Al P43355.1* MAGE-A10 P43363.1* MAGE-All P43364.1* MAGE-A12 P43365.1* MAGE-A2 P43356.1* MAGE-A2B Q6P448.1* MAGE-A3 P43357.1* MAGE-A4 P43358.1* MAGE-A5 P43359.1* MAGE-A6 P43360.1* MAGE-A8 P43361.1* MAGE-A9 P43362.1* MAGE-Bl P43366.1* MAGE-B2 015479.1* MAGE-B3 015480.1* MAGE-B4 015481.1* MAGE-B5 Q9BZ81.1* MAGE-B6 Q8N7X4.1* MAGE-CI 060732.1* MAGE-C2 Q9UBF1.1* MAGE-C3 Q8TD91.1* mammaglobin-A Q13296.1 MANF P55145.1 MAP2K2 P36507.1 MAP2K7 014733.1 MAP3K7 043318.1 MAP4K5 Q9Y4K4.1 MARTI Q16655.1 MART-2 Q5VTY9.1 MASI P04201.1 MOIR Q01726.1 MCAK Q99661.1* MCF2 P10911.1 MCF2L 015068.1 MCL1 Q07820. 1 MCTS1 Q9ULC4.1 MCSP Q6UVK1.1 MDK P21741.1 MDM2 Q00987. 1 MDM4 015151.1 MEI P48163.1 ME491 P08962.1 MESOM Q03112.1 MELK Q14680.1 MEN1 000255.1 MERTK Q12866.1 MET P08581.1 MFGE8 Q08431.1 MFHAS1 Q9Y4C4.1 MFI2 P08582.1 MGAT5 Q09328.1 Midkine P21741.1 MIF P14174.1 MKI67 P46013.1 MLH1 P40692.1 MLL Q03164.1 MLLT1 Q03111.1 MLLT10 P55197.1 MLLT11 Q13015.1 MLLT3 P42568.1 MLLT4 P55196.1 MLLT6 P55198.1 MMP14 P50281.1 MMP2 P08253.1 MMP7 P09237.1 MMP9 P14780.1 M0B3B Q86TA1.1 MORCl Q86VD1.1* MPHOSPHl Q96Q89. 1* MPL P40238.1 MRAS 014807.1 MRP1 P33527.1 MRP3 015438.1 MRPL28 Q13084.1 MRPL30 Q8TCC3.1 MRPS11 P82912.1 MSLN Q13421.1 MTA1 Q13330.1 MTA2 094776.1 MTA3 Q9BTC8.1 MTCP1 P56278.1 MTSS1 043312.1 MUC-1 P15941.1 MUC-2 Q02817.1 MUC-3 Q02505.1 MUC-4 Q99102.1 MUC-5AC P98088.1 MUC-6 Q6W4X9.1 MUM1 Q2TAK8.1 MUM2 Q9Y5R8.1 MYB P10242.1 MYC P01106.1 MYCL P12524.1 MYCLP1 P12525.1 MYCN P04198.1 MYD88 Q99836.1 MYEOV Q96EZ4.1 MY01B 043795.1 NA88-A P0C5K6.1* NAE1 Q13564.1 Napsin -A 096009. 1 NAT 6 Q93015.1 NBAS A2RRP1.1 NBPF12 Q5TAG4.1 NC0A4 Q13772.1 NDC80 014777.1 NDUFC2 095298.1 Nectin- 4 Q96NY8.1 NEK2 P51955.1 NEMF 060524.1 NENF Q9UMX5.1 NEURL1 076050.1 NFIB 000712.1 NFKB2 Q00653.1 NF-Xl Q12986.1 N.F.Y.C. Q13952.1 NGAL P80188.1 NGEP Q6IWH7.1 NKG2D-L1 Q9BZM6.1 NKG2D-L2 Q9BZM5.1 NKG2D- L3 Q9BZM4. 1 NKG2D- L4 Q8TD07.1 NKX3.1 Q99801.1 NLGN4X Q8N0W4.1 NLRP4 Q96MN2.1 NNMT P40261.1 N0L4 094818.1* N0TCH2 Q04721.1 N0TCH3 Q9UM47.1 N0TCH4 Q9 94 66.1 NOV P48745.1 NPM1 P06748.1 NR6A1 Q15406.1 N-RAS POllll. 1 NRCAM Q92823.1 NRPl 014786.1 NSE1 Q96KN4.1 NSE2 Q96KN1.1 NTRK1 P04629. 1 NUAK1 060285.1 N.U.G.G.C. Q68CJ6.1 NXF2 Q9GZY0.1* NXF2B Q5JRM6.1* NY-BR-1 Q9BXX3.1 NYD-TSPG Q9BWV7. 1 NY-ESO -1 P78358.1* NY-MEL- 1 P57729.1 0CA2 Q04671.1 ODFl Q14990.1

- 14 045699

ODF2 Q5BJF6.1* ODF3 Q96PU9.1* ODF4 Q2M2E3.1* OGGI 015527.1 OGT 015294.1 ΟΙ P5 043482.1* OS9 Q13438.1 OTOA Q05BM7.1* 0X4 0 P43489.1 OX40L P23510.1 P53 P04637.1 P56-LCK P06239.1 PA2G4 Q9UQ80.1 PAGEl 075459.1* PAGE2 Q7Z2X2.1* PAGE2B Q5JRK9.1* PAGE3 Q5JUK9.1* PAGE 4 060829.1* PAGE5 Q96GU1.1* PAK2 Q13177.1 PAN 01 T0J062.1 PAP Q06141.1 PAPOLG Q9BWT3.1 PARK 2 060260.1 PARK7 Q99497.1 PARP12 Q9H0J9.1 PASDl Q8IV76.1* PAX3 P23760.1 PAX 5 Q02548.1 PBF P00751.1 PBK Q96KB5.1* PBX1 P40424.1 PCDC1 015116.1 PCM1 Q15154.1 PCNXL2 A6NKB5.1 PDGFB P01127.1 PDGFRA P16234.1 PEPP2 Q9HAU0.1* PGF P49763.1 PGK1 P00558.1 PHLDA3 Q9Y5J5.1 PHLPP1 060346.1 PTAS1 075925.1 PTAS2 075928.1 PTK3CA P42336.1 PTK3CD 000329.1 PTK3R2 000459.1 PTM1 P11309.1 PIM2 Q9P1W9.1 PTM3 Q86V86.1 PTR 000625.1 PIWILL Q96J94.1* PIWIL2 Q8TC59.1* PTWTL3 Q7Z3Z3.1 PTWTL4 Q7Z3Z4.1 PKN3 Q6P5Z2.1 PLA2G16 P53816.1 PLACl Q9HBJ0.1* PLAG1 Q6DJT9.1 PLEKHG5 094827.1 PLK3 Q9H4B4.1 PLS3 P13797.1 PLVAP Q9BX97.1 PLXNB1 043157.1 PLXNB2 015031.1 P.M.L. P29590.1 PML-RARA Q96QH2.1 POTEA Q6S8J7.1* POTEB Q6S5H4.1* POTEC B2RU33.1* POTED Q86YR6.1* POTEE Q6S8J3.1* POTEG Q6S5H5.1* POTEH Q6S545.1* PP2A P63151.1 PPAPDC1B Q8NEB5.1 PPFTA1 Q13136.1 PPTG Q13427.1 PPP2R1B P30154.1 FRAME P78395.1* PRDX5 P30044.1 PRKAA1 013131.1 PRKCI P41743.1 PRM1 P04553.1* PRM2 P04554.1* PRMT3 060678.1 PRMT6 Q96LA8.1 PDL1 Q9NZQ7.1 PROM1 043490.1 PRSS54 Q6PEW0.1* PRSS55 Q6UWB4.1* PRTN3 P24158.1 PRUNE Q86TP1.1 PRUNE2 Q8WUY3.1 P.S.A. P07288.1 PSCA D3DWT6.1 PSMA Q04609.1 PSMD10 075832.1 PSGR Q9H255.1 PSP-94 Q1L6U9.1 PTEN P60484.1 PTH-rP P12272.1 PTK6 Q13882.1 PTPN20A Q4 JDL3.1* PTPRK Q15262.1 PTPRZ P23471.1 PTTG-1 095997.1 PTTG2 Q9NZH5.1 PTTG3 Q9NZH4.1 PXDNL A1KZ92.1 RAB11FIP3 075154.1 RAB8A P61006.1 RADI 060671.1 RAD17 075943.1 RAD51C 043502.1 RAFI P04049.1 RAGE-1 Q9UQ07.1 RAP1A P62834.1 RARA P10276.1 RASSF10 A6NK89.1 RBI P06400.1 RBL2 Q08999.1 RBM46 Q8TBY0.1* RBP4 P02753.1 RCAS1 000559.1 RCVRN P35243.1 RECQL4 094761.1 RET P07949.1 RGS22 Q8NE09.1* RGS5 015539.1 RHAMM 075330.1 RhoC P08134.1 RHOXF2 Q9BQY4.1 RL31 P62888.1 RNASET2 000584.1 RNF43 Q68DV7.1 RNF8 076064.1 RON Q04912.1 ROPN1A Q9HAT0.1* R0R1 Q01973.1 RPA1 095602.1 RPL10A P62906.1 RPL7A P62424.1 RPS2 P15880.1 RPS6KA5 075582.1 RPSA P08865.1 RQCD1 Q92600.1* RRAS2 P62070.1 RSL1D1 076021.1 RTKN Q9BST9.1 RUNX1 Q01196.1 RUNX2 Q13950.1 RYK P34925.1 SAGEl Q9NXZ1.1* SART2 Q9UL01.1 SART3 Q15020.1 SASHl 094885.1 sCLU P10909.1 SCRNl Q12765.1 SDCBP 000560.1 SDF-1 P48061.1 SDHD 014521.1 SEC31A 094979.1 SEC63 Q9UGP8.1 Semapho rin 4D SEMGl P04279.1* SFN P31947.1 SH2B2 014492.1 Q92854.1 SH2D1B 014796.1 SH3BP1 Q9Y3L3.1 SHB Q15464.1 SHC3 Q92529.1 STRT2 Q8TXJ6.1 SIVA! 015304.1 SKI P12755.1 SLBP A9UHW6.1 SLC22A1 0Q63ZE4.1 SLC25A47 Q6Q0C1.1 SLC35A4 Q96G79.1 SLC45A3 Q96JT2.1 SLC4A1AP Q9BWU0.1 SLCO6A1 Q86UG4.1* SLITRK6 Q9H5Y7.1 Sm23 P27701.1 SMAD5 Q99717.1 SMAD6 043541.1 SMO Q99835.1 Smt3B P61956.1 SNRPD1 P62314.1 SOSl Q07889.1 SOX-2 P48431.1 SOX-6 P35712.1 SOX-11 P35716.1 SPA17 Q15506.1* SPACA3 Q8IXA5.1* SPAG1 Q07617.1* SPAG17 Q6Q759.1* SPAG4 Q9NPE6.1* SPAG6 075602.1* SPAG8 Q99932.1* SPAG9 060271.1* SPANXA1 Q9NS26.1* SPANXB Q9NS25.1* SPANXC Q9NY87.1* SPANXD Q9BXN6.1* SPANXE Q8TAD1.1* SPANXNl Q5VSR9.1* SPANXN2 Q5MJ10.1* SPANXN3 Q5MJ09.1* SPANXN4 Q5MJ08.1* SPANXN5 Q5MJ07.1* SPATA19 Q7Z5L4.1* SPEF2 Q9C093.1* SPI1 P17947.1 SPINLW1 095925.1* SP011 Q9Y5K1.1* SRC P12931.1 SSPN Q14714.1 SSX-1 Q16384.1* SSX-2 QI 6385.1* SSX-3 Q99909.1* SSX-4 060224.1* SSX-5 060225.1* SSX-6 Q7RTT6.1* SSX-7 Q7RTT5.1* SSX-9 Q7RTT3.1* ST18 060284.1 STAT1 P42224.1 STEAP1 Q9UHE8.1 STK11 Q15831.1 STK25 000506.1 STK3 Q13188.1 STN Q9H668.1 SUPT7L 094864.1 Survivin 015392.1 SUV39H1043463.1 SYCE1 Q8N0S2.1 SYCP1 Q15431.1 SYCP3 Q8IZU3.1 SYT Q15532.1

- 15 045699

TA-4 Q96RI8.1 TASS! 075410.1 TAF1B Q53T94.1 TAF4 000268.1 TAF7L Q5H9L4.1* TAG-1 Q02246.1* TALI P17542.1 TAL2 Q16559.1 TARVR 015533.1 TATR P00995.1 TAKH1VRZ 014907.1 TBC1D3 Q8IZP1.1 TVR-1 R17980.1 TCL1A R56279.1 TCL1B 095988.1 TDHP Q9BT92.1 TDRD1 Q9BXT4.1* TDRD4 Q9BXT8.1* TDRD6 060522.1* TEKT5 Q96M29.1* TECH101 Q9BY14.1* TECH14 Q8IWB6.1* TECH15 Q9BXT5.1* TEX38 Q6PEX7.1* TF P02787.1 TFDP3 Q5H9I0.1* TFE3 P19532.1 TGFBR1 P36897.1 TGFBR2 R37173.1 THEG Q9P2T0.1* TTE2 Q02763.1 TIPRL 075663.1 TLR2 060603.1 TMEFF1 Q8IYR6.1* TMEFF2 Q9UIK5.1* TMEM108 Q6UXF1.1* TMEM127 075204.1 TMPRSS12 Q86WS5.1* TNC P24821.1 TNFRSF17 Q02223.1 TNFSF15 095150.1 TNK2 Q07912.1 TOMM34 Q15785.1 T0P2A P11388.1 Т0Р2В Q02880.1 T0R3A Q9H497.1 TR73 015350.1 TPA1 8N543.1 TPGS2 Q68CL5.1 THREE R60174.1 TPL2 P41279.1 TPM4 P67936.1 TRO R40225.1 TRRR2 Р59282.1* TPR P12270.1 TPTE P56180.1* TRAF5 000463.1 TRAG-3 Q9Y5P2.1* TRGC2 P03986.1 TRTM24 015164.1 TRTM37 094972.1 TRIM68 Q6AZZ1.1 TRPM8 Q7Z2W7.1 TSGA10 Q9BZW7.1* TSP50 Q9UI38.1* TSPAN6 043657.1 TSPY1 Q01534.1* TSPY2 A6NKD2.1* TSPY3 Q6B019.1* TSPYL1 Q9H0U9.1 TSSK6 Q9BXA6.1* TTC23 Q5W5X9.1 ttk Р33981.1* TULP2 000295.1* TUSC2 075896.1 TWEAK 043508.1 TXNTP Q9H3M7.1 TYMS P04818.1 TYR P14679.1 U2 snRNP B P08579.1 U2AF1 Q01081.1 UBD 015205.1 UBE 2 A P49459.1 UBE2C 000762.1 UBE2V1 Q13404.1 UBE4B 095155.1 UBR5 095071.1 UBXD5 Q5T124.1 UFL1 094874.1 URI1 094763.1 URLC10 Q17RY6.1 UROCI Q9 6N7 6.1 USP2 075604.1 USP4 Q13107.1 VAV1 P15498.1 VCX3A Q9NNX9.1 VEGFR1 R17948.1 VEGFR2 R35968.1 VHL P40337.1 VIM P08670.1 VWA5A 000534.1 WHSC2 Q9H3P2.1 WTSP1 095388.1 WNK2 Q9Y3S1.1 WNT10B 000744.1 WNT3 R56703.1 WNT-5a P41221.1 WT1 P19544.1 WWP1 Q9H0M0.1 XAGE-1 Q9HD64.1* XAGE-2 Q96GT9.1* XAGE-3 Q8WTP9.1* XAGE-4 Q8WWM0.1 XAGE-5 Q8WWM1.1* XBP1 P17861.1 XP01 014980.1 XRCC3 043542.1 YB-1 R67809.1 YEATS4 095619.1 YES1 P07947.1 YKL-40 R36222.1 ZBTB7A 095365.1 ZBTB7C A1YPR0.1 ZEB1 P37275.1 ZFYVE19 Q96K21.1 ZNF165 Р49910.1* ZNF185 015231.1 ZNF217 075362.1 ZNF320 A2RRD8.1 ZNF395 Q9H8N7.1 ZNF645 Q8N7E2.1* ZUBR1 Q5T4S7.1 ZW10 043264.1 ZWINT 095229.1

Table 3

CTA5=bold and *

List of accession numbers for viral antigens from the IEDB

Q76R62.1 P03182.1 P09258.1 P09310.1 P03227.1 P89466.1 P04601.1 P13285.1 P09991.1 P03468.1 A2T3Q0.1 P0C6X7.1 P89448.1 P12978.1 P09257.1 P50641.1 P14075.1 20178567.1 Q01023.1 P03188.1 P04585.1 P0C767.1 P12977.1 P89467.1 Q9W850.1 Q00683.1 P04591.1 P03211.1 9628706.1 P03460.1 PO 8 6 66.1 P03485.1 Q04360.1 Q913Y7.1 P89449.1 Q81871.1 P03452.1 P17763.1 P89430.1 P03410.1 P04012.1 P27958.1 Q6WB99.1 P25212.1 Q9PZT1.1 P68593.1 P03203.1 P29996.1 9629374.1 P59633.1 042053.1 P0C6L3.1 P59635.1 Q9YZN9.1 Q6WB95.1 P10233.1 P89475.1 Q6WB98.1 Q6SW67.1 Q7TFA0.1 P0CK17.1 P59594.1 1980491.1 P14079.1 P15423.1 1891762.1 P09259.1 P09269.1 Q77Q38.1 Q786F2.1 Q6SW99.1 P24771.1 F5HB98.1 9629370.1 P68336.1 P03300.1 1980486.1 Q69027.1 P28284.1 P13290.1 9626585.1 P06923.1 P14076.1 P03346.1 042062.1 P07566.1 P03204.1 Q69091.1 P09255.1 P03206.1 036634.1 P10205.1 F5HCM1.1 P0CK16.1 Q6WB97.1 Q85601.1 P89468.1 Q69467.1 P03218.1 Q786F3.1 P59637.1 1891763.1 Q6WB94.1 P03231.1 Q9IK92.1 Q6WBA1.1 P03466.1 P14335.1 P26670.1 Q9PZT0.1 1985356.1 Q2HR63.1 P59634.1 Q6SW59.1 P03277.1 P59595.1 Q69028.1 P03383.1 P03261.1 P03200.1 P04578.1 P06484.1 F5HC97.1 S5TC82.1 P18095.1 Q96895.1 P18094.1 9629372.1 P50791.1 P03230.1 P13845.1 9629712.1 P03209.1 P03129.1 Q76R61.1 P03228.1 P0C206.1 Q9WMB5.1 P03226.1 Q9QR69.1 036633.1 042049.1 P03496.1 P03428.1 P03431.1 P0C0U1.1 P03433.1 P03508.1 1980456.1 P0C739.1 P69726.1 P69723.1 1980490.1 532129755.1 P03120.1 P04020.1 P06922.1 P03114.1 P03314.1 P06790.1 P06788.1 P06927.1 P03101.1 P03107.1 P06794.1 530787712.1 P04013.1 Q80872.1 P04014.1 P03126.1 P36811.1 P06463.1 P26554.1 P04016.1 P14078.1 P03191.1 1980471.1 P06821.1 P0C797.1 F5HF49.1 P0C045.1 P04296.1 P04485.1 P10230.1 P10221.1 P06487.1 P10215.1 P04293.1 P10211.1 P10209.1 P10225.1 P10224.1 P10238.1 P10185.1 P08392.1 P10231.1 P06492.1 P04290.1 P08393.1 P08543.1 P10210.1 P08617.1 F5HB53.1 P04019.1 P04015.1 P89442.1 P89452.1 P89462.1 P59632.1 036635.1 P07210.1 Q83884.1 Q8JUX5.1 P03089.1 Q66479.1 P03185.1 P0CAP6.1 P04618.1 56160929.1 1980519.1 P08669.1 P14348.1 P03212.1 P03179.1 45617other.1 1511872.1 302317869.1 P69899.1 P09247.1 Q05127.1 P18272.1 Q9YMG2.1 Q05128.1 302371215.1 302371218.1 Q5XX08.1 302371214.1 P14336.1 138948other.1 P08292.1 1803956.1 P35253.1 1891726.1 P09308.1 P03189.1 667489389.1 P09272.1 34365530.1 Q05320.1 P59596.1 P32886.1 55097.1 P03316.1 P03276.1 Q81870.1 Q81862.1 64320.1 1933190.1

- 16045699

List of accession numbers for bacterial antigens from the IEDB

Table 4

B8ZUD1.1 P09621.1 P9WPE5.1 Q2GI62.1 Р0А5В8.1 050443.1 Q5NEZ3.1 P9WQF5.1 P9WK95.1 005311.1 P9WQD7.1 P9WKG3.1 P9WHE5.1 P0CD83.1 P9WHB9.1 P9WH91.1 P9WHE3.1 P9WNK7.1 A0A0F3MKF3.1 A1JTP3.1 B2RKS6.1 P0A1D3.1 P0A6F5.1 P0C0Z7.1 Р0С923.1 R61439.1 Q9Z708.1 Р0А521.1 P9WPE7.1 Q79FJ2.1 B8ZR84.1 I6Y3P5.1 Q2FYP2.1 P9WG41.1 P96890. 1 006625.1 16X654.1 Q8YIE1.1 P9WQ81.1 T6XWA1.1 R11311.1 053900.1 P9WTR7.1 P9WQB1.1 B8ZUC6.1 006802.1 P9WMK1.1 P9WG37.1 Q2FWC4.1 Q2GGE3.1 033347.1 P9WJ09.1 P9WJ11.1 P9WF23.1 069703.1 I6X4K0.1 B2RM93.1 R71888.1 P9WFW3.1 P9WPV1.1 P9WPU7.1 P9WPV3.1 P9WPU5.1 050391.1 P9WID7.1 P9WPC3.1 R96901.1 084848.1 Q2FUX4.1 A0A0M1YNY3.1 R49944.1 P9WPQ9.1 Q45010.1 Q2FZK7.1 P9WMN3.1 P9WPQ1.1 Q45013.1 053666.1 Q5NEH1.1 P9WHR5.1 P9WIE5.1 Q5NEQ3.1 P9WNF3.1 F2QBN0.1 B8ZTB7.1 Р0С922.1 P9WMJ9.1 Q5NGW2.1 P01556.1 Q8DMZ4.1 P33768. 1 Q2FUY2.1 Q5NG56.1 X8SE55.1 Q5NGE4.1 R94973.1 006827.1 R96872.1 I6X9Y7.1 T6XFZ8.1 050442.1 053697.1 053978.1 R95137.1 R95144.1 053519.1 Q79FZ8.1 P9WJF5.1 R71629.1 P9WJS3.1 P9WPB7.1 Q7D9T1.1 P9WHS1.1 006393.1 P9WP69.1 P9WPN5.1 P9WNX3.1 053380.1 I6YAU3.1 P0A4V2.1 P9WQP3.1 Р0С2Т2.1 P9WQP1.1 P9WQN9.1 053311.1 P9WTS7.1 006159.1 H2GU79.1 Q2G2Q0.1 P9WNV1.1 P9WNV5.1 Q8YE98.1 Q59191.1 P9WGY7.1 P9WGY9.1 Q2G2W1.1 P9WGH1.1 P9WNG9.1 P9WNG7.1 084591.1 Q9Z7A6.1 P9WGR1.1 R96404.1 I6YGS0.1 Q6MX18.1 P9WNK5.1 053692.1 P9WNK3.1 P9WNK1.1 P9WNJ9.1 P9WNJ7.1 P9WNJ5.1 P9WNJ3.1 P9WNJ1.1 P9WNT9.1 R96903.1 P9WNB1.1 P9WJE1.1 P9WJD9.1 P9WJD7.1 P9WJD3.1 P9WJC5.1 P9WJC3.1 P9WJC1.1 P9WNQ3.1 P9WJE5.1 P9WJC7.1 084646.1 T6YDV4.1 R11439.1 Q5NFJ1.1 P9WNE5.1 R14738.1 R11089.1 H7C7G3.1 L7N6B9.1 T6XFT7.1 005578.1 R96218.1 P9WN39.1 P9WN59.1 Q8YBT3.1 P9WN83.1 P9WJA9.1 P9WMY9.1 Q5NH51.1 053673.1 P9WTP9.1 P0CE15.1 R72041.1 Q5NEM8.1 Q5NT16.1 P9WJA3.1 P0A4Q1.1 P9WTP1.1 P9WTN9.1 P9WNF5.1 050846.1 Q59947.1 H7C7N8.1 Q5NEC6.1 084606.1 P9WQJ9.1 P9WQJ7.1 P9WQ71.1 053611.1 P9WKL1.1 P9WKJ7.1 D5V9Y8.1 P0SS04.1 R23700. 1 P9WJN5.1 Q5NHJ0.1 Q5NEY9.1 R15917.1 Q2G155.1 034094.1 Q8F8E1.1 069661.1 H6MMU4.1 P9WK61.1 P9WK55.1 Q8YGS9.1 050811.1 P9WQ59.1 P9WTN7.1 P9WTR1.1 050430.1 D5VCH6.1 Q5NHT7.1 P9WFU9.1 I6XFY8.1 B2RH54.1 Q46409.1 Р30690.1 АОАОJ5IWN3.1 A0PSI5.1 A4TAS4.1 В1МВ69.1 B2HSY2.1 B8ZSN3.1 E4WHS0.1 P9WK17.1 V5XE39.1 T6X7G8.1 T6Y461.1 T6YGB1.1 T6YC99.1 Q79FY7.1 T6X5Z8.1 I6Y479.1 T6YA32.1 005461.1 Q2G1E2.1 P9WK19.1 T6YAW3.1 Q5NGG4.1 051624.1 P9WJW5.1 Q50584.1 B2RHG1.1 Q5NFL7.1 P9WQN7.1 P9WHH3.1 084639.1 Q5NF24.1 P9WJH1.1 P9WJH5.1 053203.1 R55969.1 050418.1 Q5NGE0.1 N7S7K8.1 054584.1 G1UB30.1 Q5NH85.1 G1UB25.1 P0A3N8.1 E1X6Y5.1 Q5NEP7.1 Q8YHH0.1 R38006.1 R43838.1 R43839.1 P0CL67.1 P0CL66.1 QOSLZO.1 Q07337.1 G5TXT6.1 007721.1 053254.1 R75330.1 T6Y936.1 L7N649.1 L7N65 6.1 L7N693.1 Q79FK4.1 Q79FR3.1 Q79FR5.1 Q79G04.1 Q79FS8.1 Q6MWX1.1 Q79FV6.1 Q79FS5.1 Q79FQ7.1 Q79FP3.1 Q79FP2.1 Q79FK9.1 Q79FE6.1 T6XEF1.1 Q79FD4.1 Q6MX26.1 Q6MX50.1 L7N680.1 053695.1 I6X8R2.1 053246.1 I6Y0L1.1 Q2G282.1 R14283.1 P04977.1 P9WMX7.1 P9WFR1.1 P9WN09.1 086345.1 P9WGU1.1 P9WGT9.1 P9WGT7.1 P9WPF7.1 P9WTB3.1 P9WMM9.1 P9WHM5.1 P9WQE9.1 Q8DQ08.1 Q8DQ07.1 I6Y231.1 P9WHV9.1 005877.1 007236.1 086370.1 006404.1 006410.1 B8ZRL2.1 006807.1 033269.1 Q79FA9.1 Q79FK6.1 Q8VKN2.1 L7N675.1 Q79FK5.1 L0T7Y7.1 Q79FT9.1 Q79FE1.1 Q6MWX9.1 084616.1 084647.1 P9WQ27.1 084288.1 T6X9S5.1 P9WJW3.1 P9WPS9.1 R95149.1 053632.1 T6Y293.1 L0T243.1 P9WP43.1 P9WKC9.1 R96402.1 R71810.1 006417.1 Р96365.1 L0T5B2.1 R96264.1 P9WJK5.1 P9WJQ9.1 084419.1 084818.1 Q8YG32.1 00660 8.1 007175.1 P9WGA3.1 053323.1 R96354.1 P9WTM9.1 B8ZRT2.1 P9WK93.1 R13423.1 084583.1 P9WG63.1 P9WTM1.1 P9WKJ3.1 P9WNZ7.1 P9WK31.1 Q50701. 1 P9WTD3.1 Q8YC41.1 P9WPL3.1 P9WNT3.1 P9WNT7.1 P9WNT5.1 P9WQ49.1 P9WMG1.1 Q2GGR3.1 P9WK71.1 033192.1 P9WND5.1 P9WFL9.1 P9WMB7.1 P9WJ79.1 P9WND7.1 Q63RA7.1 Q63TD0.1 T6YET7.1 Q9S010.1 P9WGC9.1 Q50700.1 Q5NFR6.1 P9WGK3.1 P9WHT1.1 P9WHV3.1 Q5NTA7.1 P9WG27.1 P9WF73.1 P9WGA1.1 P9WTB9.1 P9WGL3.1 051381.1 P9WT83.1 P9WT79.1 P9WFT7.1 Q8YGS6.1 P05788.1 R17835.1 P9WTK9.1 Q5NHP7.1 P9WJU5.1 P9WGE7.1 Q2G2B2.1 P04958.1 P9WG67.1 P9WKE1.1 007226.1 P9WJ13.1 P9WHF3.1 P9WF43.1 Q7D7L0.1 P9WMF9.1 P9WGN1.1 P9WKJ9.1 P60230. 1 P9WKH7.1 053699.1 P9WHT7.1 P9WJS5.1 Q5NIT0.1 Q8YDZ3.1 Q9RPX7.1 P9WN67.1 005576.1 Q5NHL4.1 P9WN15.1 P9WMD5.1 P9WMF5.1 P9WG85.1 P9WJW7.1 P9WIH1.1 P9WIG1.1 P9WIG3.1 P9WIF5.1 P9WIF1.1 P9WIE7.1 P9WHW9.1 P9WT41.1 P9WT39.1 P9WT37.1 P9WT25.1 Q11031.1 P9WT47.1 P9WI23.1 P9WT19.1 P9WT11.1 P9WT45.1 P9WT07.1 P9WT05.1 Q79FH3.1 P9WT43.1 P9WHZ7.1 P9WHZ5.1 P9WHZ3.1 P9WHY9.1 P9WHY7.1 P9WHY5.1 Q6MX07.1 P9WHY3.1 Q6MWY2.1 Q50703.1 P9WHX3.1 P96221. 1 Q7D589.1 P9WMA3.1 P9WKW1.1 P9WKS9.1 P9WM29.1 P9WGC1.1 P9WLZ5.1 P9WLZ3.1 P9WLX1.1 P9WLV9.1 P9WLS7.1 P9WLQ1.1 P9WLJ1.1 P9WLH9.1 P9WLF3.1 P9WL97.1 P9WL87.1 P9WL85.1 P9WL83.1 P9WL67.1 P9WL63.1 P9WL51.1 P9WL47.1 P9WNH3.1 P9WGL7.1 P9WQM5.1 P9WPD9.1 A0A098A1N7.1 А0А098А2В0.1 A2RGM0.1 A5LVF6.1 A5MKZ9.1 B8ZQT8.1 B8ZQM3.1 B8ZQT5.1 B8ZR82.1 B8ZRH1.1 B8ZS71.1 B8ZS85.1 B8ZS86. 1 B8ZSJ5.1 B8ZSL3.1 B8ZSL7.1 B8ZSM6.1 B8ZT30.1 B8ZTD0.1 B8ZTS2.1 B8ZTV5.1 B8ZU53.1 B8ZUA4.1 B8ZUE5.1 B8ZUF0.1 B8ZUT6.1 B8ZUX6.1 C0R9U8.1 C6DPT8.1 C6DQ35.1 E1XJN6.1 G8W6L3.1 G8W6L7.1 G8W6U7.1 H6MNY3.1 H6MQD5.1 H8HRN0.1 H8HW90.1 H8L8K3.1 T6TQ53.1 Т6ТХ52.1 Р0С5В9.1 Q1BYS7.1 R4MDK6.1 S5F815. 1 W6GWM1.1 P9WFC9.1 P9WFJ9.1 R14916.1 Р69996.1 P9WFC5.1 Q8VKQ6.1 P9WHS3.1 A5MKT6.1

- 17 045699

List of accession numbers for fungal antigens from IEDB and UNIPROT

Table 5

Q5ANA3.1 Q5A3P6.1 Q59VM7.1 Q5A1A9.1 Q5APF0.1 Q8J0P4.1 Q4WHG0.1 Q4WQ87.1 Q59X67.1 Q59Z17.1 Q59ZT3.1 Q5AA33.1 B8N4Q9.1 Q4WAW6.1 Q4WAJ6.1 Q4X1V0.1 A0A1D8PQ86.1 Q59ZB1.1 Q873N2.1 Q59L72.1 B8NTF0.1 P46075. 1 Q4WCL1.1 Q4WRP2.1 Q59L12.1 Q59LC9.1 R48989.1 Q5AFC2.1 B8N406.1 Q4WGL5.1 Q9HEQ8.1 Q4WVT6.1 R46593.1 R82611.1 Q5ADV5.1 Q59SG9.1 R41750.1 000092.1 Q4WEN1.1 Q4WCV3.1 P0DJ06.1 094038.1 Q59WD3.1 Q59RQ0.1 B8NM71.1 Q4WLW8.1 Q4WI37.1 Q4WNT1.1 R29717.1 R46589.1 Q59W04.1 Q59RK9.1 B8MYS6.1 Q8X176. 1 Q4WZS1.1 Q4WQH4.1 Q9UW14.1 Q5AF56.1 Q59VN0.1 R31353.1 B8N8Q9.1 Q96UX3.1 Q4WDA4.1 Q4WDE1.1 Q92207.1 R83773.1 Q59WB9.1 Q5ACM4.1 B8N8R3.1 Q4WPF5.1 Q4WLS7.1 Q4WJT7.1 Q5A8T7.1 Q59YU1.1 Q59P53.1 Q5ACI8.1 B8N417.1 Q92450.1 Q4WWM6.1 Q4WLG1.1 Q5A8T4.1 Q59YV2.1 Q5A432.1 Q5AB93.1 B8N8R0.1 Q4WAW9.1 Q4WP81.1 Q4WQR6.1 R43076.1 Q5ABE5.1 Q5AK64.1 Q5ALL8.1 B8NM74.1 A4GYZ0.1 Q6MYT0.1 Q4WZS2.1 Q5AP53.1 Q59LF2.1 A0A1D8PNZ7.1 Q5A4X8.1 B8N106.1 Q4WAW3.1 Q4WTL0.1 Q4WXP0.1 Q5AL52.1 Q8NJN3.1 Q59Q30.1 Q5AD34.1 B8NHY4.1 Q70J59.1 Q4WXV2.1 Q4WU59.1 R43079.1 Q5ALN1.1 A0A1D8PN12.1 Q59V02.1 B8NJG8.1 Q4X1A4.1 Q4X0Z3.1 Q4WUG4.1 Q5AD07.1 Q59S72.1 Q5AK24.1 Q5AHC0.1 B8NM66.1 E9R876.1 Q4WN25.1 Q4WTK9.1 Q5A0E5.1 Q59K86.1 Q5AFT2.1 Q59Y11.1 B8MYL0.1 M4VQY9.1 Q4WN21.1 Q4WYP0.1 Q5AKU6.1 Q5AGD1.1 Q5A0W6.1 Q59QA5.1 B8NM62.1 Q4WF53.1 Q4X1N0.1 Q4X0B5.1 Q59RL7.1 R79023.1 Р0СВ63.1 Q5AMJ5.1 B8NGT5.1 Q4WZ64.1 Q4WQV2.1 Q4WYK9.1 G1UB61.1 Q59LP6.1 Q59U11.1 Q5AMF7.1 B8NM64.1 Q4WAZ0.1 Q4WZP2.1 Q4WY33.1 Q5ABC6.1 Q5AP87.1 Р83775.1 Q5ABW2.1 B8NV37.1 Q4WR16.1 Q4WVK2.1 Q4X1F8.1 A0A1D8PQB9.1 R22274.1 Q5APF2.1 Q5APJ9.1 B8N151.1 Q4WLB9.1 Q4WUA0.1 Q4WA45.1 R87020.1 Q5AC48.1 Q59VP2.1 Q5AM72.1 B8NEJ3.1 Q4WQS0.1 A4DA84.1 Q4WKD7.1 P0CY27.1 Q5AP59.1 Q5AEE1.1 Q5ACU3.1 B8N8M2.1 Q4WEP7.1 Q4WJX0.1 Q4WCH5.1 Q59XX2.1 Q59MV1.1 Q5AMR5.1 Q5A1V3.1 B8MYV0.1 E9R9Y3.1 Q4WP38.1 Q4WXY3.1 Q59U10.1 Q5AL27.1 Q59SU5.1 Q59RF7.1 B8N7T7.1 P41748. 1 Q4X1D7.1 Q4WPL7.1 Q59RW5.1 Q5AJD2.1 Q59VP1.1 Q5ACN3.1 B8NJG3.1 Q4WYG3.1 Q4W9Z9.1 Q4X136.1 Q59MQ0.1 P0CU38.1 Q5ADQ0.1 Q5AHE8.1 B8N8R1.1 R87184.1 Q4WE62.1 Q4WZ44.1 Q5ABU7.1 Q59QC5.1 Q5AK59.1 Q5AHA4.1 B8NJH2.1 Q4WBS1.1 Q4WZL3.1 Q4WTC7.1 Q9Y7F0.1 Q5A5N6.1 Q59RH5.1 Q5AEG7.1 B8NQ51.1 Q70DX9.1 Q4WB37.1 Q4WMK2.1 Q5AC08.1 Q59Q79.1 Q5ACW8.1 Q59V01.1 B8NM63.1 Q4WG16.1 Q4W9Z4.1 Q4WNC9.1 R30575.1 Q5AH38.1 Q5AGM0.1 Q5AK97.1 B8NM73.1 Q96X30.1 Q4WDD0.1 Q4WY67.1 Q5AAG6.1 Q5AMN3.1 Q59VN2.1 Q5A1B2.1 B8NYX0.1 Q4WV19.1 Q4WKB9.1 Q4WU12.1 074189.1 Q5A1Z5.1 094069.1 Q5AJK6.1 B8N3P7.1 Q4WAZ6.1 Q4WU07.1 Q4WA61.1 Q59W62.1 Q5A6K2.1 P0CY20.1 Q59L96.1 B8NJH1.1 Q4W944.1 Q4WBL6.1 Q4WA58.1 P0CY34.1 Q59L25.1 Q59XQ1.1 Q59MD0.1 B8MXJ7.1 Q4WTV7.1 Q4WX13.1 Q4WA60.1 Q5A1D3.1 Q5A922.1 094048.1 Q5AG46.1 B8NJB0.1 Q4WMJ9.1 Q4WV71.1 Q4WX36.1 Q5AJU7.1 Q5AFG1.1 Q5ADX2.1 Q59VW6.1 B8NPS7.1 Q4WZ65.1 Q4X0C2.1 Q4WA62.1 Q5A4H5.1 Q5ALR8.1 R46586.1 Q5A8T6.1 B8N7Z8.1 A0A067Z9B6.1 Q4WRU4.1 Q4WA59.1 Q59Y31.1 Q5AET2.1 Р83776.1 Q9UW24.1 B8NSV5.1 Q66WM4.1 Q4WGS4.1 Q4WXQ7.1 P0CY29.1 Q5AT71.1 Q5A895.1 Q59Q38.1 B8MZA3.1 Q6T267.1 Q4WP13.1 Q4WVA0.1 Q5ANJ4.1 Q5ABA6.1 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Q5AJB1.1 Q5AL13.1 Q59SI5.1 Q5A5N5.1 B8NJZ7.1 Q4WA22.1 Q4WK77.1 Q4WTT7.1 Q59UP6.1 Q59NY7.1 Q59RR3.1 Q5ADL4.1 B8N6H2.1 Q4WM60.1 Q4WCL2.1 Q4WWS3.1 Q5AMH6.1 Q5AP89.1 Q5APQ8.1 Q5AM84.1 B8NTX4.1 Q0H904.1 Q4WN75.1 Q4WVH3.1 Q59SF7.1 Q59XY0.1 P87207.1 Q5AK73.1 B8NGC8.1 R78574.1 Q4WES5.1 Q4WD95.1 Q59VX8.1 Q5ADL9.1 Q59MZ9.1 Q5A4H9.1 B8N970.1 Q4WAR8.1 Q4WVT3.1 Q4WLP1.1 Q59WG7.1 P53698.1 Q59Y41.1 Q5ALX5.1 B8MY73.1 Q4WNK7.1 Q4WVV6.1 Q4WQT6.1 Q5AFN8.1 Q5AJX2.1 Q59S52.1 Q5A748.1 B8N6W5.1 R78746.1 Q4WP83.1 Q4WCJ7.1 Q59TP1.1 Q5APS5.1 Q59U73.1 Q5ALU2.1 B8N3L3.1 Q4WPF8.1 Q4WAY7.1 Q4WTT8.1 Q5AF39.1 Q59PG6.1 Q9Y872.1 Q5A2B9.1 B8NPS8.1 Q4WX43.1 Q4WX89.1 Q4WWR2.1 Q5AP97.1 Q59NP1.1 Q5AGA9.1 Q5ALX3.1 B8NTT4.1 Q4WQL4.1 Q4WYT0.1 Q4WWL0.1 Q5A5U9.1 Q59PD3.1 Q59VL7.1 Q5A1M3.1 B8MYS7.1 Q4WBE1.1 Q4WNT9.1 Q4WZT9.1 Q5AF41.1 Q5ACW2.1 Q59KJ7.1 Q5A4H4.1 B8NM70.1 Q4WQT2.1 Q4WVS4.1 Q4X0T7.1 013318.1 Q5ANB2.1 Q5AP90.1 Q5AA26.1 B8MYS8.1 Q4WBT5.1 A4D9J5.1 Q4WU00.1 Q5AA09.1 Q5AJD0.1 Q5AD72.1 Q5ANL6.1 B8N6M6.1 Q4WQZ2.1 Q4W9B7.1 Q4WRW0.1 Q5A762.1 Q5A4P9.1 Q59S59.1 P87218.1 B8NCU7.1 Q4WD47.1 Q4WNC6.1 Q4W9V0.1 P46587.1 P78599.1 Q5APM7.1 Q59KF3.1 B8N5T6.1 Q4WCZ8.1 Q4WJW8.1 Q4WYJ7.1 Q5A287.1 Q5APC8.1 Q5A2Z1.1 Q59N29.1 B8MVS3.1 Q4WB01.1 Q4WH96.1 Q4WHY5.1 Q59X49.1 Q59LU0.1 Q59TD3.1 Q5A0L7.1 B8NCM8.1 Q4WBK6.1 Q4X0T5.1 Q4WEP0.1 Q5ADM9.1 Q5APT8.1 P84149.1 Q59UG4.1 B8NW36.1 Q4WRQ7.1 Q4WMS9.1 Q4WXD3.1 Q5AH02.1 Q59PR3.1 Q5AT97.1 Q5AHK2.1 B8NJG7.1 Q4WTQ6.1 Q4WAH4.1 Q4WJ02.1 Q5A4X5.1 Q5A2W2.1 Q5A2A2.1 Q5ADP6.1 B8N7Z6.1 Q4WJ21.1 Q4WTT3.1 Q4WP96.1 Q5A4E3.1 Q5A4E2.1 Q5A044.1 Q5AK62.1 B8NGU1.1 Q4WPQ8.1 Q4WJA1.1 Q4WN54.1 Q5A761.1 Q5A309.1 Q59P03.1 Q59YF0.1 B8NC10.1 Q4WR62.1 Q4W9R7.1 Q4WCW2.1 Q9UW23.1 A0A1D8PL26.1 Q59TU0.1 Q5AAJ7.1 B8N4P0. 1 Q4WD56.1 Q4WPP2.1 Q4WPM6.1 P53704.1 P0CU37.1 Q5APK7.1 Q5A8H7.1 B8NPN0.1 Q4WIN6.1 Q4WNQ6.1 Q4WNW3.1 Q59VR1.1 Q5AF95.1 Q59ST1.1 Q59U81.1 B8NQ08.1 Q4U3E8.1 Q4WNT0.1 Q4WST0.1 G1UB67.1 Q59MW2.1 Q5A7N3.1 Q5APB6.1 B8N3N5.1 Q4X195.1 Q4WDG1.1 Q4WNY4.1 P52496.1 Q59S50.1 Q5ANP2.1 Q59WD5.1 Q00049.1 Р0С955.1 Q4X0Z7.1 Q4WVF4.1 Q9HEW1.1 Q5AD78.1 059933.1 Q5ABA2.1 B8NDP1.1 Q4WRH9.1 Q4WMS3.1 Q4WP02.1 Q5A6B6.1 Q5AMM4.1 Q3MPQ4.1 Q5A861.1 B8NEM4.1 Q4WVD1.1 Q4WN42.1 Q4WWH6.1 Q5A1W9.1 Q5AAW3.1 Q59MP1.1 Q5AH87.1 Q9P8Z9.1 Q4WID6.1 Q4WJH6.1 Q4WVE5.1 P30418.1 Q59MG1.1 Q59MB6.1 P33181.1 B8MZJ8.1 Q4WFX9.1 Q4WYS1.1 Q4WHP3.1 Q59SN6.1 Q5ACK7.1 Q5A216.1 Q59Q43.1 B8NX10.1 Q4WRE4.1 Q4WJ01.1 Q4WRE2.1 Q5A343.1 Q5A218.1 Q9UVL1.1 Q5A860.1 B8NV05.1 Q4WC60.1 Q4WGL2.1 Q4WYX0.1 Q5ABZ2.1 Q59SJ9.1 Q59YS7.1 Q59ZW9.1 B8NET6.1 Q4WR18.1 Q4WP49.1 Q4WRB8.1 Q59MJ1.1 Q5AD49.1 Q5AGA0.1 A0A1D8PT78.1 B8MZT5.1 Q4WQY6.1 Q4WPE6.1 Q4WT88.1 Q5AJ71.1 Q59NX9.1 Q5A687.1 Q59R24.1 B8NSJ0.1 Q4WXK4.1 Q4WWW9.1 Q4WQL0.1 074201.1 Q5A119.1 Q59R28.1 Q5AHJ5.1 B8NDR8.1 Q4WT96.1 Q4WKB5.1 Q4WDZ0.1 Q5AK54.1 Q59K07.1 Q5AJS6.1 P0C0X3.1 B8NDQ2.1 Q4WVH4.1 Q4WA38.1 Q4WA70.1 093852.1 Q5AKA5.1 Q5AD59.1 Q59KL6.1 B8N9M0.1 A4D9R2.1 Q4WHL1.1 Q4WQ82.1 Q5ATR7.1 Q59QC2.1 Q5AG73.1 R43072.1 B8NLN6.1 Р0С956.1 Q4X1X0.1 Q4WMX7.1 Q5A8K2.1 Q5AL45.1 Q5AND1.1 Q5AF54.1 B8N9X2.1 Q4WR22.1 Q4WRX2.1 Q4X0V2.1 Q8TGB2.1 P0CY19.1 Q59NG5.1 Q59W44.1 B8NM08.1 Q4WQY8.1 Q4WDH9.1 Q4WT16.1 Q5A477.1 Q5AGC4.1 Q59N20.1 Р48990.1 B8NSD4.1 Q4WJJ3.1 Q4WMG1.1 Q4WXA1.1 Q5AP95.1 Q5ALP1.1 Q59WJ5.1 Q5 9U67.1 B8N122.1 Q4X265.1 Q4WDE0.1 Q4WCV5.1 Q5AF03.1 Q5AK42.1 Q5AA50.1 Q5ANB7.1 B8NCF0.1 Q9UVX3.1 Q4WCX4.1 Q4W9M7.1 Q5AMQ4.1 Q5APG7.1 Q5A319.1 Q5A3Y5.1 B8NKS1.1 Q4WR19.1 Q4X122.1 Q4WQY9.1 Q5ANT6.1 Q59Y20.1 Q5AD27.1 Q59ST2.1 B8N3R8.1 Q4WTF3.1 Q4WZF1.1 Q4WX30.1 P78595.1 Q5ALL3.1 Q5AHT7.1 Q5APA2.1 B8NG55.1 Q4WLY1.1 Q4WMU1.1 Q4WUT7.1 Q874T4.1 Q5AAT0.1 Q5ANE3.1 R12461.1 B8N0Q7.1 Q4WMU3.1 Q4WGB7.1 Q4WTQ2.1 Q9UWF6.1 Q59QD6.1 Q59S06.1 Q59TN1.1 B8N513.1 Q4WQG5.1 A4DA73.1 Q4X022.1 Q9UW12.1 Q5AML1.1 P87185.1 Q5A416.1 B8N4F5.1 Q4WPE9.1 Q4WD81.1 Q4WQZ0.1 Q5AAL9.1 Q5ACM9.1 Q5AM50.1 043133.1 B8NT06.1 Q4WAZ4.1 Q4WHG0.1 Q4WE58.1 Q5AD56.1 Q59Z14.1 Q9B8C8.1 Q59MT8.1 B8NHF2.1 Q4WLN7.1 Q4WAJ6.1 Q4WJR4.1 Q5A7S7.1 Q5AAG1.1 Q9B8C9.1 Q5A302.1 B8MWR8.1 Q4WRB0.1 Q4WCL1.1 Q4WQZ1.1 P28870.1 Q59YL9.1 Q9B8D2.1 Q5AH60.1 B8N4G0.1 Q4WC55.1 Q9HEQ8.1 Q4WQY7.1 Q59NX5.1 Q59PL9.1 Q9B8D1.1 Q5A692.1 B8N9M5.1 Q4WMV5.1 Q4WEN1.1 Q4WQY5.1 Q5ABG1.1 Q59QL0.1 Q59M69.1 Q59Q39.1 Q00278.1 Q4WAZ2.1 Q4WT37.1 Q4WXT2.1 Q5AP52.1 Q5A1U8.1 Q59VX9.1 Q59NW5.1 B8NPX1.1 Q92197.1 Q4WZS1.1 Q8J130.1 P0CY31.1 074198.1 Q59YD8.1 Q5A6Q4.1 B8NYW8.1 Q4WSE8.1 Q4WDA4.1 Q4WJX5.1 P13649.1 Q5A013.1 Q59QH0.1 R43075.1 B8N219.1 Q4WX94.1 Q4WLS7.1 Q4X1T8.1

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Q5AG77.1 R87163.1 Q5A8A2.1 Q5 9Q3 6.1 B8NQK0.1 Q4WLD0.1 Q4WWM6.1 Q4WVW4.1 Q9UW13.1 Q5AT86.1 Q9B8D7.1 Q92410.1 Q12732.1 Q4WUK5.1 Q4WP81.1 Q4WTH1.1 P0CU34.1 Q5AM80.1 Q9UW25.1 Q5A1M4.1 Q9HEY7.1 Q8TGG5.1 Q6MYT0.1 Q4WLT9.1 R40954.1 Q5A6Q7.1 Q59XY9.1 Q5ANC8.1 Q6UEG8.1 Q4WTK9.1 Q4WTL0.1 Q4WQJ5.1 Q04802.1 Q5AGV4.1 Q5A2T0.1 Q5A4K7.1 042716.1 Q4WVU5.1 Q4WXV2.1 Q4WQJ2.1 P0CY35.1 Q5AJ82.1 Q5AGW8.1 Q5ADL8.1 Q9UW95.1 Q4WLM7.1 Q4X0Z3.1 Q4WK56.1 Q5AAU5.1 Q5AIA1.1 Q5ADS3.1 Q59RQ2.1 Q9Y8D9.1 Q4W9P4.1 Q4WN25.1 Q4WJS2.1 Q59VQ8.1 Q5A9Z6.1 Q5ACR4.1 Q5APC0.1 A2SZW8.1 Q4WTT0.1 Q4WN21.1 Q4WJT9.1 Q59VF4.1 Q5AGC1.1 P0CU36.1 Q5A931.1 Q2U2U3.1 Q4WQB9.1 Q4X1N0.1 Q4WUV8.1 Q5A0X8.1 Q59ZV5.1 Q5A2Y7.1 Q59VW7.1 Q00258.1 Q4WGK6.1 Q4WQV2.1 Q4WX68.1 013426.1 Q59VP7.1 Q5A3 6 8.1 Q5AKU5.1 Q12437.1 Q4WMR0.1 Q4WZP2.1 Q4WHN8.1 Q5A0M4.1 Q5A7P3.1 Q9B8D6.1 Q59MN0.1 E9QYP0.1 Q4WYE5.1 Q4WVK2.1 Q4WJU8.1 Q59PF9.1 Q5A6K8.1 Q9B8D0.1 Q59WH7.1 Q4WS76.1 Q4WZ01.1 Q4WUA0.1 Q4WBT4.1 Q5AFP3.1 Q5AD13.1 Q5A2K0.1 Q96WL3.1 Q4WMJ7.1 Q4W930.1 A4DA84.1 Q4WZV6.1 Q5AEK8.1 Q04782.1 Q5A1Q5.1 Q59ZX6.1 R28296.1 Q4WBR0.1 Q4WJX0.1 Q4WUV9.1 Q5AFK0.1 Q5A0J9.1 Q5AEM5.1 Q59MU1.1 E9RAH5.1 Q4WHD1.1 Q4WP38.1 Q4WLV2.1 Q5APD4.1 Q59ZZ6.1 Q5AK25.1 Q5A0JO.1 Q4WW81.1 Q4WTB3.1 Q4X1D7.1 Q4WFS2.1 Q5ADQ9.1 Q5AH25.1 Q5AK10.1 Q59WK2.1 Q50EL0.1 Q4WRV9.1 Q4W9Z9.1 Q4WBM1.1 R83779.1 Q59XM1.1 Q5AT15.1 R43073.1 Q4WY82.1 Q4X267.1 Q4WE62.1 Q4WAU7.1 Q5AAH2.1 Q59NN8.1 Q5AEM8.1 R87220.1 Q4WSF6.1 Q4WVZ3.1 Q4WZL3.1 Q4WZS3.1 074254.1 Q5AP65.1 Q5A4J4.1 Q5ABD9.1 E9RCK4.1 Q4WR24.1 Q4WB37.1 Q4WPU9.1 Q5AL49.1 Q5AFF7.1 Q59YK4.1 Р83781.1 Q4WZA8.1 Q4WPM8.1 Q4W9Z4.1 Q4WVZ0.1 R53697.1 Q59VR3.1 Q59WV0.1 Q5ANB1.1 Q4WAW7.1 Q4WE86.1 Q4WDD0.1 Q4WCX9.1 Q5ACL7.1 Q5AFH3.1 Q5AHB1.1 Q5A0E2.1 Q92405.1 A4DA70.1 Q4WKB9.1 Q4WJ38.1 Q5AEM6.1 Р83780.1 Q5APK0.1 Q5AMG5.1 Q4WRY5.1 Q4WW45.1 Q4WU07.1 Q4WRC2.1 Q8TG40.1 Q5A4G9.1 Q59PW0.1 Q5A6T8.1 Q7Z7W6.1 Q4WVG2.1 Q4WBL6.1 Q4WWW5.1 Q59X38.1 Q59NQ9.1 074711.1 Q59WG5.1 Q4WZ67.1 Q4WQG9.1 Q4WX13.1 Q4WC84.1 Q59VQ3.1 A0A1D8PNP3.1 Q5ADN9.1 Q5AI80.1 Q4WZB3.1 Q4WQN1.1 Q4WV71.1 Q4WTW3.1 Q5A7Q2.1 Q5A9Z1.1 Q5ACP5.1 Q5AB49.1 Q4WLN1.1 Q4WCF1.1 Q4X0C2.1 Q4WFV6.1 Q5AJV5.1 A0A1D8PK89.1 Q5A1E1.1 Q59R32.1 Q4WR82.1 Q4WZC3.1 Q4WRU4.1 Q4WKD9.1 Q5A3Z6.1 Q59WB3.1 Q59L86.1 Q5A061.1 014434.1 Q4WYX7.1 Q4WGS4.1 Q4WP10.1 Q5A201.1 Q59ZC8.1 Q5AD23.1 Q59P50.1 Q4WMK0.1 Q4X0A5.1 Q4WP13.1 С5JZM2.1 093827.1 Q5A1L6.1 Q5A5U6.1 Q59WC6.1 Q4WPX2.1 Q4WUD3.1 Q4WHG5.1 P0DJ06.1 Q5AAT8.1 A0A1D8PN14.1 Q5ADQ7.1 Q5AT48.1 043099.1 Q4WS49.1 Q4WPF7.1 R46598.1 Q5A2J7.1 Q5A8X7.1 Q59WJ4.1 Q59ZU1.1 Q4WJ81.1 Q4WCX7.1 Q4WH83.1 R87020.1 R22011.1 Q59X39.1 Q5AGV7.1 Q5AG56.1 R67875.1 Q4WXX5.1 Q4WXW1.1 R38110.1 Q9HGT6.1 Q5ACW6.1 Q59NR8.1 Q59T36.1 Q4WZB4.1 Q4WNB5.1 Q8NJM2.1 C1GK29.1 Q9UW26.1 Р0СВ54.1 Q5A5K7.1 Q9P840.1 E9QUT3.1 042799.1 Q4WWD3.1 Q59LX5.1 A0A1D8PN88.1 Q5A210.1 Q5AHB8.1 Q4WAZ9.1 Q4WHA3.1 Q4WPU8.1 Q59PT0.1 A0A1D8PMB1.1 Q59N10.1 Q5AKU3.1 Q4WZ70.1 Q4W9M3.1 Q4WN99.1 Q3MNT0.1 Q5ABR2.1 Q5A1B3.1 Q59ZW4.1 E9RBR0.1 Q4WVH5.1 Р0С959.1

Table 6

List of accession numbers for allergens from IEDB and ALLERGENONLINE

R19594.1 R28335.1 Р29000.1 M5ECN9.1 R38948.1 P00709.1 R79085.1 R49148.1 Q6R4B4.1 R42037.1 Q9HDT3.1 R42058.1 P0C0Y4.1 R27759.1 Q2KN25.1 P00304.1 Q2KN24.1 Q2KN27.1 R43174.1 R10414.1 Q8L5L5.1 Q8GZP6.1 Q8H2B8.1 Q7Z1K3.1 A1IKL2.1 Q7M1X6.1 R49372.1 P00630.1 R43238.1 Q45W87.1 Q6PSU2.1 082580.1 Q647G9.1 Q9SQH1.1 S7EZT4.1 H6VGT3.1 Q84ZX5.1 A0PJ16.1 R67875.1 R40292.1 R28296.1 R79017.1 Q96X30.1 Q4WWX5.1 060024.1 Q92450.1 Q09072.1 Q09097.1 P04403.1 R15494.1 R25816.1 R43187.1 Q39419.1 065002.1 P05814.1 R13916.1 Q9UAM5.1 R54958.1 D0VNY7.1 R54962.1 018598.1 Q1A7B3.1 Q9NG56.1 A0ERA8.1 Q8MUF6.1 A7TZE9.1 096870.1 P02663.1 P02 66 6.1 P02668.1 Q28133.1 P00711.1 P02754.1 P02769.1 P02662.1 018873.1 R49822.1 P09582.1 B5KVH4.1 Q14790.1 E9R5X9.1 Q96385.1 Q7M1E7.1 P02229.1 Q7XCK6.1 R40108.1 R42039.1 R42040.1 R42059.1 P0C0Y5.1 P02465.1 Q6TQX2.1 R20023.1 Q08407.1 Q8S4P9.1 Q9ATH2.1 Q8W1C2.1 R18632.1 R43212.1 Q9SCG9.1 Q9M4S6.1 Q69CS2.1 Q96VP3.1 004701.1 004725.1 R94092.1 P04800.1 Q7M1X8.1 Q41183.1 R93124.1 R82946.1

-21 045699

004298.1 Q58A71.1 Q23939.1 Q967Z0.1 Q1M2P5.1 Q94507.1 Q8MVU3.1 Q86R84.1 Q00855.1 R49275.1 Q26456.1 P08176.1 Q8N0N0.1 R49278.1 Q2L7C5.1 R39675.1 Q9Y197.1 R14004.1 R49273.1 Q7Z163.1 Q9UL01.1 015315.1 Р11388.1 R30575.1 Q95182.1 R41091.1 015371.1 R25780.1 Q2PS07.1 R49327.1 R30438.1 Q5VFH6.1 Q7XAV4.1 P04075.1 Q90YL0.1 P01005.1 P01012.1 R19121.1 P02230.1 P02224.1 P02227.1 Q9NJQ6.1 065809.1 R26987.1 P04776.1 P04347.1 P04405.1 P08238.1 P12031.1 R15252.1 Q7Y1X1.1 R52407.1 082803.1 Q39967.1 P02877.1 R62805.1 P43216.1 023972.1 R24337.1 Q7Y1C1.1 R93198.1 Q9SEW4.1 Q2TPW5.1 R81294.1 P81295.1 064943.1 P07498.1 Q84UT1.1 Р80384.1 R31025.1 Q004B5.1 R14946.1 Q7M1X5.1 R14947.1 R14948.1 Q5TTW3.1 Q40237.1 R14174.1 Q5H786.1 R30440.1 P11589.1 R43211.1 R40967.1 Q01726.1 Q16655.1 Q07932.1 Q9ZNZ4.1 Q9H009.1 P12036.1 Q15233.1 Q5RZZ3.1 Q8GZB0.1 Q8NFH4.1 R19963.1 Q94G86.1 P01014.1 P22895.1 R43217.1 R55958.1 B8PYF3.1 075475.1 024554.1 Q0TX90.1 Q52PJ2.1 K7VAC2.1 Q3Y8M6.1 Q9URR2.1 Q9P8G3.1 Ά1ΚΥΖ2.1 R23284.1 Q9TZR6.1 Q25641.1 P00433.1 Q41260.1 R56164.1 Q40967.1 Q8H6L7.1 R35079.1 Q9XG86.1 R43214.1 Q5ZQK5.1 Q40960.1 R43215.1 082040.1 Q8L5D8.1 R82242.1 Q9HCM2.1 Q9ZP03.1 Q9FPR0.1 Β6Τ2Ζ8.1 Q9C5M8.1 R15722.1 R25788.1 R81651.1 024248.1 Р82534.1 E3SH28.1 065457.1 B6RQS1.1 P02761.1 R67876.1 Q9Y4W2.1 Q9ULX3.1 Р83181.1 Q8L5K9.1 C1KEU0.1 Q91482.1 Q9XHP1.1 R15322.1 Q15020.1 B9SA35.1 P01267.1 000267.1 D2T2K3.1 Q9T0P1.1 Q07283.1 Q7M3Y8.1 R25445.1 Q5NT95.1 P07101.1 015205.1 000762.1 D2KFG9.1 Η9ΆΧΒ3.1 Q8W3V4.1 R49370.1 Q05110.1 Q9ULJ6.1 Q2VST0.1 ABL09307.1 ABL09312.1 AGC39172.1 AGC39173.1 AGC39174.1 P00785.4 R85204.1 AGC39168.1 SAM31908.1 ΆΒΒ77213.1 R83958.1 AGC39176.1 CAA34486.1 ΆΆΆ32629.1 Ά5ΗΤΤ1.1 SAMZ1909.1 R85206.1 R86137.2 R85524.1 CAT38795.2 ABQ42566.1 AAR92223.1 R84527.1 AGC39164.1 AGC39165.1 AGC39166.1 AGC39167.1 4X9U V AGC39169.1 AGC39170.1 AGC39171.1 AAC37218.1 R50635.2 ХР_00165755 6.2 R18153.2 ΆΆΒ58417.1 ABF18122.1 ХР_00165346 2.1 ХР_00165414 3. 1 HR-00165429 eleven ABF18258.1 ХР_00165594 8.1 ХР_00165595 4.1 R13080.1 E37396 Q7M1X7 Q7M1X9 ΆΆΒ24432.1 CAA76831.1 ΆΆΒ47552.1 ΆΆΜ77471.1 AAS75297.1 3V0R A 4AUD B CAA55071.2 R49148.1 Q6R4B4.1 R78983.2 Q00002.2 AAV48041.1 R42037.1 Q9HDT3.2 R42058.1 OWY50380.1 ΆΆΟ91800.1 P0C0Y4.2 AGS80276.1 CAD38167.1 ΆΒΤ26088.1 ACP43298.1 AKV72168.1 R27759.1 R27760.1 R27761.1 R28744.1 ΆΆΆ32669.1 CBW30986.1 CBW30987.1 CBW30988.1 CBW30989.1 CBW30990.1 CBW30991.1 CBW30992.1 CBW30993.1 CBW30994.1 CBW30995.1 ΆΆΧ77686.1 R27762.1 CBJ24286.1 SVK52317.1 SVK62693.1 SVK62694.1 SVK62695.1 SVK62697.1 SVK62698.1 SVK62699.1 004004.1 ΆΆΡ15203.1 ΆΆΡ15202.1 ΆΆΡ15201.1 ΆΆΧ77687.1 ΆΆΧ77688.1 5EM1 A 5EV0 V ΆΆΧ77684.1 ΆΆΧ77685.1 ΆΗΆ56102.1 5EGW B P00304.2 P02878.1 ΆΆΆ20065.1 ΆΆΆ20067.1 ΆΆΆ20064.1 ΆΆΆ20066.1 ΆΆΆ2 0 0 6 8.1 R10414.2 ΆΕΚ65120.1 ΆΑΜ73729.1 ΆΑΜ73730.2 ΆΆΝ76862.1 AAL91665.1 023791.1 Q94JN2.1 CDZ09832.1 AGC60026.1 AGC60027.1 AGC60028.1 AGC60020.1 Q7Z1K3.1 AGC60035.1 AGC60036.1 Ά0Ζ95445.1 BAJ78220.1 BAJ78221.1 BAJ78222.1 BAJ78223.1 AGC60029.1 AGC60030.1 AGC60031.1 ΒΆΤ62430.1 AAF75225.1 Q9NJA9.1 Q9NAS5.1 AEQ28167.1 Р83885.1 SAC50389.1 BAF43534.1 ABL77410.1 BAF75681.1 BAF75704.1 BAF75705.1 BAF75706.1 BAF75707.1 BAF75708.1 BAF75709.1 BAF75710.1 BAF75711.1 BAF75712.1 ABV55106.1 SAV58171.1 G37396 Q7M1X6 Q7M1Y0 Ά59055 ΆΆΚ09361.1 Q7M4T5.1 P01502.1 P00630.3 ABF21077.1 ABF21078.1 Q08169.1 AST25605.1 Q5BLY5.1 CAA26038.1 MENV2 ΝΡ_00111971 5.1 NP_00103536 0. 1 ABD51779.1 ΝΡ_00101156 4.1 ΆΆΥ21180.1 CAD56944.1 ΆΗΜ25038.1 ΆΗΜ25037.1 ΆΗΜ25036.1 ΆΗΜ25035.1 R49372.1 R92918.1 ACV04796.1 AAD29409.1 R81943.3 R86809.1 ΆΆΒ22817.1 R43237.1 R43238.1 ΆΆΤ00595.1 ΆΆΤ00594.1 ΆΆΤ00596.1 ADQ53858.1 3SMH A 3S7E A B3EWP3.1 C0HJZ1.1 B3EWP4.1 ΆΆΝ77576.1 ΆΑΜ78596.1 ΆΆΚ96887.1 ACN62248.1 AAC63045.1 AAD47382.1 ΆΆΜ46958.1 ΆΆΜ93157.1 ΆΒΤ17154.1 ASN91862.1 3C3V A ADQ53859.1 AAD55587.1 ADB96066.1 AGA84056.1 AAD5 633 7.1 AAL37561.1 1W2Q A Q647G9.1 AAD56719.1 ABW17159.1 AAQ91847.1 AVR97433.1 ACA7 990 8.1 ABG85155.1 ABX56711.1 ABX75045.1 AAU21499.2 AAU21500.1 ΆΆΖ20276.1 Q45W86 CAG26895.1 2X45 A AHF71021.1 AHF71022.1 AHF71023.1 AHF71024.1 AHF71025.1 AHF71026.1 AAO24900.1 SAC50834.1 Р0С088.1 ACE07186.1 ACE07187.1 ACE07188.1 ACE07189.1 CAD12861.1 CAD12862.1 5EM0 A ΆΆΧ85388.1 ΆΆΧ85389.1 CAD23611.1 CAD23613.1 CAD23614.1 ΒΆΗ09387.1 AAD13644.1 AAD13645.1 AAD13647.1 AAD13649.1 AAD13650.1 AAD13651.1 AAD13652.1 AAV93837.1 AAV93839.1 AAD13646.1 ACN32322.1 AAV26195.1 Q06811.2 2XV9 A R46436.3 Q9UVU3 CAA06305.1 AAF86369.1 R67875.1 CAA59419.1 SAV44442.1 CAA73782.1 ΆΆΒ07620.1 R79017.2 ΆΆΚ49451.1 Q96X30.3 ΆΑΜ43909.1 Q8NKF4.2 CAT78448.1 CAT78449.1 CAT78450.1 ΆΆΒ95638.1 CAM5 4 0 6 6.1 CAA04959.1 060024.2 SAA83015.1 R46075.3

- 22 045699

AAB60779.1 Q92450.3 042799.2 CAB64688.1 Q9UUZ6.2 CAA11266.1 Q875T9.1 EAL89830.1 Q4WB37.1 KEY81716.1 KEY78748.1 AAA32702.1 CAB06417.1 AAD13106.1 P0C1B3.1 AAA32708.1 P12547.2 ADE74975.1 P29600.1 P00780.1 AAG31026.1 BAA05540.1 BAF46896.1 ATV43661.1 BAH10149.1 P04403.2 AAO38859.1 A45786 CAA54696.1 CAA54695.1 CAA54694.1 CAA96546.1 CAA96539.1 CAA96540.1 CAA96541.1 CAA96542.1 CAA96543.1 CAA96544.1 CAA96547.1 P43186.2 CAB02155.1 CAB02156.1 CAB02157.1 CAB02158.1 CAB02159.1 CAB02160.1 CAB02161.1 CAA96545.1 CAA05186.1 CAA05187.1 CAA05188.1 CAA05190.1 CAA07318.1 CAA07319.1 CAA07323.1 CAA07324.1 CAA07325.1 CAA07326.1 CAA07327.1 CAA07329.1 CAA07330.1 CAA04823.1 CAA04826.1 CAA04827.1 CAA04828.1 CAA04829.1 AAD26560.1 AAD26561.1 AAD26562.1 P43180.2 1QMR A AAP37482.1 1LLT A AAB20452.1 CAA07328.1 CAA07320.1 CAA54488.1 1B6F A 4BK7A 4B9R A 4BKC A 4BKD A 4BK6 V SAAZ 3 8 8 7.1 CAA54482.1 CAA54483.1 CAA54484.1 CAA54487.1 CAA54489.1 CAA54421.1 CAA54481.1 4BTZ A 4Z3L D B45786 1CQA A AAA16522.1 A4K9Z8.1 CAA55854.1 CAA60628.1 AAG22740.1 CAC84116.1 AHF71027.1 BAB21489.1 BAB21490.1 BAB21491.1 AAB25850.1 AAB25851.1 AJO53282.1 AAB29344.1 AAB29345.1 ACM24358.1 ABC86902.1 AAD13531.1 AAD13530.2 ABC68516.1 1YG9 A ABP35603.1 AAA86744.1 3LTZ A ACY40650.1 ACY40651.1 AAA87851.1 ABP04043.1 ACJ37389.1 ACF53836.1 ACF53837.1 ABP04044.1 AAB72147.1 ABB89296.1 ABB89297.1 ABB89298.1 AAF72534.1 ABX57814.1 AAK58415.1 AAQ24541.1 ABU97466.1 AAM83103.1 AAA78904.1 2MFK A AAC80579.1 ABH06350.1 ABH06347.1 ABH06346.1 ABH06348.1 AAX34047.1 AAM10779.1 AAQ24542.1 AAQ24543.1 AAD10850.1 ABH06352.1 ABH06359.1 2JMH A APU87558.1 APU87557.1 APU87556.1 APU87554.1 AAQ24545.1 ASX95438.1 AAP35069.1 ACV04860.1 Q7M4I6.1 Q7M4I3.1 P82971.1 P0CH88.1 ABB88514.1 XP_00590209 9.2 AAA62707.1 AAA30429.1 AAA30478.1 NP 851372.1 ABW98943.1 ABW98945.1 ABW98953.1 NP 776953.1 AAA30430.1 AAA30431.1 AAB29137.1 AAA30433.1 NP 776719.1 Q28133.1 Q28050.1 CAA29664.1 AAA30615.1 CAA32835.1 AAA30413.1 P02754.3 ACG59280.1 AAA51411.1 CAA76847.1 NP 776945.1 NP 851341.1 P80207.1 P80208.1 S65144 S65145 AAN86249.1 XP_01362321 3.1 S65143 CAA46782.1 BAA09634.1 P69199.1 P81729.1 CAA57342.1 AAN11300.1 P30575.1 AAC48794.1 CAD82911.1 CAD82912.1 AAC48795.1 AAB30434.1 CAA76841.1 BAC10663.1 ACY38525.1 AHY24648.1 CAA68720.1 CCF72371.1 CCK33472.1 CAC34055.2 CAD10376.1 AAB02650.1 CAA47357.1 CAB02206.1 CAB02207.1 CAB02208.1 CAB02215.1 CAB02216.1 CAB02217.1 AAB20453.1 ABZ81044.1 ABZ81040.1 ABZ81043.1 ABZ81042.1 ABZ81041.1 AAB34907.1 AAB34908.1 AAB34909.1 CAA47366.1 CAB02209.1 CAB02213.1 CAA47367.1 AAO32314.1 ABW86978.1 ABW86979.1 ABV49590.1 5E1R F ABM53030.1 CAD10374.1 ACJ23862.1 ACJ23861.1 ACJ23863.1 CAA64868.1 ADN39439.1 2MC9A P83507.1 CAX62129.1 CAX62130.1 BAA08246.1 Q7M1E7.1 BAF32143.1 AAF35431.1 AAL07319.1 AAL92870.1 ACR77509.1 AAL92871.1 A2V735.1 CAA09938.2 P02229.2 P02230.1 P02221.2 P84296.1 P02227.1 P12548.1 P84298.1 P12549.1 P12550.1 P02226.2 P02222.2 P02223.2 P02224.2 P02231.1 P02228.1 AAU43733.1 P84160.1 P84159.1 CAT23765.1 P84161.1 CAH03799.1 ADK47394.1 ABQ59329.1 CAQ72970.1 CAQ72971.1 CAQ72972.1 AAK67491.1 AAK67492.1 ACF19589.1 ABC88428.1 AGL34968.1 ADH10372.1 AGL34967.1 CAB39376.1 CAA50325.1 CAA50326.1 CAA50328.1 CAA96548.1 CAA96549.1 AAD48405.1 AAG40329.1 AAG40330.1 AAG40331.1 CAA50327.1 AAL86739.1 AAO67349.2 AAO65960.1 ACO56333.1 AAK01235.1 AAK01236.1 A4KA41.1 A4KA40.1 A4KA44.1 A4KA43.1 A4KA45.1 A4KA39.1 AAK28533.1 AAL73404.1 AHA36627.1 ACR43473.1 ACR43474.1 ACR43475.1 ACR43477.1 ACR43478.1 ACR43476.1 BAH10152.1 ARX70262.1 AAC61869.1 AAW81034.1 BAD77932.1 BAA05543.1 BAA05542.1 BAA07020.1 P43212.1 BAC23082.1 BAC23083.1 BAC23084.1 BAF32105.1 BAF32110.1 BAF32116.1 BAF32119.1 BAF32122.1 BAF32128.1 BAF32130.1 BAF32133.1 BAF32134.1 BAA06172.1 BAF45320.1 AAK27264.1 BAT94503.1 BAJ04354.1 BAF51970.1 BAA06905.1 CAD92666.1 AAW69549.1 P83834.1 ACB45874.1 AAP13533.2 CAB62551.1 CAC37790.2 ABK78766.1 ACY01951.1 CAC05258.1 AAF72625.1 AAF72626.1 AAF72627.1 AAF72628.1 AAF72629.1 AAR21074.1 AAR21073.1 AAB28566.1 AAB28567.1 AAB32317.1 AAF80379.2 AAK96255.1 AAL14077.1 AAL14078.1 AAL14079.1 AAB50734.2 CAA69670.1 CAA01909.1 CAA01910.1 CAA62634.1 AAS02108.1 CAC83658.1 CAC83659.1 CAD20406.1 AAP96759.1 2103117A CAA10345.1 AAB42200.1 P82946.1 AAK62278.1 CAD20405.1 AEY79726.1 AAB01092.1 BAA13604.1 CAB03715.1 CAB03716.1 CAB06416.1 AAL76932.1 BAB88129.1 ADL32660.1 ADL32661.1 ADL32662.1 ADL32663.1 ADL32664.1 ADL32665.1 ADL32666.1 AAL76933.1 AEY79728.1 AEY79727.1 CAA55072.2 CAA55067.2 CAA55070.1 P42040.2 CAA55068.1 AAO91801.1 AAX14379.1 P40918.1 CAD42710.1 ABA42918.1 CAD38166.1 ATT08931.1 L7UZ85.1 AAP35078.1 AAD52672.1 AAM64112.1 AAP57094.1 ABU97470.1 ATO08850.1 AGT78542.1 AGC56216.1 ATO08860.1 AAP35082.1 AIO08851.1 AGC56218.1 AIO08848.1 AAP35065.1 AGC56219.1 AIO08870.1 AIO08861.1 BAX34757.1 BAE45865.1 AAP35068.1 ABO84970.1 ABO84971.1 ABO84972.1 ABO84973.1 P16311.2 BAC53948.1 ABA39436.1 ABU49605.1 AAP35075.1 AFJ68066.1 ADM52184.1 ABL84749.1 ABL84750.1 ABL84751.1 BAA04557.1 AAK39511.1 ATO08864.1 P39673.1 BAA04558.1 BAA01240.1 BAA01241.1

- 23 045699

AAL47677.1 CAI05850.1 CAI05849.1 CAI05848.1 AVA39438.1 BAD74060.2 AAR35073.1 AFJ68072.1 BAA01239.1 ABN14313.1 AAA99805.1 ABY28115.1 ASK76291.1 ASK76292.1 VAA09920.1 AAV27594.1 ACK76296.1 ACK76297.1 AAF28423.1 AAP35077.1 ASK76299.1 ATO08853.1 AAM19082.1 AVO84963.1 ABO84964.1 ABO84966.1 AVO84967.1 AVO84968.1 AVO84969.1 ANS94806.1 BAV90601.1 ANH03180.1 ATP86946.1 AIP86945.1 ATP86944.1 ATP86943.1 ATP86942.1 ATP86941.1 ATP86940.1 ATP86939.1 AJF93907.1 AAP35080.1 ATO08867.1 ATO08866.1 R16312.1 ATT08932.1 ΑΑΥ84565.1 ΑΑΥ84564.2 ACD50950.1 ALA65345.1 AAG02250.1 CAD38361.1 CAD38362.1 CAD38363.1 CAD38364.1 CAD38365.1 CAD38366.1 CAD38367.1 CAD38368.1 CAD38369.1 CAD38370.1 CAD38371.1 AAX47076.1 2AS8 V ABV66255.1 3F5V V ACG58378.1 CAQ68250.1 AAA28296.1 AAV60215.1 AFJ68065.1 AVA39435.1 AAB69424.1 CAA75141.1 АВВ52642.1 AST32128.1 AAO73464.1 ADK92390.1 AAM21322.1 1KTJ A CAD38372.1 CAD38373.1 CAD38374.1 CAD38375.1 CAD38376.1 CAD38377.1 CAD38378.1 CAD38379.1 CAD38381.1 CAD38382.1 CAD38383.1 AVA39437.1 SAC22338.1 ABG76196.1 1A9V A ΑΒΥ53034.1 AAF86462.1 CAQ68249.1 AFJ68070.1 AFJ68067.1 ABC73706.1 ASV46292.1 4ZCE A ALA22869.1 ALA22868.1 AAA19973.1 AAD38942.1 R49274.1 AAV32842.1 CAD69036.1 CAA35692.1 R49277.1 AAA80264.1 SAS09234.1 AAV35977.1 AAV32224.1 AAX37326.1 ΑΑΥ84563.1 ABC96702.1 AAA28303.1 P53357.1 CAA47341.1 AAA68279.1 AAA28301.1 AAA28302.1 Р83340.1 AAC48691.1 R81216.1 P81217.1 CAA52194.1 AAM09530.3 BAF47268.1 BAF47269.1 AAO73305.1 AVO71783.1 BAF76431.1 BAF76430.1 AAC82351.1 AAC82352.1 AAC82350.1 AAC82349.1 VAK09233.1 VAK09232.1 VAV79444.1 BAO50872.1 VAO50870.1 AAX57578.1 ABC18306.1 023878.1 023880.1 Q9XFM4.1 ABQ10638.1 BAT21117.1 AVO93594.1 ADW27428.1 AVT32184.1 ACJ23865.1 ACJ23864.1 ACJ23866.1 ΑΑΖ76743.1 CAA44343.1 CAA44344.1 R30438.2 AAC37318.1 ΝΡ_00104161 8.1 CAA44345.1 AAC41616.1 CAA59279.1 AAL49391.1 AAS77253.1 ADK56160.1 ADM15668.1 AAS98889.1 AAS98890.1 AGT20779.1 AEM89226.1 ACD65080.1 ACD65081.1 CAJ85646.1 CAJ85644.1 CAJ85642.1 CAJ85641.1 ABD39049.1 ACX47057.1 ACX47058.1 4С9С В SAS86258.1 AAY83342.1 ΑΑΥ83341.1 ΑΑΥ83345.1 AHL24661.1 AHL24660.1 AAQ83588.1 AAV74343.1 AAQ08947.1 VAN10153.1 ΑΑΝ73248.1 AAL79930.1 AAL79931.1 ΑΗΥ02994.1 P02622.1 AAK63086.1 AAK63087.1 SAM56785.1 SAM56786.1 B3A0L6.1 Р86980.1 ΝΡ 990450.1 P01005.1 ACJ04729.1 CAA23681.1 P01012.2 CAA23682.1 1JTI A 1UHG D CAA26040.1 P02789.2 P00698.1 AAA48944.1 CAA23711.1 CAA43098.1 VAA13973.1 P02604.3 SAH32963.1 ADD18879.1 ADD19985.1 ADD19989.1 AAF82096.1 ACS49840.1 R24337.1 SAA11755.1 ABU97472.1 CAA11756.1 CAA42646.1 CAA35691.1 AAA33947.1 VAA23360.2 AAV01374.1 VAV64303.1 VAA74452.2 BAB64306.1 R25974.1 CAA26723.1 AAA33966.1 SAA2 657 5.1 VAA00154.1 CAA33217.1 CAA37044.1 CAA26478.1 VAA74953.1 AAA33964.1 AAA33965.1 VAV15802.1 AAD09630.1 ΝΡ_00123844 3.1 ACD36976.1 ACD36975.1 ACD36974.1 ACD36978.1 VAV21619.2 R22895.1 AAV09252.1 VAA25899.1 R82947.1 CAA45777.1 CAA45778.1 AAV23464.1 AAV23482.1 AAV23483.1 CAA56343.1 CAA60533.1 CAB59976.1 CAB76459.1 AAQ54603.1 VAN10148.1 BAJ61596.1 AAG08987.1 APG42675.1 CAA75506.1 AAP47226.1 P23110.1 CAB38044.1 CAA39880.1 AAA16792.1 CAB53458.1 SAS13961.1 SAS42881.1 AAL25839.1 AAP37470.1 ADR82196.1 CCW27997.1 AAA87456.1 AAP87281.1 ΑΒΝ03965.1 ΑΒΝ03966.1 ΑΒΝ09653.1 ABN09654.1 ABN09655.1 ACY91851.1 ACZ74626.1 AEV41413.1 AFJ97275.1 AFJ97274.1 AAC82355.1 AAR98518.1 AAS49447.1 CAA05978.1 1WKX A ABW34946.1 AAS27724.1 CAA11041.1 CAA11042.1 AAF25553.1 CAE85467.1 CAA75312.1 1G5U A AAF34341.1 AAF34342.1 AAF34343.1 SAV51914.1 CAB96215.1 SAS00532.1 Q9LET9.1 CAD24068.1 CAA81610.1 CAA93121.1 CAA10140.1 Q7M262 CAB10766.1 CAB10765.1 AAG42255.1 AAC48288.1 AAS48287.1 R32936.2 Р80198.1 CAA51204.1 CAA42832.1 AAA32970.1 SAAZ 518 8.1 CAA08836.1 CAA41956.1 CAA49555.1 CAA45085.1 CAA46705.1 AAP94213.1 AAR15200.1 AAR15199.1 AAM54365.1 AAM54366.1 APR62629.1 AAV41308.1 AAF18269.1 ACI47547.1 AAW29810.1 SAS05582.1 R81295.1 AAD03608.1 SAS48400.1 AAS15474.2 AAR21072.1 AAR21071.1 Q9LD79.2 AAF80164.1 AAF80166.1 AAV97933.1 AAT45383.1 AAX35807.1 CAD87730.1 CAD87731.1 AAQ55550.1 SAV71342.1 SAV62213.1 CAD32313.1 CAD32314.1 2118249B 2118249A AAQ73484.1 AAQ73486.1 AAQ73487.1 AAQ73488.1 AAQ73489.1 AAQ73490.1 AAQ73491.1 AAQ73492.1 CAA57160.1 CAA58755.1 AAQ73493.1 AAQ73494.1 SAV62212.1 CAB65963.1 CAP17694.1 SAS84590.2 CAC84593.2 SAA5 4 818.1 SAA5 4 819.1 AAZ91659.1 BAW03243.1 BAW03242.1 AAL07320.1 ABC02750.1 ACM89179.1 ASV38288.1 AVT98020.1 ACC76803.1 R14946.2 AAA63278.1 AAA63279.1 CAB63699.1 Q7M1X5.1 R14947.1 CAA51775.1 R14948.1 SAN92637.1 AAD20386.1 CAB64344.1 AAA33405.1 Q40240.2 CAT84850.2 Q53HY0.2 Q6EBC1.1 ABR21771.1 ABR21772.1 ASV05815.1 F5B8W5.1 F5B8W4.1 F5B8W3.1 F5B8W2.1 F5B8W1.1 F5B8W0.1 F5B8V9.1 Β3Α0Ν2.1 ADC55380.1 AHA85706.1 Р86739.1 R86741.1 Р86740.1 R86742.1 VAA32435.1 VAA32436.1 AAD25927.1 CAA65341.1 CAD20981.3 CAD68071.1 CAT43283.4 CAA09883.1 CAA09884.1 CAA09885.1 CAA09886.2 CAA09887.4 CCU97864.1 CCV00099.1 CCU98198.1 CCU99457.1 SHO79205.1 CCU99206.1 CAA96534.1 CAA96535.1 CAA96536.1 CAA96537.1 AAD13683.1 AAD26546.1 AAD26547.1 AAD26548.1 AAD26552.1 AAD2 65 53.1 AAD26554.1 AAD2 65 55.1 AAD26558.1 CAD32318.1 AAO25113.1 AAD29671.1 AAV01362.1

- 24 045699

CAA88833.1 CAA58646.1 AAK13029.1 AAK13030.1 AAK13027.1 AAB35897.1 AAX19848.1 AAX19851.1 Q9FSG7.1 CAT99612.1 CAT99611.1 AFM77001.1 AAC36740.1 029330.1 AAT80665.1 AAT80664.1 AAT80662.1 AAT80659.1 AAT80649.1 AAR22488.1 Q9M5X7.1 CAD46559.1 CAD46561.1 CAD46560.1 AAX19854.1 AAX19856.1 AAX19858.1 AAX19860.1 CAK93713.1 CAK93753.1 CAK93757.1 CAT99618.1 CAT99619.1 CAT99617.1 AAD29412.1 AAD29413.1 AAD29414.1 AAM55492.1 AEE98392.1 B3EWS0.1 B3EWE5.3 G5DC91.2 BAF47263.1 AGF86397.1 CAA73720.1 P86745.1 P86749.1 P86750.1 P86752.1 P86753.1 P86754.1 P86757.1 P86761.1 P86760.1 P02620.1 P86765.1 P86768.1 P86769.1 P86770.1 P86771.1 P86772.1 P86774.1 P86775.1 AAD55792.2 Q99MG7.1 AAA60330.1 AAG08989.1 AHW81906.1 AAV33670.1 AAV33672.1 P85894.1 P02762.2 CAA26953.1 A2BTM8.1 AAA39768.1 AAK54834.1 2CYG A 1Z3QA GAG 81811.1 AAB82772.2 BAD36780.1 AAB50883.1 CAA49760.1 2206305A AAB36316.1 BAH10150.1 CAE17317.1 CAE17316.l· BAE54433.1 P19963.2 T53806 E53806 F53806 C53806 A38968 G53806 B53806 H53806 CAA7 3 0 3 8.1 CAA73037.1 CAA73036.1 AAB32652.2 AAO22133.1 AAO22132.1 AAN18044.1 AAQ10281.1 AAQ10280.1 AAQ10279.1 AAQ10278.1 AAQ10277.1 AAQ10276.1 AAQ10274.1 AAQ10271.1 AAQ10268.1 AAQ08190.1 ABP58632.1 ABP58633.1 ABP58635.1 ABP58636.1 ABP58637.1 AAL92578.1 AAY88919.1 ACZ57582.1 E1U332.1 E3SU11.1 024170.1 024171.1 A4GFC0.1 A4GFC3.1 CAA73035.1 AAD05375.1 AAO33897.1 P80740.2 CAD21706.2 ABP58627.1 ABX26131.1 ABX26132.1 ABX26134.1 ABX26138.1 ABX26139.1 ABX26140.1 ABX26141.1 ABX26143.1 ABX26145.1 ABX26147.1 ABX54842.1 ABX54844.1 ABX54849.1 ABX54855.1 ABX54859.1 ABX54862.1 ABX54864.1 ABX54866.1 ABX54869.1 ABX54876.1 ABX54877.1 AAB66909.1 P81430.2 AAF31152.1 AAF31151.1 AAK58515.1 2 JON A BAE54432.1 Q25632.1 BAJ07603.1 P86431.1 P86432.1 BAF95206.1 AFV53352.1 AAG42806.1 AAG42802.1 Q948T6.2 AAA86533.1 AAF72991.1 BAB71741.1 Q40638.2 BAD13150.1 BAC20657.1 BAA01998.1 BAA01996.1 BAA07772.1 BAA07773.1 BAA07774.1 BAA07710.1 BAA07711.1 BAA07712.1 BAA07713.1 AAB99797.1 Q01882.2 Q01883.2 BAC19997.1 BAC20650.1 ADK39021.1 ACA96507.1 CBY17558.1 AAC38996.1 BAF47265.1 BAF47266.1 2008179A CAA65123.1 CAA54587.1 CAT94601.1 CAA59370.1 CAA65122.1 P55958.1 Q9T0M8.1 Q9XG85.1 CCP19647.1 CAP05019.1 Q7M1E8 AAB36008.1 AAB36009.1 AAB36010.1 AAB36011.1 AAB36012.1 AAB46820.1 AAB46819.1 AKF12278.1 CBM42667.1 CBM42666.1 CBM42665.1 CBM42664.1 CBM42663.1 CBM42662.1 CBM42661.1 CBM42660.1 ACA23876.1 AAX37288.1 AAO15713.1 C7E3T4.1 ADV17342.1 ADV17343.1 AAX11194.1 AAF71379.1 AAG44693.2 AAF23726.1 AAM33821.1 AAB34785.1 ADK27483.1 AAD25995.1 AAG44480.1 Q92260.1 AAK51201.1 AAR17475.1 AAD42074.1 ABB89950.1 ABM60783.1 AAD25926.1 AEX34122.1 AAG44478.1 AKH04310.1 AKH04311.1 AAX33729.1 AEV23867.1 AAD19606.1 CAB38086.1 ACS14052.1 AAC34736.1 AAC34737.1 AAB82404.1 AAC34312.1 AAD13533.1 AAP13554.1 ADB92492.1 AAX33734.1 AAX33727.1 ADR82198.1 AAB09632.1 AAB62731.1 AAB63595.1 Q25641.1 ADB92493.1 ADD17628.1 AAX33728.1 3EBW A ACJ37391.1 AAX33730.1 AAT77152.1 ACA00204.1 AAL86701.1 AAG08988.1 CAB01591.1 AAB27445.1 Q41260.1 P56164.1 P56165.1 P56166.1 P56167.1 ADC80502.1 ADC80503.1 CAA55390.1 CAA81613.1 1N10A CAG24374.1 2118271A AAN32987.1 CAA7 0 6 0 9.1 ABG81289.1 ABG81290.1 ABG81291.1 ABG81292.1 ABG81293.1 ABG81294.1 ABG81295.1 CAA7 0 6 0 8.1 CAA54686.1 CAB42886.1 CAA53529.1 CAD54670.2 CAF32567.2 CAF32566.2 CAQ55938.1 CAQ55939.1 CAQ55940.1 CAQ55941.1 3TSH A CAD54671.2 CAA52753.1 S32101 S38584 Q7M1L8 2023228A CAB05371.1 CAB05372.1 CAA50281.1 AAC16525.1 AAC16526.1 AAC16527.1 AAC16528.1 AAC25994.1 AAC25995.1 AAC25997.1 AAC25998.1 AAK25823.1 CAD38384.1 CAD38385.1 CAD38386.1 CAD38387.1 CAD38388.1 CAD38389.1 CAD38390.1 CAD38391.1 CAD38392.1 CAD38393.1 CAD38394.1 CAD38395.1 CAD38396.1 CAD38397.1 1L3P A CAD87529.1 CAA81609.1 CCD28287.1 CAA7 6556.1 CAA7 6557.1 CAA7 65 5 8.1 1NLX N CAA76887.1 3FT1 A AGT28425.1 CAD10390.1 AHC94918.1 CEJ95862.1 CTQ87571.1 ABU42022.1 ABG73109.1 ABG73110.1 ABG73108.1 ABO36677.1 ABR29644.1 CAF25233.1 CAF25232.1 CAB82855.1 AJG44053.1 A0A158V755. 1 A0A158V976. 1 2Ν81_A CAC41633.1 CAC41634.1 CAC41635.1 CAD80019.1 ABY21305.1 ABY21306.1 ALF39466.1 ALF00099.1 CAD20556.1 CAE52833.1 CAC85911.1 CBW45298.1 A60372 F37396 CAA10520.1 AAG42254.1 P22284.1 P22286.1 A60373 P22285.1 AAA29793.1 AAD52615.1 AAD52616.1 AAT95010.1 AAS67044.1 AAS67043.1 AAS67042.1 AAS67041.1 AAP37412.1 AAT95009.1 P35780.1 P83377.1 P83542.1 A2VBC4.1 ADT89774.1 ADL09135.1 P86687.1 ADD63684.1 P86686.1 Q7Z156.2 P05946.1 AGE44125.1 ABL89183.1 ABS12234.1 AFA45339.1 ACN87223.1 AKV72167.1 AHY24177.1 BAH59276.1 AAB97141.1 ADR66945.1 ADR66946.1 ADR66947.1 ADR66948.1 AAC02632.1 AAS47037.1 AAS47036.1 AAS47035.1 1H2O A AAF26449.1 ADR66943.1 ADR66944.1 AAD29411.1 AAB38064.1 P82534.1 ACE80974.1 AAL91662.1 3EHK A AGR27935.1 ADN39440.1 ADN39441.1 P82952.1 ACE80939.1 ACE80956.1 ACE80958.1 ACE80957.1 ACE80959.1 ACE80955.1 ACE80972.1 P83332.1 P83335.1 AEV57471.1 ABB78006.1 AJE61291.1 AJE61290.1 P81402.1 AAV40850.1 ADR66939.1 AGW21344.1 CAD37201.1 CAD37202.1 P86888.1 BAH10154.1 COHKCO.1 AHB19227.1 AHB19226.1 AHB19225.1 AAF26451.1 AET05733.1 AET05732.1 AET05730.1 065200.1 AAD29410.1 AAC24001.1

- 25 045699

ABZ81045.1 ABZ81047.1 ABZ81046.1 SAS83046.1 SAS95152.1 SAS83047.1 CAC95153.1 P02761.1 Q63213 AAA41198.1 ATS82657.1 AAP30720.1 AAT37679.1 CAA38097.1 ABG54495.1 ABG54494.1 Q91483.3 AS T 6 810 3.1 CAA66403.1 CBL79146.1 ASN70931.1 CBL79147.1 ΝΡ_00113318 eleven AHL24657.1 ARS33724.1 AAT99258.1 AAX11261.1 AAX11262.1 ASO34813.1 Р83181.1 ASO34814.1 ACS34771.1 AHL24658.1 ADK22841.1 ADK22842.1 SAH32966.1 SAH32967.1 SHD75397.1 AAO15613.1 AAS93669.1 AAS93674.1 AAS93675.1 AAS93676.1 AAO15607.1 AAX37321.1 AGM48615.1 CAQ68366.1 VAN10151.1 Q7M1Y1 S37396 D37396 AAR06493.1 AAC67308.1 ХР_00303059 eleven BAW32538.1 BAW32537.1 BAW32536.1 BAW32535.1 VAS66618.1 SAH32965.1 AFA45340.1 AFJ80778.1 ABS12233.1 CAQ72968.1 CAQ72969.1 AAV37403.1 AAV37406.1 AAV34365.1 SAN92630.1 SAN92627.1 Q7M263 CBG76811.1 BAE54429.1 BAE54430.1 ASV55491.1 AAK15088.1 AST41244.1 AAD42943.1 AAK15089.1 AAG23840.1 ASN85188.1 AAD42942.1 AAD42944.1 AAK15087.1 CAA62909.1 CAA62910.1 CAA62911.1 CAA62912.1 CAA62908.1 R15322.2 AAX77383.1 AAX77384.1 ABU95411.1 ABU95412.1 ABU53681.1 NP-00130688 3. 1 NP_00131612 3.1 CAD10377.1 AAL29690.1 AAL75449.1 AAL75450.1 CAJ19705.1 AAV42069.1 CAA75803.1 ANS08074.1 ANS08073.1 ABA81885.1 АВВ16985.1 CAA31575.1 CAA27571.1 CAA27588.1 AAA33819.1 R15476.2 R16348.1 R20347.3 AAV63099.1 VAA04149.1 VAN10156.1 AAF65312.1 AAF65313.1 AAS97370.1 AAC97369.1 AAV36117.1 AAV36119.1 AAV36120.1 AAV36121.1 AAT95008.1 R35775.1 AAV65434.1 R35776.2 R35779.2 ADD74392.1 ATL01319.1 ATL01318.1 ATL01316.1 ATL01317.1 ATL01320.1 ATL01321.1 AST37324.1 1ESF B CAJ43561.1 R34071.1 R20723.1 P06886.1 AAT6 65 67.1 ABS29033.1 AAT6 65 66.1 AAD46493.1 AAS75831.1 P00791.3 AAA30988.1 ΝΡ_00100520 8 . 1 R58171.1 S43242 S43243 S43244 ADX78255.1 ADM18346.1 ADM18345.1 ADK47876.1 Р86360.1 CEE03319.1 CEE03318.1 AAK63089.1 AAK63088.1 CBL79145.1 R86978.1 CAX62602.1 R86979.1 BAE54431.1 BAE46763.1 VAN10155.1 AAF07903.2 AAD52013.1 AAD52012.1 Q8J077.1 CAD23374.1 R24296.2 CAA42453.1 ACG59281.1 AKJ77988.1 AKJ77986.1 AKJ77987.1 CAT64398.1 AKJ77990.1 AKJ77985.1 CAA35238.1 CAA25593.1 CAA26383.1 CAA26384.1 CAA26385.1 AAA34275.1 AAA34276.1 AAA34279.1 AAA34280.1 AAA34281.1 AAA34282.1 AAA34283.1 AAA34284.1 VAA12318.1 R81496.1 ACE82289.1 BAE20328.1 CAR82265.1 CAR82266.1 CAR82267.1 BAN29067.1 CAT64397.1 CAI64396.1 P08819.2 R27357.1 ACE82291.1 SAA61945.2 SAA61943.2 SAA61944.2 CAQ57979.1 SVA13560.1 AAA34272.1 AAA34274.1 AAA34288.1 AAA34289.1 VAA11251.1 CAT78902.1 BAN29066.1 CAY54134.1 SAV96931.1 CAA43331.1 CAA31396.1 CAA26847.1 CAA24934.1 CAA43361.1 AAV02788.1 CAA27052.1 CAA24933.1 BAN29068.1 CAA31395.4 AAZ23584.1 VAS76688.1 CAT84642.1 CAA35598.1 CAZ76052.1 SVA13559.1 CAA35597.1 SAS14917.1 ACE82290.1 Q6W8Q2.1 CAA72273.1 CAB52710.1 CAZ76054.1 CAA31685.1 CAA30570.1 AAA34285.1 AAA34286.1 AAA34287.1 022116 CAA59338.1 CAA59339.1 CAA59340.1 022108 CAT79052.1 AEN31546.1 BAN29069.1 CAA65313.1 ABS58503.1 R82977.2 SSK33471.1 APY24042.1 CAA34709.1 CAA39099.1 CAA36063.1 CAA44473.1 AAA34290.1 AAX34057.1 AAX34058.1 AAX34059.1 AOD75395.1 AOD75396.1 AOD75399.1 ABQ96644.1 ABU97479.1 AAT40866.1 AAU11502.1 AVM53751.1 ABU97480.1 SAA73221.1 ACL36923.1 ABZ81991.1 AGG10560.1 AAT66607.1 AAT66609.1 ASN42744.1 AAT66610.1 ACJ65 836.1 AGC36415.1 ASN42743.1 AST44002.1 ABQ59259.1 ABQ59258.1 ABQ59255.1 ACJ54737.1 ASN42741.1 AGC36416.1 AKV72166.1 ATV43662.1 VAN10157.1 P0DMB5.1 P0DMB4.1 Р0СН87.1 R35781.1 R35782.1 CBY83816.1 CBY93636.1 R81657.1 R35783.1 CAJ28931.1 R35784.1 CAJ28930.1 CAL59818.1 CAL59819.1 R51528.1 R35760.1 ABC73068.1 Р0СН89.1 R35785.1 R35786.1 Р0СН86.1 R35787.1 AAV48072.1 AAAZZZZ.1 SAV42887.1 1QNX A R49370.1 CAI77218.1 2ATM A ACA00159.1 AAX19889.1 ABG02262.1 ABW23574.1 VAA74451.1 SAA5 0 0 0 8.1 R80273.2 R80274.1 R33556.1 CAR48256.1 ABD79096.1 ABD79097.1 ABD79098.1 ACX37090.1 R29022.1 2209273A AAO45607.1 AAO45608.1 AAK56124.1 2HCZ X ABD79094.1 ABD79095.1 ABF81661.1 ABF81662.1 Q1ZYQ8.2 P0C1Y5.1 AAV86960.1 ABG81312.1 ABG81313.1 ABG81314.1 ABG81315.1 ABG81316.1 ABG81317.1 ABG81318.1 CAA51718.1 CAA51719.1 CAA51720.1 AAG35601.1 5FEF A AAA33493.1 AAA33494.1 CAT64400.1 AAX40948.1

Table 7

List of accession numbers for autoimmune antigens from the IEDB

I7HKY1.1 Q9P0J1.1 R61604.1 Q9NUQ2.1 Q9P212.1 R16885.1 P09543.1 R17980.1 Q9 94 60.1 000231.1 000487.1 R48556.1 Q61733.1 R82909.1 R21953.1 Q9CHK3.1 Q9BYD6.1 Q9BYC9.1 Q96A35.1 Q9P0J6.1 P04035.1 Q99714.1 B2RLH8.1 R62277.1 P08708.1 R62269.1 R63220.1 R62851.1 R62273.1 R62861.1 R46781.1

- 26 045699

P08865.1 P17643.1 Q9H0D6.1 F5HCM1.1 E5RK45.1 A0A0B7JKK9 . 1 A1JIP3.1 B2RKS6.1 P0A6F5.1 P0C0Z7.1 Q49375.1 Q9Z708.1 Ρ0Α521.1 Ρ42384.1 Ρ0Α520.1 P9WPE7.1 P10809.1 P10155.1 Ρ05388.1 Ρ05386.1 Ρ05387.1 Ρ27635.1 Ρ62906.1 Ρ40429.1 P35268.1 A8MUS3.1 Ρ62750.1 Ρ61353.1 Ρ46776.1 Ρ46779.1 Ρ47914.1 Ρ39023.1 P62888.1 Q02878.1 Ρ18124.1 Ρ62917.1 Ρ32969.1 Q6SW59.1 Ρ08253.1 Ρ11021.1 Q969T7.1 Q76LX8.1 C6AV76.1 Q2FWL5.1 B1RDC1.1 Q2G2D8.1 Ρ42684.1 Q8TZT6.1 Q9Y4K1.1 P02709.1 Ρ02710.1 Ρ02711.1 Ρ04756.1 Ρ02708.1 Ρ02712.1 Ρ11230.1 Q07001.1 P02715.1 Q04844.1 Ρ07510.1 Ρ13536.1 F1N690.1 M9YGB9.1 043427.1 P68133.1 P62736.1 Ρ60709.1 Ρ63261.1 Q9NQW6.1 015144.1 Q9H981.1 Q8N3C0.1 Q6VMQ6.1 Q6JQN1.1 Q5T8D3.1 Ρ82987.1 Q6ZMM2.1 Q9NZK5.1 Q8TUX7.1 Q9NP61.1 Q9UJY4.1 043488.1 Ρ07897.1 Ρ16112.1 Q73ZL3.1 Q92667.1 Ρ49588.1 C9JKR2.1 F8ELD9.1 P15121.1 F5HF49.1 Ρ05186.1 Ρ55008.1 Q5STX8.1 Ρ02763.1 Ρ01009.1 P35368.1 P04217.1 Ρ25100.1 Ρ08697.1 Ρ18825.1 Ρ02765.1 Ρ01023.1 Ρ12814.1 043707.1 P35611.1 Q9UBT7.1 Ρ61163.1 Ρ02489.1 Ρ02511.1 Ρ06733.1 Ρ06280.1 Q16352.1 Q96Q83.1 Ρ37840.1 Q9UJX4.1 Ρ01019.1 Q9P2G1.1 Q9H8Y5.1 Q8N6D5.1 H0YKS4.1 P04083.1 Ρ50995.1 Ρ07355.1 Ρ08758.1 Ρ08133.1 Q9NQ90.1 Q03518.1 P01008.1 Q10567.1 Q9BXS5.1 Q96CW1.1 000203.1 Ρ02647.1 Ρ02652.1 Ρ06727.1 P04114.1 P02655.1 C9JX71.1 Ρ05090.1 Ρ02649.1 Q9BZR8.1 Ρ03182.1 Q9BRQ8.1 Q9ATL6.1 P47863.1 Ρ55087.1 Ρ55064.1 Ρ20292.1 Q15057.1 Q96P48.1 Ρ35869.1 Q5VUY2.1 P03928.1 Ρ25705.1 Ρ06576.1 Ρ56385.1 Q9DB20.1 Ρ18859.1 Q9BZC7.1 Q8WWZ7.1 Q9NUT2.1 Ρ61221.1 Ρ53396.1 A1JNN2.1 P0A6G7.1 Q9H2U1.1 Q14562.1 084848.1 P78508.1 Q99712.1 Ρ17342.1 Q99856.1 Q8TVW6.1 Q96GD4.1 Q8WXX7.1 015392.1 P02730.1 Ρ98160.1 F8W034.1 Ρ20749.1 Ρ41182.1 Q9NYF8.1 Q6W2J9.1 Q8NFU0.1 P15291.1 Ρ07550.1 Ρ02749.1 Ρ61769.1 Q13425.1 Q562R1.1 Ρ42025.1 P13929.1 F0K2P6.1 043252.1 Q13057.1 Q8TUF8.1 Q8NFC6.1 Ρ18577.1 Q5VSJ8.1 Q02161.1 P02663.1 Ρ02769.1 Q9NWK9.1 095415.1 Q7Z569.1 Q99728.1 Q9P287.1 Q9NRL2.1 Q9UTF9.1 Q58F21.1 Ρ25440.1 Q15059.1 060885.1 Ρ18892.1 Q8NCU7.1 P04003.1 075844.1 Ρ12830.1 Ρ33151.1 Q8NE86.1 Ρ62158.1 Ρ07384.1 Ρ17655.1 P20810.1 P27797.1 094985.1 Ρ10644.1 Ρ31321.1 Ρ13861.1 070739.1 Q8QVL3.1 Q8QVL6.1 Q8QVL9.1 Q91CY5.1 Q91CZ6.1 Q98Y63.1 Q99AQ9.1 Q9DTD4.1 Q9DUB7.1 Q9DUC1.1 Q9JG76.1 Q9QU30.1 Q9QUB8.1 Q80AR5.1 Q80QT8.1 Q8UZK7.1 Ρ14348.1 Q9H2A9.1 P00918.1 Ρ16870.1 075339.1 015519.1 Q14790.1 Ρ04040.1 Ρ35221.1 P49913.1 P07858.1 Ρ07339.1 Ρ25774.1 Q03135.1 Q16663.1 Q9H9A5.1 Q9Y5K6.1 P09326.1 P14209.1 Q99741.1 000311.1 075794.1 Ρ04637.1 B2RD01.1 Q03188.1 P49454.1 Q9HC77.1 Q02224.1 Ρ00450.1 Ρ08622.1 Ρ35514.1 Q05980.1 P9WMJ9.1 Q9H444.1 P36222.1 000299.1 Ρ05108.1 015335.1 Q6UVK1.1 Q9P2D1.1 Ρ10645.1 075390.1 014503.1 Q00610.1 Ρ09497.1 075508.1 Ρ56750.1 Q9P2T0.1 Q7Z460.1 075122.1 075153.1 Ρ10909.1 Q7Z401.1 Ρ00451.1 Ρ00488.1 Ρ48444.1 Ρ61923.1 E9PP50.1 P23528.1 Q8WUD4.1 Q49A88.1 Q16204.1 Ρ38432.1 Ρ02452.1 Ρ02458.1 P05539.1 P02462.1 G1K238.1 Q7STB2.1 Ρ20908.1 Q02388.1 Ρ27658.1 Ρ12107.1 Q99715.1 Q05707.1 Ρ39059.1 Q9UMD9.1 Ρ08123.1 Ρ08572.1 Q7STB3.1 Ρ05997.1 P12110.1 P13942.1 F1MZU6.1 Q01955.1 Ρ12111.1 Ρ02745.1 Ρ02746.1 Ρ09871.1 P01024.1 P0C0L5.1 Ρ01031.1 Q07021.1 Ρ13671.1 Ρ02748.1 Ρ08603.1 Q03591.1 Q6PUV4.1 W1Q7Z5.1 Q15021.1 Q15003.1 Ρ42695.1 Q14746.1 Q9NZB2.1 Q12860.1 Q02246.1 P78357.1 Q9UBW8.1 Ρ36717.1 Ρ02741.1 Ρ12277.1 Ρ06732.1 H0Y8U5.1 Q13618.1 Q86VP6.1 Ρ25024.1 Ρ16220.1 Ρ06493.1 Ρ11802.1 Q00534.1 Ρ50750.1 P41002.1 P04080.1 Ρ50238.1 Ρ52943.1 014957.1 Ρ20674.1 Ρ10606.1 Ρ14854.1 P15954.1 P10176.1 Q16678.1 Ρ10635.1 Q14008.1 Q9Y5Y2.1 Q96KP4.1 Ρ14416.1 Q5QP82.1 P07585.1 E5RFJ0.1 Q86SQ9.1 Q9Y394.1 Ρ49366.1 Q5QJE6.1 Ρ24855.1 Q02413.1 P32926.1 Ρ15924.1 Q16760.1 Ρ19572.1 A9NHS5.1 Q9JZ09.1 Ρ06959.1 P08461.1 P10515.1 Ρ20285.1 P0AFG6.1 Q5F875.1 Ρ19262.1 Ρ36957.1 Q16555.1 P53634.1 Q14689.1 Q13443.1 Q12959.1 Q15398.1 Q16531.1 Ρ40692.1 Ρ43246.1 P09884.1 P03198.1 Ρ04293.1 Q9NRF9.1 Q9UGP5.1 Ρ89471.1 Q13426.1 Ρ49736.1 P33992.1 P11387.1 Q02880.1 Q9UBZ4.1 Ρ24928.1 014802.1 Q9NW08.1 Ρ31689.1 P25686.1 060216.1 095793.1 Ρ55265.1 Q6P0N6.1 Q13202.1 Q8TVF4.1 Ε9ΡΕΒ9.1 Q9UTT4.1 Ρ11161.1 Q14258.1 Q9ULT8.1 095714.1 Q7Z6Z7.1 Q9Y4L5.1 043567.1 Q63HN8.1 Q969K3.1 Q8TUQ4.1 P19474.1 Q6AZZ1.1 Q9C026.1 Q14669.1 Q5T4S7.1 P18146.1 Q05BV3.1 Q6ZMW3.1 095967.1 Ρ15502.1 Q9BY07.1 Ρ13804.1 Q6PJG2.1 A6PW80.1 Ρ68104.1 Ρ13639.1 Q96RP9.1 Q9BW60.1 Q9UT08.1 Ρ17813.1 Q9NZ08.1 P14625.1 Q14511.1 Q6P2E9.1 B2RLL7.1 084591.1 Q9Z7A6.1 Ρ03188.1 Ρ04578.1

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P14075.1 Q6SW67.1 Q92817.1 R12724.1 Q12929.1 R61916.1 P07099.1 Ρ03211.1 P12978.1 P12977.1 P03203.1 P03204.1 Q99808.1 R27105.1 P03372.1 Ρ32519.1 Q15723.1 P60842.1 Q14240.1 R38919.1 R41567.1 Q14152.1 V5ME19.1 Ρ60228.1 075821.1 Q13347.1 Q9Y262.1 F1TTN3.1 Q96KP1.1 Q9 6Ά65.1 084646.1 Q01780.1 P30822.1 014980.1 R41180.1 R15311.1 Q08945.1 R52907.1 Q9BXW9.1 Q14296.1 Q16658.1 Q7L8L6.1 Q7L5A8.1 R49327.1 Q8TX29.1 Q8TB52.1 Q7Z6M2.1 Q7L513.1 Q9BZ67.1 A1ZL39.1 P02792.1 R35555.1 P02671.1 P02675.1 P02679.1 Q06828.1 P02751.1 Q4ZHG4.1 R20930.1 R21333.1 R30043.1 075955.1 Q14254.1 Ρ49771.1 Q12841.1 Q13461.1 R32314.1 095954.1 P04075.1 P09972.1 P07954.1 Q9H0Q3.1 Q7Z6J4.1 P30279.1 R30281.1 096020.1 095067.1 R14078.1 R51570.1 Q08380.1 000214.1 Q3B8N2.1 R34903.1 P09104.1 A4D1B5.1 Р17900.1 P06396.1 Q12789.1 Q8WUA4.1 P03300.1 P08292.1 R27958.1 P03995.1 R14136.1 R47871.1 Q8TDQ7.1 P35575.1 Q9NQR9.1 Q9Z186.1 R11413.1 P06744.1 R48318.1 Q99259.1 Ρ48320.1 Q05329.1 Q05683.1 P00367.1 Q05586.1 Q5VSF9.1 Q12879.1 S0G235.1 Ρ15104.1 Q06210.1 R35754.1 R18283.1 P09211.1 P04406.1 Q9NPB8.1 R11216.1 Ρ06737.1 P11217.1 Q31BS5.1 P04921.1 043292.1 R30419.1 D6RB28.1 Q96S52.1 Q969N2.1 Q86SQ4.1 Q9HC97.1 K7EQ05.1 R28799.1 Ρ0Ά6Ρ5.1 R44536.1 Q8WWP7.1 Ρ62826.1 P16520.1 P09471.1 Q9BVP2.1 Q9NVN8.1 P00738.1 Q9Y6N9.1 Q96CS2.1 Ρ48723.1 Q0VDF9.1 P08107.1 R34931.1 R11142.1 P04792.1 P07900.1 Q14568.1 Ρ08238.1 P54652.1 Q15477.1 P03452.1 R69905.1 R68871.1 P02042.1 Ρ69892.1 Ρ02790.1 Q14CZ8.1 P09651.1 Q32P51.1 R14866.1 Q8WVV9.1 043390.1 Q1KMD3.1 088569.1 P22626.1 Q9Y241.1 095263.1 R12314.1 P09429.1 R26583.1 Ρ25021.1 Ρ49773.1 Q9NQE9.1 R12081.1 Q9NVP2.1 Q8WUT4.1 Q9H0E3.1 P07305.1 Q02539.1 Ρ16403.1 P16402.1 R10412.1 R16401.1 P0CE15.1 Q92522.1 P0C0S8.1 P0C0S9.1 Q93077.1 Q9BTM1.1 Q71UI9.1 P0C0S5.1 R16104.1 R62808.1 R33778.1 Ρ62807.1 Ρ10853.1 P06899.1 060814.1 Q99877.1 Q16778.1 Q5QNW6.1 R57053.1 Ρ68431.1 Ρ68432.1 Q16695.1 Q71DT3.1 R49450.1 R62803.1 R62805.1 R62806.1 Q99525.1 Ρ02259.1 Q9NR48.1 P01892.1 P04439.1 R16188.1 R10314.1 P01891.1 Ρ10316.1 Ρ13747.1 P30464.1 P03989.1 R30685.1 R18463.1 Q95365.1 R30480.1 Ρ30484.1 Ρ30486.1 P18464.1 Р30490.1 R30495.1 P01889.1 Q31612.1 R30460.1 Q07000.1 Q29960.1 F8W9Z8.1 Q29963.1 R10321.1 R28068.1 R20036.1 P04440.1 Ρ01909.1 Ρ01906.1 E9PTB1.1 P01920.1 Q5Y7D6.1 P01903.1 R79483.1 R13762.1 Q30154.1 Ρ04229.1 P20039.1 Q95TE3.1 Q5Y7A7.1 P01911.1 Q29974.1 P01912.1 Ρ13760.1 Q9GZN2.1 Q9H2X6.1 Q9H422.1 R51610.1 R50502.1 295441875 . 1 295413967. 1 295413927 . 1 295413946 . 1 295441907 295441886 295413949 312192955 295413970 295413952. 295413922 295413835 . 1 . 1 . 1 . 1 . 1 1 . 1 . 1 295413838 . 1 295413935 . 1 295413976 . 1 P01880.1 Q9Y6R7.1 Q9Y5U9.1 Q5VY09.1 014498.1 P78318.1 000410.1 R11314.1 Q9BY32.1 P01317.1 A6XGL2.1 Ρ01308.1 F8WCM5.1 P01325.1 P01326.1 015503.1 Q13429.1 P01344.1 Q9Y287.1 060478.1 Q8N201.1 P23229.1 Q13349.1 P08514.1 P05106.1 R16144.1 Q9H0C8.1 Q14624.1 Q9UMF0.1 P01562.1 P01563.1 P01574.1 R38484.1 R14316.1 Q15306.1 Q13568.1 Ρ20591.1 P20592.1 Q9BYX4.1 014879.1 Q12905.1 Q12906.1 R42701.1 Q5TF58.1 Q9NZM3.1 P03956.1 Q9Y547.1 Q13099.1 060306.1 084606.1 Q9Y283.1 Ρ10997.1 Q05084.1 Q9P266.1 Q53G59.1 R13645.1 P02533.1 P08779.1 Q04695.1 Ρ05783.1 Ρ35527.1 P04264.1 R35908.1 R12035.1 R48668.1 P08729.1 QO 7 6 66.1 Q96EK5.1 Ρ52732.1 Q96Q89.1 Q99661.1 P01042.1 Q6NY19.1 Q13601.1 Q04760.1 Ρ42166.1 P19137.1 P11047.1 043813.1 P0SS04.1 Р23700.1 R46379.1 Q6SW84.1 Ρ13285.1 075845.1 P40126.1 Q99538.1 R29536.1 P02750.1 Q15345.1 Q8NHL6.1 Q8NHJ6.1 Q6GTX8.1 Q9NPC1.1 Q14847.1 R61968.1 R11182.1 R18428.1 R50851.1 Ρ06858.1 P0A5J0.1 P9WK61.1 Q86W92.1 P05451.1 R23141.1 P07195.1 R31994.1 Ρ31995.1 Ρ01130.1 Q7Z4F1.1 A4QPB2.1 R20132.1 P05455.1 R18627.1 Q13094.1 Ρ01374.1 Q8NHM5.1 060341.1 R10253.1 000754.1 Р10619.1 Q13571.1 R11279.1 Q9UQV4.1 Ρ22897.1 P14174.1 R34810.1 Q8NDA8.1 P06491.1 P07199.1 F5HDQ6.1 Ρ03227.1 Q14764.1 P08392.1 R40925.1 R40926.1 Q8N5Y2.1 Q9ULC4.1 Q96TJ6.1 Η3ΒΤ46.1 Ρ11226.1 Q8WXG6.1 Q92585.1 R43243.1 R50281.1 R51512.1 Q9NPA2.1 Ρ03485.1 Q96RN5.1 Ά6ΖJ87.1 Q99705.1 R40967.1 Q01726.1 Q16655.1 R15529.1 190341000 . 1 F5HB52.1 000562.1 R16035.1 R56192.1 Q9UBB5.1 Q29983.1 Q16891.1 Ρ55082.1 Ρ55083.1 P46821.1 R27816.1 Q9UPY8.1 Q9Y2H9.1 Q504T8.1 Q8N183.1 Ρ03107.1 Ρ26539.1

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P36745.1 P50799.1 Q81023.1 Q8TCT9.1 Q9H2D1.1 060830.1 094826.1 Q8IWA4.1 P28482.1 Q16584.1 043318.1 043683.1 Q9Y3D0.1 P08571.1 E7EWX8.1 Q99549.1 Q04360.1 Q96T58.1 Q8WXT7.1 Q9H8L6.1 P11229.1 P20309.1 Q5VZF2.1 000499.1 P01106.1 P02687.1 P25188.1 P25274.1 P81558.1 F7A0B0.1 P02686.1 P02689.1 P25189.1 P60201.1 P60202.1 P20916.1 Q13875.1 E9PG44.1 Q16653.1 Q5SUK5.1 P24158.1 P41218.1 Q969H8.1 Q8WXC6.1 P05164.1 Q9NPC7.1 Q9H1R3.1 P35749.1 P35579.1 Q09013.1 095248.1 014745.1 084639.1 P15586.1 P54450.1 Q8TXJ6.1 095167.1 095298.1 P19404.1 075251.1 Q6N069.1 Q73WP1.1 Q86VF7.1 Q9BT67.1 075113.1 Q15843.1 Q13564.1 Q8TXH7.1 P58400.1 P58401.1 Q09666.1 P12036.1 P07196.1 P07197.1 Q8NEJ9.1 Q13491.1 P59665.1 P08246.1 Q9Y6K9.1 Q9NV10.1 P43490.1 Q14112.1 Q5JPE7.1 P69849.1 095897.1 Q13253.1 P05114.1 P05204.1 P80272.1 Q15233.1 P29597.1 P23497.1 P08651.1 Q14938.1 Q16236.1 P19838.1 Q6P4R8.1 075694.1 P52948.1 P11654.1 Q8TEM1.1 Q9QY81.1 B4DW92.1 Q9Y6Q9.1 Q9H1E3.1 P67809.1 Q9H8H0.1 P78316.1 000567.1 Q9Y2X3.1 Q9NR30.1 P19338.1 075607.1 Q8NFH5.1 P03466.1 P0C025.1 Q99733.1 Q12830.1 Q96RS6.1 Q9H209.1 A6NMS3.1 P23515.1 Q9HD40.1 295413917 . 1 295413964 . 1 295441897. 1 Q9PWU2.1 P0C675.1 P11926.1 P54368.1 P10451.1 A2T3P5.1 A2T3T2.1 Q8TAD7.1 Q9BXB4.1 Q9UBL9.1 P03262.1 Q96ST3.1 P50897.1 Q8IXS6.1 Q6ZV29.1 Q6ZW49.1 Q9UBV8.1 Q15154.1 060664.1 Q01453.1 060437.1 P32119.1 043808.1 Q13794.1 Q9H2J4.1 Q8TZ21.1 Q92903.1 095674.1 Q9UKL6.1 P04180.1 P30086.1 000329.1 P42356.1 014986.1 P57054.1 095394.1 E4NG02.1 P00558.1 P18669.1 P15259.1 Q96FE7.1 Q9Y263.1 Q13393.1 P26276.1 Q2FZ93.1 B2RTD6.1 Q9Y617.1 P05155.1 P00747.1 025249.1 P13796.1 P07359.1 P16234.1 Q96CS7.1 Q9H7P9.1 Q15149.1 043660.1 Q8TUK5.1 Q6UX71.1 P09874.1 Q460N5.1 Q9UKK3.1 Q15365.1 Q15366.1 Q9BY77.1 A6Q6E9.1 B2RGP7.1 295413956 . 1 T6XH73.1 Q96FM1.1 043525.1 P19156.1 P18434.1 P0CG38.1 Q16633.1 084616.1 075915.1 084647.1 P68950.1 P02545.1 Q6P2Q9.1 043143.1 Q9HCS7.1 Q96TZ0.1 P9WQ27.1 084288.1 Q92841.1 Q15751.1 Q7Z333.1 084419.1 084818.1 B2RJ72.1 Q8N0Y7.1 060312.1 Q9UHA3.1 P89479.1 Q9H3G5.1 Q02809.1 P07737.1 Q8WUM4.1 Q53EL6.1 P49683.1 P12004.1 Q9UQ80.1 Q7Z6L0.1 Q07954.1 P13674.1 C9JTZ6.1 Q9H7Z7.1 P40306.1 P49720.1 P28074.1 060678.1 014744.1 P03189.1 P78543.1 075629.1 084583.1 060888.1 P30101.1 Q14554.1 Q96JJ7.1 P03129.1 Q9H8V3.1 Q96PZ2.1 Q8WU58.1 Q96TP4.1 Q92636.1 Q96JP0.1 Q4ZG55.1 Q9ULT3.1 Q96ST2.1 Q7Z3U7.1 P33215.1 Q8NHV4.1 Q9UFN0.1 060502.1 Q6UWS5.1 Q86U86.1 P23297.1 P60903.1 P06702.1 P04271.1 Q9UPN6.1 Q6PT26.1 Q6ZMD2.1 Q9BVV6.1 P14079.1 Q8WUY1.1 P50616.1 015027.1 Q15436.1 Q15437.1 D4ACF2.1 Q9QJ57.1 Q9QJ42.1 Q70J99.1 Q9GZT5.1 B1AQ67.1 Q9UM07.1 P21980.1 Q92954.1 Q96JQ0.1 Q9JZQ0.1 A6NMY6.1 Q6FDV9.1 Q5VTE0.1 548558395. 1 Q2VTR3.1 Q58FF8.1 Q9HCE1.1 P13985.1 A2RGE9.1 Q8TXJ9.1 Q6P2P2.1 D3HT40.1 P42588.1 56160925. 1 Q53H96.1 P08559.1 H0YD97.1 000330.1 P14618.1 Q9BXR0.1 Q9H974.1 Q9H2M9.1 P35241.1 Q14699.1 P0DJD1.1 Q9BYM8.1 A6NK89.1 P61106.1 B2RHG7.1 P04626.1 Q13546.1 Q92932.1 Q16849.1 P78509.1 P03209.1 P35249.1 P15927.1 P27694.1 075678.1 Q14257.1 Q9NQC3.1 Q9BZR6.1 P10276.1 P10826.1 P49788.1 Q8TC12.1 P10745.1 P02753.1 P52566.1 Q7Z6T6.1 Q9BRR9.1 Q15052.1 Q8TY67.1 P11908.1 Q15418.1 Q9UK32.1 043159.1 Q9ULK6.1 Q7L0R7.1 Q9C0B0.1 Q9H0A0.1 000472.1 P18333.1 Q6PD62.1 Q9NTZ6.1 Q5T481.1 Q96EV2.1 Q9BQ04.1 P35637.1 Q9UKM9.1 P22087.1 Q9Y230.1 P31153.1 Q9NSC2.1 094885.1 Q93084.1 P08168.1 P10523.1 Q9BQB4.1 014828.1 Q13018.1 Q9UHJ6.1 Q9H4L4.1 Q9GZR1.1 Q15019.1 Q14141.1 015270.1 Q92743.1 043464.1 P49842.1 Q9BZL6.1 015075.1 Q96GX5.1 Q8TD19.1 Q13153.1 F5GWT4.1 P63151.1 A6PVN5.1 Q06190.1 P53041.1 Q8N8A2.1 Q13315.1 P49591.1 Q86SQ7.1 P02787.1 P36952.1 Q14140.1 B7WNR0.1 P02768.1 Q9BYB0.1 Q5T123.1 Q9BZZ2.1 P67812.1 Q9BY50.1 P61009.1 P37108.1 P42224.1 Q92783.1 Q96FS4.1 Q9UTB8.1 075094.1 Q55732.1 000193.1 Q7Z3B0.1 P62304.1 P62306.1 P62308.1 P62314.1 P62316.1 P62318.1 P63162.1 P14678.1 P53814.1 Q13573.1 Q63008.1 P05023.1 Q96K37.1 Q9NQZ2.1 Q96L92.1 Q14515.1 Q13813.1 Q01082.1 P63208.1 P21453.1 P23246.1 M5JGM9.1 Q9NY15.1 Q7KZF4.1 Q9NQZ5.1 P16949.1 P05093.1 P08686.1 P36956.1 Q12772.1 Q7Z7C7.1 Q96BY9.1 P38646.1 P08254.1 Q14683.1 095347.1 Q8TY18.1 P07566.1 P51649.1 P14410.1 000391.1 075897.1 Q8NDZ2.1 P00441.1 014512.1 Q8TWZ8.1 Q6UWL2.1 Q8TAQ2.1 015056.1 P60880.1 Q9UQF0.1 015400.1 Q9UNK0.1 B4DHN5.1 000560.1 Q16635.1 Q9Y490.1 Q8N9U0.1 095271.1 D3YTG3.1 Q7Z7G0.1 Q9ULW0.1 P13686.1 Q86VP1.1 Q96F92.1 Q4KMP7.1 Q9UL17.1 P01730.1 Q99832.1 F5H7V9.1 P24821.1 Q9UKZ4.1 Q5VYS8.1 Q92563.1 Q8N6V9.1 Q9Y6M0.1 P04958.1 P05452.1 Q8NBS9.1 Q86V81.1 Q86YJ6.1 Q5VV42.1 P40225.1 P07996.1 P35442.1 P04818.1 P63313.1 P62328.1 P01266.1 H7C1F5.1 P07202.1 P16473.1 P21463.1 Q07157.1 Q9NR96.1 J3KNT7.1 Q9Y2L5.1 P03206.1 P37837.1 P20062.1 P51532.1 Q14241.1 Q7KZ85.1 P05412.1 A0AVK6.1 Q14469.1 P31629.1 P17275.1 Q8NHW3.1 Q9ULX9.1 P35716.1 Q06945.1 P57073.1 Q02447.1 Q02446.1 015164.1 Q9BWW7.1 Q04726.1 Q04727.1 P02786.1 Q9Y4A5.1 P01137.1 Q15582.1 P61586.1 P37802.1 P29401.1 P69222.1 Q92616.1 P51571.1 Q14956.1 Q96GE9.1 P57088.1 Q9BXS4.1 Q9C0B7.1 Q9Y5L0.1 P02766.1 Q13428.1 Q5T2D2.1 Q07283.1 P22102.1 A2RCL1.1 Q8NDV7.1 Q6P9F5.1 Q6ZTA4.1 P04295.1 014773.1 Q97HE9.1 Q9Y3T0.1 P17752.1 Q8TWU9.1 Q0VAP8.1 P68363.1 Q9BQE3.1 P07437.1 Q9H4B7.1 Q13885.1 Q13509.1 P04350.1 P68371.1 Q9BUF5.1 Q14679.1 075347.1 014788.1 P48023.1 P43489.1 P25445.1 Q8N726.1 Q99816.1 Q15672.1 P14679.1 P07101.1 P23458.1 Q9Y2R2.1 P29350.1 P78324.1 P08621.1 P17133.1 Q62376.1 P09012.1 P09234.1 075643.1 Q9UMX0.1 Q9UHD9.1 Q9Y4E8.1 Q9UPT9.1 Q8NFA0.1 Q86T82.1 Q86UV5.1 015205.1 P62979.1 H0Y5H6.1 Q14157.1 000762.1 Q96LR5.1 P62253.1 P22314.1 A0AVT1.1 Q15386.1 Q92575.1 I6ZLG2.1 015294.1 Q9DUC0.1 Q6ZRT6.1 Q9NSG2.1 Q9BWL3.1 Q9NZ63.1 P0C727.1 Q9ZDE9.1 Q89882.1 P39999.1 Q12965.1 A2A306.1 A2RGM0.1 A6NG79.1 B8ZS71.1 B8ZUA4.1 E7EPZ9.1 F8W7G7.1 H0Y335.1 J3KP29.1 M7PC26.1 M7PDR8.1 M7Q4Y3.1 Q5T8M8.1 Q7TWS5.1 S5U6K1.1 S5UMF6.1 S5USV8.1 W5Z3U0.1 Q9BSU1.1 Q49AR2.1 P69996.1 P06132.1 Q709C8.1 075436.1 Q9UBQ0.1 Q96AX1.1 P32241.1 Q3ASL6.1 Q00341.1 P08670.1 P03180.1 P02774.1 P04004.1 Q01668.1 000555.1 P27884.1 043497.1 P04275.1 Q9Y279.1 Q16864.1 075348.1 Q2M389.1 075083.1 Q9UNX4.1 C9J016.1 Q8TWA0.1 Q6UXN9.1 Q2TAY7.1 P13010.1 P12956.1 Q9Y2T7.1 A1JUA3.1 095625.1 Q8NAP3.1 Q96K80.1 Q9Y6R6.1 Q01954.1 Q9P243.1 Q96KR1.1 Q8TWU4.1 P25311.1

- 29 045699

Prediction of an individual's immunological response to a polypeptide antigen.

Specific polypeptide antigens induce immune responses in only a subset of human subjects. There is currently no diagnostic test that can predict whether a polypeptide antigen is likely to induce an immune response in an individual. In particular, there is a need for a test that can predict whether a person is a patient with an immune response to a vaccine or immunotherapeutic composition.

In accordance with the present invention, the polypeptide antigen-specific T lymphocyte response of an individual is determined by the presence in the polypeptide of one or more fragments that may be presented by multiple HLA class I molecules or multiple HLA class II molecules of the individual.

In some cases, the invention provides a method for predicting whether a subject will have an immune response to administration of a polypeptide, wherein the immune response is predicted if the polypeptide is immunogenic, in accordance with any method described herein. A cytotoxic T lymphocyte response is predicted if the polypeptide contains at least one amino acid sequence that is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject. A T helper cell response is predicted if the polypeptide contains at least one amino acid sequence that is a T lymphocyte epitope capable of binding to at least two HLA class II molecules of the subject. A cytotoxic T lymphocyte response is predicted to be absent if the polypeptide does not contain any amino acid sequence that represents a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject. A lack of T helper response is predicted if the polypeptide does not contain any amino acid sequence that represents a T lymphocyte epitope capable of binding to at least two HLA class II molecules of the subject.

In some cases, the polypeptide is an active component of a pharmaceutical composition, and the method includes predicting the formation or production of anti-drug antibodies (ADA) to the polypeptide. The pharmaceutical composition may be a drug selected from those listed in table. 8. According to the present invention, ADA formation will occur if or to the extent that the active ingredient polypeptide is recognized by multiple HLA class II molecules of the subject, resulting in a T helper cell response to support an antibody response to the active ingredient. The presence of such epitopes (PEPI) can predict the formation of ADA in a subject. The method may further include selecting or recommending treatment by administering to the subject a human subject-specific pharmaceutical composition that is predicted to induce low or no ADA formation, and, if necessary, further administering the composition to the subject. In other cases, the method predicts that the pharmaceutical composition will induce unacceptable ADAs, and the method further includes selecting or recommending, or treating the subject with another treatment or therapy. The polypeptide may be a checkpoint inhibitor.

The method may include predicting whether a subject will respond to treatment with a checkpoint inhibitor.

Table 8

Examples of medications associated with ADA-related side effects

Medicine Side effects associated with ADA Abciximab anaphylaxis Adalimumab Anti-drug antibodies and treatment failure Basiliximab anaphylaxis Cetuximab immunoglobulin E, anaphylaxis Epoetin Antibody-mediated pure red cell aplasia Erythropoietin pure red cell aplasia Etanercept does not have a noticeable impact on safety Clotting factor IX anaphylaxis Infliximab anaphylaxis oktz anaphylaxis Pegloticase Antibodies to a drug, treatment failure rIFN-beta anaphylaxis Recombinant factor VIII anaphylaxis Thrombopoietin thrombocytopenia Ustekinumab antibodies to ustekinumab, affecting the effectiveness of treatment

There is also currently no test that can predict the likelihood that a person will have a clinical response or experience clinical benefit from a vaccine or immunotherapy composition. This is important because T cell responses currently

- 30 045699 measured in a group of individuals participating in clinical trials of vaccines or immunotherapy do not correlate well with clinical responses. Thus, the subpopulation of patients with a clinical response is significantly smaller than the subpopulation of patients with an immune response. Therefore, to enable the personalization of vaccines and immunotherapies, it is important to predict not only the likelihood of an immune response in a given subject, but also whether the immune response generated by the drug will be clinically effective (for example, whether it can kill cancer cells or pathogen-infected cells or pathogens ).

The inventors have discovered that the presence in a vaccine or immunotherapeutic composition of at least two polypeptide fragments (epitopes) that can bind to at least three of an individual's HLA class I (>2 PEPI3+) is predictive of clinical response. In other words, if >2 PEPI3+ can be identified in the active ingredient polypeptide(s) of a vaccine or immunotherapeutic composition, then the individual is likely to be a clinical responder. The term clinical response or clinical effectiveness as used herein can mean preventing or delaying the onset of a disease or condition, reducing one or more symptoms, inducing or prolonging remission, or delaying recurrence or recurrence or worsening, or any other improvement or stabilization of a disease state. subject. When appropriate, clinical response may be correlated with disease control or objective response as defined by the Response Evaluation Criteria In Solid Tumors (RECIST) manual.

Therefore, in some cases, the invention provides a method for predicting whether a subject will have a clinical response to administration of a pharmaceutical composition, such as a vaccine or immunotherapeutic composition, containing one or more polypeptides as active ingredients. The method may include determining whether one or more polypeptides together contain at least two different sequences, each of which is a T lymphocyte epitope capable of binding to at least two or, in some cases, at least three HLA class I molecules of the subject; and predicting that the subject will have a clinical response to administration of the pharmaceutical composition if one or more polypeptides together contain at least two different sequences, each of which is a T lymphocyte epitope capable of binding to at least two, or in some cases by at least three HLA class I molecules of the subject; or that the subject will not have a clinical response to administration of the pharmaceutical composition if one or more polypeptides together contain no more than one sequence that is a T lymphocyte epitope capable of binding to at least two, or in some cases at least three molecules Subject's HLA class I.

In the context of this method, two T-lymphocyte epitopes are different from each other if they have different sequences, and in some cases also if they have the same sequence that is repeated in the target polypeptide antigen. In some cases, different T lymphocyte epitopes in the target polypeptide antigen do not overlap with each other.

In some cases, all fragments of one or more active ingredient polypeptides or polypeptides that are immunogenic in a particular human subject are identified using the methods described herein. Identification of at least one fragment of polypeptide(s) that is a T lymphocyte epitope capable of binding to at least two or at least three HLA class I molecules of the subject suggests that the polypeptide(s) will induce or is likely to induce , induces a cytotoxic T lymphocyte response in a subject. Identification of at least one fragment of polypeptide(s) that is a T lymphocyte epitope capable of binding to at least two or at least three or at least four HLA class II molecules of the subject suggests that the polypeptide(s) ) will induce or is likely to induce a T helper cell response in the subject. The identification of the absence of fragments of the polypeptide(s) that represent T lymphocyte epitopes capable of binding to at least two or at least three HLA class I molecules of the subject suggests that the polypeptide(s) will not, or are likely not to induce induces a cytotoxic T lymphocyte response in a subject.

Identification of the absence of fragments of the polypeptide(s) that are T lymphocyte epitopes capable of binding to at least two or at least three or at least four HLA class II molecules of the subject suggests that the polypeptide(s) will not induce or are not likely to induce a T helper cell response in the subject. Identification of at least two fragments of one or more polypeptides of an active ingredient of a vaccine or immunotherapeutic composition, in which each fragment is a T lymphocyte epitope capable of binding to at least two or at least three HLA class I molecules of a subject, suggests that the subject is most likely

- 31 045699 has or will have a clinical response to administration of the composition. Identification of less than two fragments of one or more polypeptides that are T lymphocyte epitopes capable of binding to at least two or at least three HLA class I molecules of the subject suggests that the subject is less likely to have or not to have clinical response to the administration of the composition.

Without being bound by any theory, one reason for the increased likelihood of obtaining clinical efficacy from a vaccine/immunotherapy comprising at least two PEPIs binding to multiple HLAs is that the population of affected cells, such as cancer or tumor cells, or cells , infected by viruses or pathogens such as HIV, are often heterogeneous both within and among affected subjects. A particular cancer patient, for example, may or may not express or overexpress a particular cancer-associated polypeptide vaccine target antigen, or the cancer may contain heterogeneous cell populations, some of which (over-)express the antigen and some of which do not. In addition, the likelihood of developing resistance is reduced when more PEPIs that bind to multiple HLAs are included or targeted by the vaccine/immunotherapy because the patient is less likely to develop resistance to the composition due to mutation of the PEPI target(s).

Therefore, the likelihood that a subject will respond to treatment is increased by (i) the presence of more multi-HLA binding PEPIs in the active ingredient polypeptides; (ii) the presence of PEPI in a larger number of target polypeptide antigens; and (iii) (over)expression of target polypeptide antigens in the subject or in affected cells of the subject. In some cases, the expression of target polypeptide antigens in a subject may be known, for example, if the target polypeptide antigens are present in a sample obtained from the subject. In other cases, the likelihood that a particular subject or affected cells from a particular subject (over-)express a specific or any combination of target polypeptide antigens can be determined using population expression frequency data. Data on the frequency of expression in a population may relate to the population corresponding to the subject and/or disease, or the population to be treated. For example, the frequency or likelihood of expression of a particular cancer-associated antigen in a particular cancer or by a subject having a particular cancer, such as breast cancer, can be determined by detecting the antigen in the tumor, such as breast cancer samples. In some cases, such expression frequencies can be determined from published graphics and scientific publications. In some cases, the method of the invention includes the step of determining the frequency of expression of the relevant target polypeptide antigen in the relevant population.

A number of pharmacodynamic biomarkers have been disclosed to predict vaccine potency/efficacy in individual human subjects as well as in populations of human subjects. Biomarkers have been developed specifically for cancer vaccines, but similar biomarkers could be used for other vaccines or immunotherapy formulations. These biomarkers speed up the development of a more effective vaccine, as well as reduce the cost of development, and can be used to evaluate and compare different formulations. Typical biomarkers are:

AG95 - vaccine efficacy: the number of antigens in a cancer vaccine that a specific tumor type has a 95% probability of expressing. AG95 is an indicator of vaccine effectiveness and does not depend on the immunogenicity of the antigens used to prepare the vaccine. AG95 is calculated from the frequency of tumor antigen expression. Such data can be obtained from experiments published in peer-reviewed scientific journals. Technically, AG95 is determined by the binomial distribution of antigens in the vaccine and takes into account all possible variations and frequencies of expression.

Amount of PEPI3+ - Vaccine Immunogenicity in Subject: Vaccine-derived PEPI3+ are individual epitopes that bind to at least 3 HLAs of the subject and induce T-lymphocyte responses. PEPI3+ can be determined using the PEPI3+ test in subjects for whom the full 4-digit HLA genotype is known.

The number of AR is the antigenicity of the vaccine in the subject: the number of antigens with PEPI3+ used to prepare the vaccine. Vaccines contain sequences from target polypeptide antigens expressed by affected cells. The AP number represents the number of antigens in the vaccine that contain PEPI3+, and the AP number represents the number of antigens in the vaccine that can induce T lymphocyte responses in a subject. The amount of AR characterizes the vaccine antigen-specific T-lymphocyte responses of the subject, since it depends only on the HLA genotype of the subject and is independent of the disease, age and drug treatment of the subject. The correct value is between O (no PEPI presented by the antigen) and the maximum number of antigens (all antigens present PEPI).

AP50 - antigenicity of the vaccine in the population: The average number of antigens with PEPI used to prepare the vaccine in the population. AP50 is suitable for characterizing vaccine antigen-specific T cell responses in a given population because

- 32 045699 they depend on the HLA genotype of subjects in the population.

AGP quantity - vaccine effectiveness in a subject: the number of antigens used to prepare the vaccine expressed in the tumor with PEPI. The amount of AGP indicates the number of tumor antigens that the vaccine recognizes and induces a T-lymphocyte response to them (target damage). The amount of AGP depends on the frequency of expression of the antigen used to prepare the vaccine in the subject's tumor and the HLA genotype of the subject. The correct value is between 0 (no PEPI presented by the expressed antigen) and the maximum number of antigens (all antigens expressed and presented by PEPI).

AGP50 - effectiveness of a cancer vaccine in a population: The average number of vaccine antigens expressed in a specified tumor with PEPI (i.e. AGP) in a population. AGP50 indicates the average number of tumor antigens that can recognize vaccine-induced T lymphocyte responses. AGP50 depends on the frequency of antigen expression in the tumor type indicated and the immunogenicity of the antigens in a given population. AGP50 can evaluate vaccine effectiveness in different populations and can be used to compare different vaccines in the same population. The calculation of AGP50 is similar to that used for AG50, except that expression is weighted by the prevalence of PEPI3+ in the subject on the expressed antigens used to prepare the vaccine. In a theoretical population in which each subject has a PEPI for each vaccine antigen, the AGP50 will be equal to the AG50. In another theoretical population in which no subject has a PEPI from any vaccine antigen, the AGP50 would be 0. In general, the following statement is true: 0 < AGP50 < AG50.

mAGP is a potential biomarker for selecting likely responders: The likelihood that a cancer vaccine will induce T cell responses to multiple antigens expressed in a specified tumor. mAGP is calculated from the expression frequencies of the antigens used to prepare the vaccine, for example, in the tumor, and the presence of vaccine-derived PEPI in the subject. Technically, based on the AGP distribution, mAGP is the sum of the probabilities of multiple AGPs (>2 AGPs).

The prognostic results outlined above can be used to inform a physician's decisions regarding the treatment of a subject. Accordingly, in some cases, the polypeptide is an active ingredient, such as a vaccine or immunotherapeutic composition, wherein the method of the invention predicts that the subject will have, is likely to have, or is more likely than a minimum threshold probability to have an immune response and/or a clinical response for a treatment comprising administering the active ingredient polypeptide to a subject, and the method further includes selecting a treatment or selecting a vaccine or immunotherapeutic composition for treating a particular human subject. Also provided is a method of treating with a subject-specific pharmaceutical composition, set or panel of polypeptides containing one or more polypeptides as active ingredients, wherein the pharmaceutical composition, set or panel of polypeptides is determined to have a threshold minimum likelihood of inducing a clinical response in a subject, wherein response probability was determined using the method described herein. In some cases, the minimum threshold is determined by one or more pharmacodynamic biomarkers described herein, for example, a minimum amount of PEPI3+ (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or more PEPI3+ ), a minimum number of AGPs (e.g. AGP=at least 2 or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more) and/or a minimum mAGP (e.g. AGP=at least 2 or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more). For example, in some cases, a subject is selected for treatment if its likelihood of responding to a predetermined number of target polypeptide antigens, wherein the target polypeptide antigens are not necessarily expressed, exceeds a predetermined threshold (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or more). Alternatively, the method may predict that one or more polypeptide(s) of the composition will not induce a T lymphocyte response and/or clinical response in the subject, and further includes selecting another treatment for the particular human subject.

Prediction of an autoimmune or toxic immune response to a polypeptide antigen.

Differences between HLAs can influence the likelihood of developing an autoimmune disease, condition, or response. In some cases, the method of the invention can be used to identify a polypeptide or polypeptide fragment that is immunogenic and/or associated with an autoimmune disorder or response. In some cases, the method includes determining whether the polypeptide contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three, or at least four, or at least five HLA class I of the subject; or in other cases, a sequence that is a T lymphocyte epitope capable of binding to at least four, or at least five, or at least six HLA class II of the subject; and identifying the polypeptide or said sequence as immunogenic or associated with

- 33 045699 an autoimmune disorder or autoimmune response in the subject.

Differences between HLAs may also influence the likelihood that a subject will experience immunotoxicity from a drug or polypeptide administered to the subject. A toxic immune response may occur if the polypeptide administered to a subject contains a fragment that corresponds to a fragment of an antigen expressed in normal healthy cells of the subject and that contains an amino acid that is a T lymphocyte epitope capable of binding to multiple HLA class I molecules of the subject. Thus, in some cases in accordance with the invention, the method is used to identify a toxic immunogenic site or fragment of a polypeptide or to identify subjects who are likely to experience immunotoxicity in response to administration of one or more polypeptides or fragments thereof. The polypeptide may be an active ingredient in a vaccine or immunotherapeutic composition.

The method may include determining whether the polypeptide(s) contains a sequence that is a T lymphocyte epitope capable of binding to at least two or, in other cases, at least three HLA class I molecules of the subject. In some cases, the method includes determining whether the polypeptide contains a sequence that is a T lymphocyte epitope capable of binding to at least four or at least five HLA class I molecules of the subject; or an amino acid sequence that is a T lymphocyte epitope capable of binding to at least four, or at least five, or at least six, or at least seven HLA class II of the subject. The method may further include identifying the sequence as toxic immunogenic to the subject or predicting a toxic immune response in the subject. In other cases, the absence of a specified amino acid sequence is identified, and the method further includes predicting the absence of a toxic immune response in the subject. The method may further include selecting or recommending treatment of the subject by administering one or more polypeptides or a pharmaceutical composition predicted to induce no or low immunotoxicity, and, if necessary, further treating the subject by administering the polypeptide. The invention also provides a method of treating a subject in need thereof by administering said polypeptide or composition to the subject.

In some cases, the method described herein further includes mutating a polypeptide that is predicted to be immunogenic in a particular human subject, or that is predicted to be immunogenic in a subset of subjects in a human population. Also provided is a method of reducing the immunogenicity of a polypeptide that has been identified as immunogenic in a particular human subject or a portion of a human population using any of the methods described herein. The polypeptide may be mutated to reduce the amount of PEPI in the polypeptide or to reduce, in a subject or a subset of a population, the number of HLA class I or class II molecules that bind to a fragment of a polypeptide identified as immunogenic in a subject or a portion of a subpopulation. In some cases, the mutation may reduce or prevent a toxic immune response, or may enhance effectiveness by preventing the formation of ADA in a subject or a portion of a specified population. The mutated polypeptide may further be selected or recommended for treatment of the specified subject or subject of the specified population. The subject may further be treated by administering the mutated polypeptide. The invention also provides a method of treating a subject in need thereof by administering such a mutated polypeptide to the subject.

Predicting an Individual's Response to Checkpoint Inhibitor Treatment Typically, some or all of the tumor-specific T cell clones that are induced by the tumor are inactive or perform poorly in patients with metastatic cancer. Inactive tumor-specific T lymphocytes cannot destroy tumor cells. A portion of these inactive T lymphocytes can be reactivated by checkpoint inhibitors (such as ipilimumab), such as monoclonal antibodies that recognize checkpoint molecules (eg, CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, BTLA ). According to the present invention, treatment of a subject with a checkpoint inhibitor will be effective only if or to the extent that the expressed cancer antigens can be adequately recognized by the individual's HLA, i.e. if the cancer or disease associated antigens contain epitopes that are recognized by multiple, preferably at least three, HLA class I molecules of the subject. Thus, in some cases, the methods of the present invention can be used to identify one or more, or subsets of T lymphocyte lineages that may be reactivated by a checkpoint inhibitor, or to predict patients likely to respond to checkpoint inhibitor (immuno)therapy.

Accordingly, in some cases, the invention provides a method for predicting whether a subject will respond to cancer treatment with a checkpoint inhibitor. In some cases, the method includes the step of identifying or selecting one or more polypeptides or polypeptide fragments that are associated with the disease or condition to be treated, or

- 34 045699 which is associated with obtaining an immune or clinical response to treatment with a checkpoint inhibitor. In some cases, the polypeptide is a tumor-associated and/or mutational antigen. The polypeptide may be present in a sample obtained from the subject. The polypeptide may be a polypeptide that is frequently (over-)expressed in a population corresponding to a subject and/or disease. The polypeptide may consist of or contain PEPI (or PEPI3+) identified from a subject known to have responded positively to administration of a checkpoint inhibitor. The polypeptide may contain or consist of an amino acid sequence that is stored or recorded or retrieved from a database.

In some cases, the method includes determining whether the polypeptide(s) contains a sequence that is a T lymphocyte epitope capable of binding to multiple HLA class I molecules of the subject. In some cases, the presence of at least two, or at least three, or four, or five, or six, or seven, or eight of the specified different amino acid sequences is determined, and/or the presence of the specified amino acid sequence in at least two, or in at least three, four or five different target polypeptide antigens. In some cases, the method may include determining whether the polypeptide(s) contains a sequence that is a T lymphocyte epitope capable of binding to at least two, or in other cases, at least three, or at least four HLA molecules class II subject. Response to checkpoint inhibitor treatment can be predicted if the above requirement(s) are met. Neither response nor clinical response can be predicted if the above requirement(s) are not met.

The invention also provides a method for identifying a polypeptide fragment or T lymphocyte epitope in a polypeptide that may be a target of a subject's immune response following checkpoint inhibitor treatment, or that will be a target of T lymphocytes reactivated by checkpoint inhibitor treatment.

The method may further include selecting, recommending, and/or administering a checkpoint inhibitor to a subject predicted to respond, or selecting, recommending, and/or administering another treatment to a subject predicted to not respond to the checkpoint inhibitor. In other cases, the invention provides a method of treating a human subject in need thereof, comprising administering to the subject a checkpoint inhibitor, wherein the subject is predicted to respond to administration of the checkpoint inhibitor in a manner described herein.

Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors and CTLA-4 inhibitors. Costimulatory antibodies deliver positive signals through immunoregulatory receptors including, but not limited to, ICOS, CD137, CD27 OX-40 and GITR. In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor.

Formulation and preparation of pharmaceutical compositions for an individual human subject.

In some aspects, the invention provides a method of formulating or producing a polypeptide or polynucleic acid that encodes a polypeptide to induce an immune response, a cytotoxic T lymphocyte response, or a T helper cell response in a particular human subject. The invention also provides a human subject-specific drug, immunogenic composition or pharmaceutical composition, set or panel of peptides, methods for formulating or producing them, compositions that can be prepared by these methods, and their use in a method of inducing an immune response, a cytotoxic T-response. lymphocyte or T helper response in a subject, or a method of treating, vaccinating, or providing immunotherapy to a subject. A pharmaceutical composition, set or panel of peptides contains as active ingredients one or more polypeptides that together contain two or more T-lymphocyte epitopes (PEPIs) capable of binding to a plurality of HLA class I molecules or a plurality of HLA class II molecules of a subject that are immunogenic for a subject as described herein, or have been identified as immunogenic for a subject in a manner described herein.

The composition/kit may, if necessary, further contain at least one pharmaceutically acceptable diluent, carrier or preservative and/or additional polypeptides that do not contain any PEPI. Polypeptides may be engineered or non-naturally occurring. The kit may contain one or more individual containers, each containing one or more active ingredient peptides. The composition/kit may be a personalized medicinal product for the prevention, diagnosis, amelioration, treatment or elimination of a disease in an individual, such as cancer.

Typically, each PEPI is a fragment of a target polypeptide antigen, and polypeptides that contain one or more PEPIs are target polypeptide antigens for treatment, vaccination, or immunotherapy. The method may include the step of identifying one or more suitable target polypeptide antigens. Typically, each target polypeptide antigen

- 35 045699 will be associated with the same disease or condition, pathogenic organism or group of pathogenic organisms, or virus, or type of cancer.

The composition, set or panel may contain, or the method may include selecting for each PEPI a sequence of up to 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 consecutive amino acids of a polypeptide target antigen, such as a polypeptide described herein, which consecutive amino acids constitute the amino acid sequence of PEPI.

In some cases, the amino acid sequence is flanked at the N- and/or C-terminus by additional amino acids that are not part of the rigid sequence of the target polypeptide antigen. In some cases the sequence is flanked by 41, or 35, or 30, or 25, or 20, or 15, or 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 additional amino acid HaN-and/or C-terminal or between fragments of target polypeptides. In other cases, each polypeptide either consists of a fragment of a target antigen polypeptide, or consists of two or more such fragments, end-to-end (arranged sequentially in the peptide end-to-end) or overlapping in a single peptide (in which two or more fragments partially contain overlapping sequences, for example when two PEPIs in the same polypeptide are within 50 amino acids of each other).

When fragments of different polypeptides or from different regions of the same polypeptide are assembled into a designed peptide, there is a possibility of formation of neo-epitopes near the junction or junction. Such neo-epitopes span at least one amino acid from each fragment on each side of the junction or junction, and may be referred to herein as constituent amino acid sequences. Neoepitopes can induce unwanted T lymphocyte responses against healthy cells (autoimmunity). Peptides can be formulated, or peptides can be screened, to destroy or eliminate neoepitopes that correspond to a protein fragment expressed in normal healthy human cells, and/or neoepitopes that are capable of binding to at least two, or in some cases at least at least three, or at least four HLA class I molecules of the subject, or in some cases at least two, or at least three, four or five HLA class II molecules of the subject. The methods of the present invention can be used to identify or screen for such neoepitopes as described herein. The alignment can be determined using known methods such as BLAST algorithms. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

At least both of two multi-HLA PEPI-binding polypeptides of the composition may target a single antigen (e.g., a polypeptide vaccine comprising two multi-HLA PEPI-binding polypeptides derived from a single antigen, e.g., a tumor-associated antigen, that is the target of a vaccine/immunotherapy ), or may target different antigens (e.g., a polypeptide vaccine containing one multi-HLA-binding PEPI derived from one antigen, e.g., a tumor-associated antigen, and a second multi-HLA-binding PEPI, derived from another antigen, e.g. another tumor-associated antigen, both of which are vaccine/immunotherapy targets).

In some cases, the active ingredient polypeptide(s) are contained together, or the method includes selecting all or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more different PEPI. PEPIs may be fragments of one or more different target polypeptide antigens. By identifying specific fragments of each target polypeptide antigen that are immunogenic for a particular subject, a plurality of such fragments, optionally from a plurality of different target polypeptide antigens, can be combined into a single active ingredient polypeptide or a plurality of active ingredient polypeptides for use in combination, or to maximize the number of T lymphocyte clones that can be activated by one or more polypeptides of a certain length.

Currently, most vaccines and immunotherapeutic compositions target only one polypeptide antigen. However, according to the present invention, in some cases it is useful to provide a pharmaceutical composition or active ingredient polypeptide that targets two or more different polypeptide antigens. For example, most cancers or tumors are heterogeneous, in the sense that different cancer or tumor cells in a subject (over-)express different antigens. Tumor cells from different cancer patients also express different combinations of tumor-associated antigens. The most effective anti-cancer immunogenic compositions are likely those that target multiple antigens expressed by a tumor, and therefore a greater number of cancer or tumor cells in an individual human subject or population.

The beneficial effect of combining multiple PEPIs in one therapeutic product (administration of one or more pharmaceutical compositions that together contain multiple PEPIs) can be

- 36 045699 illustrated using the personalized vaccine polypeptides described below in Examples 17 and 18. Examples of CTA expression probabilities in ovarian cancer are as follows: BAGE: 30%; MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. If patient XYZ received a vaccine containing PEPI only in the BAGE and MAGE A9 family, then the probability of having mAGP (multiple expressed antigens with PEPI) would be 11%. If Patient XYZ were to receive a vaccine containing only PEPI for MAGE A4 and MAGE A10 CTAs, there would be a 19% chance of having multiple AGPs. However, if the vaccine contained all 4 of these CTAs (BAGE, MAGE A9, MAGE A4 and MAGE A10), then the probability of having mAGP would be 50%. In other words, the effectiveness will be greater than the combined mAGP probabilities for both drugs with two PEPIs (mAGP probability for BAGE/MAGE+ mAGP probability for MAGE A4 and MAGE A10). Patient XYZ's PIT vaccine described in Example 17 contains an additional 9 PEPI and thus has a greater than 99.95% probability of having mAGP.

Similarly, examples of CTA expression probabilities in breast cancer are as follows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. Treating patient ABC with a vaccine containing PEPI only in MAGE C2: 21% and MAGE A1 has a mAGP probability of 7%. Treatment of patient ABC with a vaccine containing PEPI only in SPC1: 38%; MAGE A9 has a mAGP probability of 11%. Treatment of an ABC patient with a vaccine containing PEPI in MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 has a mAGP probability of 44% (44>7+11). Patient ABC's PIT vaccine described in Example 18 contains an additional 8 PEPI and thus has a greater than 99.93% probability of mAGP.

Accordingly, in some cases, the PEPIs of the active ingredient polypeptides are derived from two or more different target polypeptide antigens, for example different antigens associated with a particular disease or condition, for example different antigens or cancer- or tumor-associated antigens expressed by the target pathogen. In some cases, PEPIs occur in total or at least from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 ,21,22,23,24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or more different target polypeptide antigens. The different target polypeptide antigens may be other polypeptides that are useful to target, or that may be selectively targeted by different PEPI3+. In some cases, different target polypeptide antigens are non-homologues or non-paralogues, or have less than 95%, or 90%, or 85%, or 80%, or 75%, or 70% sequence identity across the entire length of each polypeptide, or 60%, or 50%. In some cases, the different polypeptides are polypeptides that do not share PEPI3+. Alternatively, in some cases, PEPI3+ are derived from different target polypeptide antigens when they are not shared with other polypeptide antigens that are the target of the active ingredient polypeptides.

In some cases, one or more or each of the immunogenic polypeptide fragments is derived from a polypeptide that is present in a sample taken from a particular human subject. This indicates that the polypeptide is expressed in the subject, for example, a cancer- or tumor-associated antigen, or a cancer-testis antigen expressed by cancer cells of the subject. In some cases, one or more or each of the polypeptides is a mutational neoantigen or expression neoantigen of the subject. One or more or each fragment may contain a neoantigen-specific mutation. Because mutational neoantigens are specific to a subject, a composition targeting one or more neoantigen-specific mutations is personalized to both his particular disease and specific HLA set.

In other cases, one or more or each of the immunogenic polypeptide fragments is derived from a target polypeptide antigen that is not ordinarily expressed or minimally expressed in normal healthy cells or tissues, but is expressed at a high rate in subjects or diseased individuals. cells of a subject having a particular disease or condition, as described above. The method may include identifying or selecting such a target polypeptide antigen. In some cases, two or more or each of the immunogenic polypeptide fragments/PEPIs are derived from different cancer or tumor associated antigens, each of which is (over-)expressed at high frequency in subjects having a type of cancer or cancer derived from a particular type cells or tissues. In some cases, immunogenic polypeptide fragments are derived from a total of or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 different associated with cancer or tumor polypeptides. In some cases, one or more, or each, or at least one, at least two, at least three, at least four, at least five, or at least six, or at least seven polypeptides are selected from antigens listed in any of the tables. 2-7.

In some cases, one or more or each of the target polypeptide antigens is a cancer-testis antigen (CTA). In some cases, immunogenic fragments/PEPI

- 37 045699 polypeptides are derived from at least 1 or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 CTAs, or a total of 3 or more different target polypeptide antigens, optionally, of which 1, 2 or all three or at least three are CTAs, or 4 or more different polypeptide antigens, optionally, of which 1, 2, 3 or all four or at least 1, 2, 3 or 4 are CTA, or from 5 or more different polypeptide antigens, optionally, of which 1, 2, 3 , 4 or all five or at least 1, 2, 3, 4 or 5 are CTAs, or from 6 or more different polypeptide antigens, optionally of which 1, 2, 3, 4, 5 or all six or at least 1, 2, 3, 4, 5 or 6 are CTAs, or from 7 or more different polypeptide antigens, optionally of which 1, 2, 3, 4, 5, 6 or all 7 or at least 1, 2, 3, 4, 5, 6 or 7 are CTAs, or from 8 or more different polypeptide antigens, optionally of which 1, 2, 3, 4, 5, 6, 7 or all 8 or at least 1, 2, 3, 4, 5, 6, 7 or 8 represent a CTA. In some cases, one or more or each of the target polypeptide antigens is expressed by a bacterium, virus or parasite.

In some cases, one or more polypeptide fragments contains an amino acid sequence that represents a T lymphocyte epitope capable of binding to at least two or at least three HLA class I of the subject, and one or more polypeptide fragments contains an amino acid sequence that represents A T lymphocyte epitope capable of binding to at least two, or at least three, or at least four HLA class II of a subject, wherein the HLA class I and HLA class II binding fragments may not necessarily overlap. A composition prepared in this manner can induce both a cytotoxic T lymphocyte response and a helper T cell response in a particular human subject.

Immunogenic and pharmaceutical compositions, methods of treatment and methods of administration.

In some aspects, the invention relates to a pharmaceutical composition, set or panels of polypeptides described above having one or more polypeptides as the active ingredient(s). They may be used in a method of inducing an immune response, treating, vaccinating, or providing immunotherapy to a subject, and the pharmaceutical composition may be a vaccine or immunotherapeutic composition. Such treatment involves administering to a subject one or more polypeptides or pharmaceutical compositions that together contain all of the polypeptides of the active ingredient for treating the subject. Multiple polypeptides or pharmaceutical compositions may be administered together or sequentially, for example, all pharmaceutical compositions or polypeptides may be administered to a subject over a period of 1 year, or 6 months, or 3 months, or 60, or 50, or 40, or 30 days.

The immunogenic or pharmaceutical compositions or kits described herein may contain, in addition to one or more immunogenic peptides, a pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilizer, preservative, adjuvant, or other materials well known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient(s). The pharmaceutical carrier or diluent may be, for example, aqueous solutions. The exact nature of the carrier or other material may depend on the route of administration, for example, oral, intravenous, dermal or subcutaneous, nasal, intramuscular, intradermal and intraperitoneal routes.

The pharmaceutical compositions of the invention may contain one or more pharmaceutically acceptable carriers. Typically these are large, slowly metabolized macromolecules such as proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose (WO 00/56365), lactose and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. Pharmaceutical compositions may also contain diluents such as water, saline, glycerin, and the like. In addition, there may be auxiliary substances such as wetting or emulsifying agents, pH buffers, and the like. Sterile, pyrogen-free, phosphate-buffered saline is a typical vehicle (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472).

The pharmaceutical compositions according to the invention can be lyophilized or in aqueous form, that is, in solutions or suspensions. Liquid compositions of this type allow the compositions to be administered directly from the packaged form without the need for reconstitution in an aqueous medium and are thus ideal for injection. Pharmaceutical compositions can be presented in vials or in ready-filled syringes. Syringes may come with or without needles. A syringe will contain a single dose, while a vial may contain a single dose or multiple doses.

The liquid compositions of the invention are also suitable for reconstituting other medicinal products from lyophilized form. When a pharmaceutical composition is to be used for such immediate reconstitution, the invention provides a kit that may contain two vials or may contain one pre-filled syringe and one vial, the contents of the syringe being used to reconstitute the contents of the vial prior to injection.

The pharmaceutical compositions according to the present invention may contain an antimicrobial agent, in particular when it is packaged in the form of multiple doses. Antimicrobials such as 2-phenoxyethanol or parabens (methyl, ethyl, propylparabens) may be used. Some preservative is preferably present at low levels. The preservative may be added exogenously and/or may be a component of a body of antigens that are mixed to form the composition (eg, present as a preservative in pertussis antigens).

The pharmaceutical compositions according to the invention may contain a detergent, for example Tween (polysorbate), DMSO (dimethyl sulfoxide, dimethyl sulfoxide), DMF (dimethylformamide, dimethylformamide). Detergents are typically available at low levels, eg <0.01%, but can also be used at higher levels, eg 0.01-50%.

The pharmaceutical compositions of the invention may contain sodium salts (eg sodium chloride) and free phosphate ions in solution (eg using a phosphate buffer).

In some embodiments, the pharmaceutical composition may be encapsulated in a suitable carrier either to deliver peptides to antigen presenting cells or to enhance stability. As will be appreciated by one skilled in the art, a variety of carriers are suitable for delivering the pharmaceutical composition of the invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

To enhance the immunogenicity of the composition, the pharmacological compositions may contain one or more adjuvants and/or cytokines.

Suitable adjuvants include an aluminum salt such as aluminum hydroxide or aluminum phosphate, but may also be a calcium, iron or zinc salt, or may be an insoluble suspension of acylated tyrosine or acylated sugars, or may be cationically or anionically derivatized saccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid (MPL), lipid A derivatives (eg, reduced toxicity), 3-O-deacylated MPL [3D-MPL], quil A adjuvant, saponin, QS21, Freund's incomplete adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), AS-2 (Smith-Kline Beecham, Philadelphia, PA), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether compositions , polyoxyethylene ester compositions, muramyl peptides or imidazoquinolone compounds (for example, imiquamod and its homologues). Human immunomodulators suitable for use as adjuvants in the invention may also be used as adjuvants, including cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL- 7, IL-12, etc.), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocytes, macrophage colony-stimulating factor (GM-CSF).

In some embodiments, the compositions contain an adjuvant selected from the group consisting of Montanide ISA-51 (Seppic, Inc., Fairfield, NJ, USA), QS-21 (Aquila Biopharmaceuticals, Inc., Lexington, Mass., USA), GM -CSF, cyclophosamide, Bacillus Calmette-Guerin (BCG), corynebacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanins (KLH, keyhole limpet hemocyanins), Freund's adjuvant (complete and incomplete), mineral gels, hydroxide aluminum (alum), lysolecithin, pluronyl polyols, polyanions, peptides, oil emulsions, dinitrophenol, diphtheria toxin (DT, diphtheria toxin).

By way of example, the cytokine may be selected from the group consisting of, but not limited to, transforming growth factor (TGF) such as TGF-α and TGF-β; insulin-like growth factor-I and/or insulin-like growth factor-II; erythropoietin (EPO, erythropoietin); osteoinductive factor; interferon, such as, but not limited to, interferon-α, -β and -γ; colony-stimulating factor (CSF), such as, but not limited to, macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). In some embodiments, the cytokine is selected from the group consisting of nerve growth factors such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as, but not limited to, TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor II; erythropoietin (EPO); osteoinductive factor; interferon (IFN), such as, but not limited to, IFN-α, IFN-β and IFN-γ; colony stimulating factor (CSF), such as macrophage-CSF (MCSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukin (II), such as, but not limited to, IL-1, IL-1.alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 , IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18; LIF; set of ligands or FLT-3; angiostatin; thrombospondin;

- 39 045699 endostatian; tumor necrosis factor (TNF, tumor necrosis factor); and L.T.

It is expected that the adjuvant or cytokine may be added in an amount of about 0.01 to 10 mg per dose, preferably in an amount of about 0.2 to 5 mg per dose. Alternatively, the adjuvant or cytokine may be present at a concentration of from about 0.01 to 50%, preferably at a concentration from about 2% to 30%.

In certain aspects, the pharmaceutical compositions of the present invention are prepared by physically mixing the adjuvant and/or cytokine with PEPI under appropriate sterile conditions in accordance with known techniques to obtain the final product.

Examples of suitable polypeptide fragment compositions and routes of administration are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). The preparation of vaccines and immunotherapeutic compositions is generally described in Vaccine Design (The subunit and adjuvant approach (ed. Powell MF & Newman MJ (1995), Plenum Press, New York). Encapsulation in liposomes, which also provided for, described by Fullerton, US patent 4235877.

In some embodiments, the compositions disclosed herein are provided as a nucleic acid vaccine. In some embodiments, the nucleic acid vaccine is a DNA vaccine. In some embodiments, DNA vaccines or gene vaccines comprise a plasmid with a promoter and suitable transcriptional and translational control elements and a nucleic acid sequence encoding one or more polypeptides of the invention. In some embodiments, plasmids also contain sequences to enhance, for example, expression levels, intracellular targeting, or proteasomal processing. In some embodiments, DNA vaccines comprise a viral vector containing a nucleic acid sequence encoding one or more polypeptides of the invention. In additional aspects, the compositions disclosed herein comprise one or more nucleic acids encoding peptides determined to be immunoreactive with a biological sample. For example, in some embodiments, the compositions contain one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more peptides containing a fragment that is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules and/or at least three HLA class II molecules of the patient. In some embodiments, the peptides are derived from an antigen that is expressed in cancer. In some embodiments, the DNA or gene vaccine also encodes immunomodulatory molecules to manipulate the resulting immune responses, such as increasing vaccine efficacy, stimulating the immune system, or reducing immunosuppression. Strategies to enhance the immunogenicity of DNA or gene vaccines include encoding xenogeneic versions of antigens, fusing antigens with molecules that activate T cells or trigger associative recognition, priming with DNA vectors followed by amplification with a viral vector, and the use of immunomodulatory molecules. In some embodiments, the DNA vaccine is administered via a needle, gene gun, aerosol injector, patch, microneedle, abrasion, among other forms. In some forms, the DNA vaccine is included in liposomes or other forms of nanobodies. In some embodiments, the DNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; protamine liposome; polysaccharide particles; cationic nanoemulsion; cationic polymer; cationic polymer liposome; cationic nanoparticles; cationic lipid and cholesterol nanoparticles; cationic lipid, cholesterol, and PEG nanoparticles (polyethyleneglycol, polyethylene glycol); dendrimer nanoparticles. In some embodiments, DNA vaccines are administered by inhalation or oral administration. In some embodiments, the DNA vaccine is administered to the blood, thymus, pancreas, skin, muscle, tumor, or other sites.

In some embodiments, the compositions disclosed herein are provided as an RNA-based vaccine. In some embodiments, the RNA is a non-replicating mRNA or a self-amplifying RNA of viral origin. In some embodiments, the non-replicating mRNA encodes the peptides disclosed herein and contains 5' and 3' untranslated regions (UTRs). In some embodiments, the virally derived self-amplifying RNA encodes not only the peptides disclosed herein, but also a viral replication machinery that allows intracellular RNA to be amplified and protein overexpressed. In some embodiments, the RNA is directly administered to the individual. In some embodiments, the RNA is chemically synthesized or transcribed in vitro. In some embodiments, the mRNA is produced from a linear DNA template using phage T7, T3, or Sp6 RNA polymerase, and the resulting product contains an open reading frame that encodes the peptides disclosed herein flanking the UTR, 5' cap, and poly(A ) tail. In some embodiments, various 5' cap variants are added during or after a transcription reaction using a vaccinia virus capping enzyme, or by including synthetic cap analogues or an anti-reverse cap. In some embodiments, an optimal length of poly(A) tail is added to the mRNA either directly from the coding DNA template or using poly(A) polymerase. The RNA encodes one or more peptides containing a fragment that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules and/or at least three HLA class II molecules of the patient. In some embodiments, the fragments are derived from an antigen that is expressed in cancer. In some embodiments, the RNA includes signals to enhance stability and translation. In some embodiments, the RNA also includes non-naturally occurring nucleotides to increase half-life or modified nucleosides to alter the immunostimulatory profile. In some embodiments, the RNA is administered via a needle, gene gun, aerosol injector, patch, microneedle, abrasion, among other forms. In some forms, the RNA vaccine is incorporated into liposomes or other forms of nanobodies that facilitate cellular uptake of the RNA and protect it from degradation. In some embodiments, the RNA vaccine includes a delivery system selected from the group consisting of a transfection agent; protamine; protamine liposome; polysaccharide particles; cationic nanoemulsion; cationic polymer; cationic polymer liposome; cationic nanoparticles; cationic lipid and cholesterol nanoparticles; nanoparticles of cationic lipid, cholesterol and PEG; dendrimer nanoparticles; and/or naked mRNA; naked mRNA with in vivo electroporation; mRNA with protamine complex; mRNA bound to a positively charged cationic oil-in-water nanoemulsion; mRNA bound to a chemically modified dendrimer and forming a complex with polyethylene glycol (PEO)-lipid; mRNA with protamine complex in PEG-lipid nanoparticle; mRNA bound to a cationic polymer such as polyethylenimine (PEI); mRNA associated with a cationic polymer such as PEI and a lipid component; mRNA bound to a polysaccharide (eg chitosan) particle or gel; mRNA in cationic lipid nanoparticles (eg, 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP, 1,2-dioleoyloxy-3-trimethylammoniumpropane) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA, which forms a complex with cationic lipids and cholesterol; or mRNA in complex with cationic lipids, cholesterol and PEG lipid. In some embodiments, RNA vaccines are administered by inhalation or oral administration. In some embodiments, the RNA is administered to the blood, thymus, pancreas, skin, muscle, tumor, or other sites and/or through an intradermal, intramuscular, subcutaneous, intranasal, intranodal, intravenous, intrasplenic, intratumoral, or other delivery route.

Polynucleotide or oligonucleotide components can be naked nucleotide sequences or in combination with cationic lipids, polymers or targeting systems. They can be delivered by any available method. For example, the polynucleotide or oligonucleotide may be administered by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide or oligonucleotide can be delivered directly through the skin using a delivery device, such as particle-mediated gene delivery. The polynucleotide or oligonucleotide can be administered topically to the skin or mucosal surface, for example, by intranasal, oral or intrarectal administration.

The uptake of polynucleotide or oligonucleotide constructs can be improved by several known transfection methods, such as those that involve the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-dextran (diethylaminoethyldextran), as well as lipofectants, such as lipofectam and transfectam. The dosage of the administered polynucleotide or oligonucleotide can be changed.

Administration is typically in a prophylactically effective amount or a therapeutically effective amount (as appropriate, although prophylaxis may be considered therapy) sufficient to result in a clinical response or demonstrate clinical effectiveness in the individual, such as an effective amount to prevent or delay the onset of disease. or condition, to relieve one or more symptoms, to induce or prolong remission, or to delay recurrence or recurrence.

The dose can be determined according to various parameters, especially depending on the substance used; the age, weight and condition of the person being treated; routes of administration; and the required mode. The amount of antigen in each dose is chosen as the amount that induces an immune response. The doctor will be able to determine the required route of administration and dosage for any particular person. The dose may be delivered as a single dose or may be delivered as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered hourly. Conventional peptides, polynucleotides or oligonucleotides are typically administered in the range of 1 pg to 1 mg, more typically 1 pg to 10 μg for particle mediated delivery and 1 μg to 1 mg, more typically 1 to 100 μg, more typically 5 to 50 mcg for other routes. Typically, each dose is expected to contain 0.01-3 mg of antigen. The optimal amount for a particular vaccine can be determined through studies involving observation of immune responses in subjects.

Examples of the methods and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub) Lippincott, Williams & Wilkins.

In some cases, more than one peptide or composition of peptides is administered in accordance with the present invention. Two or more pharmaceutical compositions may be administered together/simultaneously and/or at different times or sequentially. Thus, the invention includes kits of pharmaceutical compositions and their use. The use of a combination of different peptides, not necessarily targeting different antigens, is important to overcome the problems of genetic heterogeneity of tumors and HLA heterogeneity in individuals. Multiple pharmaceutical compositions of PEPI formulated for use in a single regimen can form a drug product.

Routes of administration include, but are not limited to, intranasal, oral, subcutaneous, intradermal, and intramuscular. Subcutaneous administration is particularly preferred. Subcutaneous administration may be, for example, an injection into the abdomen, the lateral and anterior aspects of the shoulder or thigh, the scapular region of the back, or the upper ventrodorsal gluteal region.

One skilled in the art will appreciate that the compositions of the present invention may also be administered in one or more doses, as well as other routes of administration. For example, such other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intracardial, intralobally, intramedullary, intrapulmonary, and intravaginal. Depending on the desired duration of treatment, the compositions of the invention may be administered one or more times, also periodically, for example monthly for several months or years, and at different dosages.

Solid dosage forms for oral administration include capsules, tablets, capsule tablets, pills, powders, pellets and granules. In such solid dosage forms, the active ingredient is typically combined with one or more pharmaceutically acceptable excipients, examples of which are described in detail above. Oral medications may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweeteners or flavoring agents, coloring agents and, if desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin and combinations thereof.

One or more compositions of the invention may be administered, or methods and uses for treatment of the invention may be performed, alone or in combination with other pharmacological compositions or treatments, for example chemotherapy and/or immunotherapy, and/or a vaccine. Other therapeutic compositions or treatments may, for example, be one or more of those described herein, and may be administered either simultaneously or sequentially (before or after) the composition or treatment of the present invention.

In some cases, treatment may be given in combination with checkpoint blockade/checkpoint inhibitor therapy, costimulatory antibodies, cytotoxic or noncytotoxic chemotherapy and/or radiotherapy, targeted therapy or monoclonal antibody therapy. Chemotherapy has been demonstrated to sensitize tumors to killing by tumor-specific cytotoxic T lymphocytes induced by vaccination (Ramakrishnan et al. J Glin Invest. 2010; 120(4): 1111-1124). Examples of chemotherapeutic agents include alkylating agents, including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysine) and chlorambucil; anthracyclines; epothilones; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide), dimethyltriazenoimidazole-carboxamide, ethylenimines/methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan, antimetabolites including folic acid analogues such as methotrexate (amethopterin); alkylating agents, antimetabolites, pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside), purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin); epipodophyllotoxins; enzymes such as L-asparaginase; biological response modifiers such as IFNa, IL-2, G-CSF and GM-CSF; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone and anthracycline, substituted ureas such as hydroxyurea, methylhydrazine derivatives including procarbazine (N-methylhydrazine, MIH) and procarbazine; adrenocortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and analogues/derivatives; hormones/hormonal therapy and agonists/antagonists, including adrenocorticosteroid hormone antagonists such as prednisone and its equivalents, dexamethasone and aminoglutethimide, progestins such as hydroxyprogesterone caproite, medroxyprogesterone acetate and megestrol acetate, estrogen, such as diethylstilbestrol and ethinyl estradiol equivalents, antie strict, for example ,

- 42 045699 tamoxifen, androgens including testosterone propionate and fluoxymesterone/equivalents, antiandrogens such as flutamide, gonadotropin-releasing hormone analogues and leuprolide and non-steroidal antiandrogens such as flutamide; natural products including vinca alkaloids such as vinblastine (VLB) and vincristine, epipodophyllotoxins such as etoposide and teniposide, antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C), enzymes such as L asparaginase, and biological response modifiers such as interferon alphanomes.

In some cases, the treatment method is a vaccination method or a method of providing immunotherapy. As used herein, immunotherapy is the treatment of a disease or condition by inducing or enhancing an immune response in an individual. In some embodiments, immunotherapy refers to therapy that includes administering one or more drugs to an individual to induce T lymphocyte responses. In a specific embodiment, immunotherapy refers to a therapy that involves administering or expressing polypeptides that contain one or more PEPIs to an individual to induce a T lymphocyte response to recognize and kill cells that present one or more PEPIs on the cell surface, in combination with HLA class I. In another specific embodiment, the immunotherapy includes administering one or more PEPIs to an individual to induce a cytotoxic T lymphocyte response to cells that present tumor associated antigens (TAA) or cancer testicular antigens (CTAs) containing one or more PEPI on the cell surface. In another embodiment, immunotherapy refers to a therapy comprising administering or expressing polypeptides that contain one or more HLA class II-presented PEPIs to an individual to induce a T helper cell response to provide co-stimulation of cytotoxic T lymphocytes that recognize and destroy diseased cells, presenting one or more cell surface PEPIs in combination with HLA class I. In yet another specific embodiment, immunotherapy refers to a therapy that includes administering to an individual one or more drugs that reactivate existing T lymphocytes to kill target cells. It is theoretically believed that the cytotoxic T lymphocyte response will eliminate cells representing one or more PEPIs, thereby improving the clinical condition of the individual. In some cases, immunotherapy may be used to treat tumors. In other cases, immunotherapy may be used to treat intracellular pathogenic diseases or disorders.

In some cases, the invention relates to the treatment of cancer or the treatment of solid tumors. Treatment can be directed against cancer or malignant or benign tumors of any type of cell, tissue or organ. The cancer may or may not be metastatic. Typical types of cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors or blastomas. The cancer may or may not be a hormone-related or hormone-dependent cancer (eg, estrogen- or androgen-related cancer).

In other cases, the invention relates to the treatment of a viral, bacterial, fungal or parasitic infection or any other disease or condition that can be treated with immunotherapy.

Systems.

The present invention provides a system comprising a memory module configured to store data comprising the HLA class I and/or class II genotype of a subject and the amino acid sequence of one or more test polypeptides; and a computing module configured to identify and/or quantify amino acid sequences in one or more test polypeptides that are capable of binding to multiple HLAs of the subject. The system may be configured to obtain data from at least one sample from at least one subject. The system may comprise an HLA genotyping module to determine the HLA class I and/or class II genotype of a subject. The memory module may be configured to store data output from the genotyping module. The HLA genotyping module may receive a biological sample obtained from a subject and determine the HLA class I and/or class II genotype of the subject. The sample usually contains the subject's DNA. The sample may be, for example, blood, serum, plasma, saliva, urine, exhalation, cellular or tissue sample. The system may further comprise an output module configured to display the sequence of one or more fragments of one or more polypeptides predicted to be immunogenic for a subject, or any output prediction or selection or treatment recommendation described herein document, or the value of any pharmacodynamic biomarker described herein.

Additional embodiments of the invention

1. A human subject-specific pharmaceutical composition for treating a disease or disorder in a specific human subject, comprising:

(a) at least two different polypeptides, each of at least two different

- 43 045699 polypeptides are 10-50 amino acids in length and contain a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three molecules

The HLA class II of the subject, and wherein the T lymphocyte epitope of each of the at least two polypeptides is different from one another, and (b) a pharmaceutically acceptable adjuvant.

2. The human subject-specific pharmaceutical composition according to claim 1, containing at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides.

3. The human subject-specific pharmaceutical composition according to claim 1, containing 3-40 different polypeptides.

4. The human subject-specific pharmaceutical composition according to claim 1, wherein a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject contains from 7 to 11 amino acids, and/or a T-lymphocyte epitope that binds to with at least three HLA class II molecules, contains from 13 to 17 amino acids.

5. The human subject-specific pharmaceutical composition according to claim 1, wherein the epitopes of at least two different polypeptides are derived from the same antigen.

6. The human subject-specific pharmaceutical composition of claim 1, wherein the epitopes of at least two different polypeptides are derived from two or more different antigens.

7. The human subject-specific pharmaceutical composition according to claim 5, wherein the antigen is a cancer cell-expressed antigen, a cancer cell-expressed neo-antigen, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disorder, or is an allergen.

8. The human subject-specific pharmaceutical composition according to claim 7, wherein the cancer cell is obtained from the subject.

9. The human subject-specific pharmaceutical composition according to claim 5, wherein the antigen is selected from the antigens listed in table. 2-7.

10. The human subject-specific pharmaceutical composition of claim 1, wherein the at least two different polypeptides further comprise up to 10 amino acids flanking a T lymphocyte epitope that are part of a rigid sequence flanking the epitope in the corresponding antigen.

11. The human subject-specific pharmaceutical composition of claim 1, wherein the at least two different polypeptides further comprise up to 10 amino acids flanking the T lymphocyte epitope that are not part of the rigid sequence flanking the epitope in the corresponding antigen.

12. The human subject-specific pharmaceutical composition of claim 1, wherein two of at least two polypeptides are end-to-end or overlapping in the composite polypeptide.

13. The human subject-specific pharmaceutical composition of claim 12, comprising two or more different constituent polypeptides, wherein the two or more different constituent polypeptides contain epitopes that are different from each other.

14. The human subject-specific pharmaceutical composition of claim 13, wherein the constituent polypeptides have been screened to eliminate substantially all neo-epitopes encompassing the junction between the two polypeptides, and which:

(i) corresponds to a human polypeptide fragment expressed in healthy cells of the subject;

(ii) is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (iii) meets the requirements of both (i) and (ii).

15. The human subject-specific pharmaceutical composition of claim 1, wherein at least two of the polypeptides do not contain any of the amino acid sequences that:

(i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject.

16. The human subject-specific pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable diluent, carrier, preservative, or a combination thereof.

17. The human subject-specific pharmaceutical composition according to claim 1, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillus

- 44 045699

Calmette-Guerin (BCG), Corynebacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanins (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gels, aluminum hydroxide (alum), lysolecithin , pluronyl polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT, diphtheria toxin) and combinations thereof.

18. A set containing one or more individual containers, each of which contains:

(i) one or more polypeptides 10-50 amino acids in length containing an amino acid sequence representing a T lymphocyte epitope that binds to at least three HLA class I molecules of a subject and/or at least three HLA class II molecules of a subject, and (ii) a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or a combination thereof.

19. The set according to claim 18, containing at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, in which the amino acid sequence of the T-lymphocyte epitope of each of the different polypeptides differs from one another.

20. The set according to claim 18, additionally containing a package insert.

21. A pharmaceutical composition containing a nucleic acid molecule expressing two or more polypeptides, each polypeptide 10-50 amino acids in length containing a T lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class I molecules II subject, wherein each of the two or more polypeptides contains a different T-lymphocyte epitope, and the polypeptides do not contain amino acid sequences that are adjacent to each other in the corresponding antigen.

22. The pharmaceutical composition according to claim 21, in which the nucleic acid molecule expresses at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, each 10-50 amino acids in length, containing an amino acid sequence that is a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T-lymphocyte epitope of each of the different polypeptides differs from one another.

23. A human subject-specific pharmaceutical composition for treating a disease or disorder in a specific human subject, comprising at least one of different polypeptides, wherein each of the at least one of the different polypeptides contains at least a first region and a second region, wherein:

(i) the first region of 10-50 amino acids in length contains an amino acid sequence representing a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, (ii) the second a stretch of 10-50 amino acids in length, containing an amino acid sequence representing a T-lymphocyte epitope that binds to at least three HLA class I molecules of a subject and/or at least two HLA class II molecules of a subject, wherein the amino acid sequence is T-lymphocyte the epitope of each of the first and second regions of each of the at least three different polypeptides contains different sequences.

24. The human subject-specific pharmaceutical composition according to claim 23, comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides.

25. The human subject-specific pharmaceutical composition according to claim 23, containing 2-40 different polypeptides.

26. The human subject-specific pharmaceutical composition according to claim 23, wherein a T lymphocyte epitope that binds to at least three HLA class I molecules of the subject contains from 7 to 11 amino acids, and/or a T lymphocyte epitope that binds to at least three HLA class II molecules, contains from 13 to 17 amino acids.

27. The human subject-specific pharmaceutical composition of claim 23, wherein the epitopes of at least the first and second regions are derived from the same antigen.

28. The human subject-specific pharmaceutical composition of claim 23, wherein the epitopes of at least the first and second regions are derived from two or more different antigens.

29. The human subject-specific pharmaceutical composition according to claim 27, wherein the antigen is an antigen expressed by a cancer cell, a neoantigen expressed by a cancer cell,

- 45 045699 cell-associated antigen, tumor-associated antigen, or antigen expressed by a target pathogen, antigen expressed by a virus, antigen expressed by a bacterium, antigen expressed by a fungus, antigen associated with an autoimmune disorder, or is allergen.

30. The human subject-specific pharmaceutical composition of claim 29, wherein the cancer cell is obtained from the subject.

31. The human subject-specific pharmaceutical composition according to claim 27, wherein the antigen is selected from the antigens listed in table. 2-7.

32. The human subject-specific pharmaceutical composition of claim 23, wherein the polypeptides have been screened to eliminate substantially all neo-epitopes spanning the junction between the two regions, and which:

(i) corresponds to a human polypeptide fragment expressed in healthy cells of the subject;

(ii) is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (iii) meets the requirements of both (i) and (ii).

33. The human subject-specific pharmaceutical composition of claim 23, wherein the at least one polypeptide does not contain any of the amino acid sequences that:

(i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject.

34. The human subject-specific pharmaceutical composition of claim 23, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or a combination thereof.

35. The human subject-specific pharmaceutical composition of claim 34, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus Calmette-Guerin (BCG), Corynebacterium parvum, levamisole , azimezone, isoprinisone, dinitrochlorobenzene (DNCB), cochlear lymph hemocyanins (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gels, aluminum hydroxide (alum), lysolecithin, pluronyl polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT, diphtheria toxin) and combinations thereof.

36. A method of preparing a human subject-specific pharmaceutical composition for use in a method of treating a specific human subject, comprising:

(i) selecting a polypeptide fragment that has been identified as immunogenic in the subject by:

a) determining whether the polypeptide contains:

1) an amino acid sequence that is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, or

2) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class II molecules of the subject, or

3) or meets the requirements of both (1) and (2), and

b) identifying said sequence as a fragment of a polypeptide that is immunogenic for a subject;

(ii) selecting a first sequence of up to 50 consecutive amino acids of polypeptides, wherein the consecutive amino acids include the amino acid sequence of the fragment selected in step (i), and (iii) preparing a subject-specific pharmaceutical composition containing as active ingredients one or more polypeptides, which together have all the amino acid sequences selected in the previous steps.

37. The method of claim 36, further comprising, prior to the production step, repeating steps (i)-(ii) to select a second amino acid sequence from up to 50 consecutive amino acids of the same or a different polypeptide relative to the first amino acid sequence;

38. The method of claim 37, further comprising, prior to the production step, further repeating steps (i)-(ii) one or more times to select one or more additional amino acid sequences from up to 50 consecutive amino acids of the same or different polypeptides relative to the first and second amino acid sequences.

39. The method of claim 36, further comprising, prior to the production step, selecting a longer fragment of the polypeptide if the fragment selected in step (i) is an HLA class I binding epitope, wherein the longer fragment comprises the fragment selected in step ( i); and is a T-lymphocyte epitope capable of binding to at least three molecules

- 46 045699 HLA class II of the subject.

40. The method of claim 36, wherein each polypeptide either consists of one of the selected amino acid sequences or contains or consists of two or more selected amino acid sequences arranged end to end or overlapping in a single composite polypeptide.

41. The method of claim 36, wherein any neoepitopes formed at the junction between any two of selected amino acid sequences located end to end in one composite polypeptide are screened to eliminate substantially all polypeptides containing the amino acid sequence of the neoepitope , which:

(v) corresponds to a human polypeptide fragment expressed in healthy cells;

(vi) is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (vii) meets the requirements of both (i) and (ii).

42. The method of claim 36, wherein the one or more polypeptides have been screened to eliminate polypeptides containing an amino acid sequence that:

(i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject.

43. The method of claim 36, further comprising determining an HLA class I genotype and an HLA class II genotype from a biological sample of the subject before step (i).

44. The method according to claim 43, in which the determination of the HLA class I genotype and the HLA class II genotype is performed by sequence based typing (SBT) methods.

45. The method of claim 43, wherein determination of the HLA class I genotype and the HLA class II genotype is carried out by sequencing, next generation sequencing, sequence specific primer (SSP) methods or sequence specific oligonucleotide (SSO) methods specific oligonucleotide).

46. The method of claim 43, wherein the biological sample is blood, serum, plasma, saliva, buccal swab, urine, exhalation, cell or tissue.

47. The method of claim 43, wherein the biological sample is saliva or a buccal swab.

48. A method of treating cancer in a particular human subject in need thereof, comprising administering to the particular human subject a pharmaceutical composition comprising at least one polypeptide, wherein each of the at least one polypeptide, 10-50 amino acids in length, contains a first amino acid sequence that is a T lymphocyte epitope that binds to at least three HLA class I molecules of a subject and/or at least three HLA class II molecules of a subject, wherein the T lymphocyte epitope of each of the at least one polypeptide is derived from an antigen that is specific for cancer.

49. The method according to claim 48, in which the composition contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, wherein the amino acid sequence of the T lymphocyte epitope of each of the different polypeptides is different from one another and is derived from one or more antigens expressed by a cancer cell from the subject.

50. The method of claim 48, wherein the composition contains 2-40 different polypeptides.

51. The method of claim 48, wherein the T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject contains from 7 to 11 amino acids, and/or the T-lymphocyte epitope that binds to at least three HLA molecules class II, contains from 13 to 17 amino acids.

52. The method of claim 48, wherein the composition comprises at least two different polypeptides and the amino acid sequence epitopes of the at least two different polypeptides are derived from the same antigen.

53. The method of claim 48, wherein the composition comprises at least two different polypeptides and the epitopes of the at least two different polypeptides are derived from two or more different antigens.

54. The method of claim 48, wherein the one or more antigens is a neoantigen expressed by a cancer cell, a cancer-associated antigen, or a tumor-associated antigen.

55. The method according to claim 48, in which one or more antigens is selected from the antigens listed in table. 2.

56. The method of claim 48, wherein the at least one different polypeptide further comprises up to 10 amino acids flanking a T lymphocyte epitope that are part of a rigid sequence flanking the epitope in the corresponding antigen.

- 47 045699

57. The method of claim 48, wherein the at least one different polypeptide further comprises up to 10 amino acids flanking the T lymphocyte epitope that are not part of the rigid sequence flanking the epitope in the corresponding antigen.

58. The method of claim 48, wherein the composition comprises at least two different polypeptides, wherein two of the polypeptides are end-to-end or overlapping in the composite polypeptide.

59. The method of claim 58, comprising two or more different constituent polypeptides, wherein the two or more different constituent polypeptides contain epitopes that are different from each other.

60. The method of claim 59, wherein the composite polypeptides have been screened to eliminate substantially all neo-epitopes spanning the junction between the two polypeptides, and which:

(i) corresponds to a human polypeptide fragment expressed in healthy cells of the subject;

(ii) is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (iii) meets the requirements of both (i) and (ii).

61. The method of claim 48, wherein the at least one polypeptide does not contain any of the amino acid sequences that:

(i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T-lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject.

62. The method of claim 48, wherein the composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or a combination thereof.

63. The method of claim 62, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus Calmette-Guérin (BCG), Corynebacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole lymph hemocyanins (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gels, aluminum hydroxide (alum), lysolecithin, pluronyl polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT, diphtheria toxin) and their combinations.

64. The method of claim 48, further comprising administering a chemotherapeutic agent, targeted therapy, radiotherapy, a checkpoint inhibitor, other immunotherapy, or combinations thereof.

65. A human subject-specific pharmaceutical composition for the treatment of a disease or disorder in a specific human subject, comprising: (a) a polypeptide 10-50 amino acids in length and containing a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject; and (b) a pharmaceutically acceptable adjuvant.

66. The human subject-specific pharmaceutical composition according to claim 65, comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different polypeptides, wherein each of the different polypeptides, 10-50 amino acids in length, contains a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequence of the T-lymphocyte epitope of each of the different polypeptides differs from one another.

67. The human subject-specific pharmaceutical composition of claim 66, comprising 2-40 different polypeptides.

68. The human subject-specific pharmaceutical composition of claim 65, wherein a T lymphocyte epitope that binds to at least three HLA class I molecules of the subject contains from 7 to 11 amino acids, and/or a T lymphocyte epitope that binds to at least three HLA class II molecules, contains from 13 to 17 amino acids.

69. The human subject-specific pharmaceutical composition of claim 66, comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are derived from the same antigen.

70. The human subject-specific pharmaceutical composition of claim 66, comprising at least two different polypeptides, wherein the epitopes of the at least two different polypeptides are derived from two or more antigens.

71. The human subject-specific pharmaceutical composition of claim 69, wherein the antigen is a cancer cell-expressed antigen, a cancer cell-expressed neo-antigen, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogen, antigen expressed by a virus, antigen expressed by a bacterium, antigen expressed by a fungus, antigen associated with an autoimmune

- 48 045699 violation, or is an allergen.

72. The human subject-specific pharmaceutical composition of claim 71, wherein the cancer cell is obtained from the subject.

73. The human subject-specific pharmaceutical composition according to claim 69, wherein the antigen is selected from the antigens listed in table. 2-7.

74. The human subject-specific pharmaceutical composition of claim 69, comprising at least two different polypeptides, wherein two of the polypeptides are end-to-end or overlapping in the composite polypeptide.

75. The human subject-specific pharmaceutical composition of claim 65, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus Calmette-Guerin (BCG), Corynebacterium parvum, levamisole , azimezone, isoprinisone, dinitrochlorobenzene (DNCB), cochlear lymph hemocyanins (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gels, aluminum hydroxide (alum), lysolecithin, pluronyl polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT, diphtheria toxin) and combinations thereof.

76. The human subject-specific pharmaceutical composition of claim 65, comprising at least two different polypeptides, wherein two of the at least two polypeptides are end-to-end or overlapping in the composite polypeptide.

77. The human subject-specific pharmaceutical composition of claim 76, comprising two or more different constituent polypeptides, wherein the two or more different constituent polypeptides contain epitopes that are different from each other.

78. The human subject-specific pharmaceutical composition of claim 77, wherein the constituent polypeptides have been screened to eliminate substantially all neo-epitopes spanning the junction between the two polypeptides, and which:

(i) corresponds to a human polypeptide fragment expressed in healthy cells of the subject;

(ii) is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject, or (iii) meets the requirements of both (i) and (ii).

79. The human subject-specific pharmaceutical composition of claim 66, wherein at least two of the polypeptides do not contain any of the amino acid sequences that:

(i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least two HLA class I molecules of the subject.

80. Set containing:

(a) a first human subject-specific pharmaceutical composition comprising (i) a first polypeptide of 10-50 amino acids in length, and comprising a T-lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three the subject's HLA class II molecules; and (ii) a pharmaceutically acceptable adjuvant;

(b) a second human subject-specific pharmaceutical composition comprising (i) a second polypeptide of 10-50 amino acids in length, and comprising a T lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three the subject's HLA class II molecules; and (ii) a pharmaceutically acceptable adjuvant, wherein the first and second polypeptides contain different T lymphocyte epitopes.

81. The kit of claim 77, wherein the first composition and/or the second composition comprise one or more additional polypeptides, wherein each additional polypeptide, 10-50 amino acids in length, contains an amino acid sequence that is a T lymphocyte epitope that binds to at least three HLA class I molecules of the subject and/or at least three HLA class II molecules of the subject, wherein the amino acid sequences contain different T lymphocyte epitopes.

Examples

Example 1: Process for Predicting and Confirming HLA Epitope Binding.

Prediction of binding between specific HLAs and epitopes (9-mer peptides) was based on the Immune Epitope Database epitope prediction tool (www.iedb.org).

The HLA I epitope binding prediction process was validated by comparison with HLA I epitope pairs determined in laboratory experiments. The dataset was compiled from HLA I-epitope pairs described in expert publications or publicly available immunological databases.

A measure of agreement with the experimentally determined data set was determined (Table 9). The binding of HLA I-epitope pairs for the data set was correctly predicted with a probability of 93%. By coincidence, unrelated HLA I-epitope pairs were also correctly sprog

- 49 045699 are nosed with a probability of 93%.

Table 9 Analytical specificity and sensitivity of the HLA epitope binding prediction process _______HLA-epitope pairs _______True epitopes (n=327) False epitopes (n=100)

(Binding Match) (Matching non-binding) HIV 91% (32) 82%(14) Viral 100% (35) 100% (11) Tumor 90% (172) 94% (32) Other (fungi, bacteria, etc.) 100% (65) 95% (36) Total 93% (304) 93% (93)

The accuracy of prediction of multi-HLA binding epitopes was determined) Based on analytical specificity and sensitivity using 93% probability for true positive and true negative prediction and 7% probability (= 100%-93%) for false positive and false negative prediction, can be calculated the likelihood of an epitope that binds to multiple HLAs in an individual. The probability of multiple HLAs binding to an epitope shows the relationship between the number of HLAs binding to the epitope and the expected minimum amount of actual binding. According to the PEPI definition, three is the expected minimum amount of HLA for epitope binding (bold).

Table 10

Prediction accuracy of epitopes binding to multiple HLA____

Expected minimum amount of actual HLA binding Predicted number of HLA bindings to epitope 0 1 2 3 4 5 1 35% 95% 100% 100% 100% 100% 2 6% 29% 90% 99% 100% 100% 3 1 % 4% 22% 84% 98% 100% 4 0% 0% 2% 16% 78% 96% 5 0% 0% 0% 1 % 10% 71% 6 0% 0% 0% 0% 0% 5 %

A validated HLA-epitope binding prediction process was used to determine all of the binding HLA-epitope pairs described in the examples below.

Example 2: Epitope presentation by multiple HLA predicts cytotoxic T lymphocyte (CTL) response.

Presentation of one or more polypeptide antigen epitopes by one or more of an individual's HLA I has been determined to be predictive of CTL response.

The study was conducted by retrospectively analyzing six clinical trials conducted on 71 patients with cancer and 9 HIV-infected patients (Table 11) ¹⁷ . Patients from these studies were treated with an HPV vaccine, three different NY-ESO-1 cancer vaccines, one HIV-1 vaccine, and a specific monoclonal antibody CTLA-4 (ipilimumab), which has been shown to reactivate CTL against the NY-ESO-1 antigen. 1 in patients with melanoma. All of these clinical trials measured antigen-specific CD8+ CTL responses (immunogenicity) in study subjects following vaccination. In some cases, a correlation between CTL responses and clinical responses has been reported.

No patients were excluded from the retroactive study for any reason other than data availability. The 157 patient data sets (Table 11) were randomized using a standard random number generator to create two independent groups for the training and evaluation studies. In some cases, groups contained multiple data sets from the same patient, resulting in a training group of 76 data sets from 48 patients and a test/validation group of 81 data sets from 51 patients.

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Table 11

Summary table of patient datasets

Clinical trial Immunotherapy Antigen target Diseases e Number of patients* Number of data sets (number of antigen x number of patients) Immunoassays performed in clinical trials** JHEA genotyping method Link 1 VGX-3100 HPV16-E6 HPV16-E7 HPV18-E6 HPV18-E7 HPV16|18 Cervical cancer 17|18 5 x 17 IFN-γ ELISPOT High Definition SBT 1 2 HIV vaccine HIV-1 Gag HIV-1 RT AIDS 9/12 2x9 IFN-γ ELISPOT SSO with low-medium resolution 2 3 rNY-ESO-1 NY-ESO-1 Breast and ovarian cancer, melanoma and sarcoma 18/18 1x18 In vitro and Ex vivo IFN-r ELISPOT High Definition SBT 3 4 4 Ipilimumab NY-ESO-1 Metastatic melanoma 19/20 1 x 19 ICS after T lymphocyte stimulation Low-medium resolution typing, genomic DNA SSP, high-resolution sequencing 5 5 NY-ESO-lf NY-ESO-1 (91-110) Esophageal cancer, non-small cell lung cancer and gastric cancer 10/10 1x10 ICS after T lymphocyte stimulation SSO study and SSP genomic DNA 6 6 Overlap with other peptides NY-ESO-1 NY-ESO-1 (79-173) Esophageal cancer, lung cancer, malignant melanoma 7/9 1x7 ICS after T lymphocyte stimulation SSO study and SSP genomic DNA 7 Total 6 7 80 157 Not applicable

^Number of patients used in retrospective analysis compared to original number of patients from clinical trials.

**Immunoassays are based on stimulation of T lymphocytes with antigen-specific peptide pools and determination of the amount of cytokines released by various methods.

CT: clinical trial; SBT: sequence-based typing; SSO: sequence-specific oligonucleotide; ICS: intracellular cytokine staining; SSP: site-specific priming.

The recorded CTL responses of the training dataset were compared to the HLAI-restricted epitope profile (9-mers) of the antigens used to prepare the vaccine. Antigen sequences and HLA I genotype of each patient were obtained from publicly available protein sequence databases or expert publications, and the HLA I epitope binding prediction process was performed blind to the patients' clinical CTL response data. The number of epitopes from each antigen predicted to be associated with at least 1 (PEPI1+), or at least 2 (PEPI2+), or at least 3 (PEPI3+), or at least 4 (PEPI4+), or at least at least 5 (PEPI5+) or all 6 (PEPI6) HLA class I molecules of each patient, and the number of associated HLAs were used as classifiers for the recorded CTL responses. The true positive rate (sensitivity) and true negative rate (specificity) were determined from the training data set for each classifier (number of associated HLAs) separately.

ROC curve analysis was performed for each classifier. In the ROC curve, the true positive rate (sensitivity) was plotted against the false positive rate (1-specificity) for various cut-off points (Fig. 1). Each point on the ROC curve represents a sensitivity/specificity pair corresponding to a certain decision threshold (number of epitopes (PEPI)). The area under the ROC curve (AUC) is a measure of how well the classifier can distinguish between two diagnostic groups (patient with CTL response or patient without response).

The analysis unexpectedly revealed that predicted epitope presentation by multiple HLA class I subjects (PEPI2+, PEPI3+, PEPI4+, PEPI5+, or PEPI6) in each case was a better predictor of CTL response than epitope presentation by only one or more HLA class I (PEPI1+, AUC=0 ,48, Table 12).

Table 12

Determining the diagnostic value of PEPI biomarker using ROC curve analysis AUC classifiers

PEPI1+ 0.48 PEPI2+ 0.51 PEPI3+ 0.65 PEPI4+ 0.52 PEPI5+ 0.5 PEPI6+ 0.5

An individual's CTL response was best predicted by taking into account antigen epitopes that could be presented by at least 3 of the individual's HLA class I (PEPI3+, AUC=0.65, Table 12). The threshold number of PEPI3+ (the number of antigen-specific epitopes presented by 3 or more of an individual's HLA) that best predicted a positive CTL response was 1 (Table 13). In other words, when at least one antigen-derived epitope is presented to at least 3 HLA class I of a subject (>1 PEPI3+), then the antigen can initiate the formation of at least one CTL clone, and the subject is likely to be a patient with a CTL response. Using a cutoff value of >1 PEPI3+ to predict probable patients with a CTL response (test >1 PEPI3+) provided a diagnostic sensitivity of 76% (Table 13).

Table 13

Determination of a cutoff value >1 PEPI3+ to predict likely patients with a CTL response in the training dataset. Quantity of PEPI3+

1 2 3 4 5 6 7 8 9 eleven 12 Sensitivity: 1- 0.76 0.60 0.31 0.26 0.14 0.02 0 0 0 0 0 specificity: 0.59 0.24 0.21 0.15 0.09 0.06 0.06 0.03 0.03 0.03 0.03

Example 3: Test confirmation > 1 PEPI3+.

A test set of 81 data from 51 patients was used to test a cutoff value of >1 PEPI3+ to predict antigen-specific CTL response. For each data set in the test group, it was determined whether a threshold value of >1 PEPI3+ (at least one antigen-derived epitope presented by at least three of the individual's class I HLAs) was reached. It was compared with experimentally determined CTL responses obtained in clinical trials (Table 14).

Clinical validation showed that the PEPI3+ peptide induces a CTL response in humans with an 84% success rate. 84% is the same value that was determined by analytical confirmation of the PEPI3+ prediction, epitopes that bind to at least 3 HLAs of an individual (Table 10). These data provide compelling evidence that PEPIs induce an immune response in humans.

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Table 14

Diagnostic test performance characteristics > 1 PEPI3+ (n=81)

Performance characteristics

Positive 100% [A/(A + B)] predictive value, PPV)

Sensitivity 100%[A/(A+C)]

Specificity 100%[D/(B + D)]

Negative 100%[D/(C +D)] predictive value, NPV)

Overall Percentage of Agreement (OPA) 100%[(A + D)/ N]

Fisher's exact test (p) „ Resul

Description of tat

The probability that an individual who meets the >1 PEPI3+ threshold has antigen-specific CTL responses after treatment with immunotherapy.

Proportion of subjects with antigen-specific responses

CTLs after treatment with immunotherapy that 75% meet the threshold of >1 PEPI3+.

Proportion of subjects without antigen-specific CTL responses after treatment with immunotherapy that do not meet the 55% threshold of >1 PEPI3+.

The likelihood that an individual who does not meet the >1 PEPI3+ threshold will not have antigen-specific CTL responses after treatment with immunotherapy.

Percentage of predictions based on a cutoff value >1 PEPI3+ that match the experimentally determined outcome, either positive or negative.

0.01

ROC curve analysis determined diagnostic accuracy using PEPI3+ counts as cutoff values (Figure 2). AUC value=0.73. For ROC curve analysis, an AUC value of 0.7 to 0.8 is generally considered a reliable diagnosis.

A PEPI3+ count of at least 1 (>1 PEPI3+) best predicted the CTL response in the test data set (Table 15). This result confirmed the threshold value determined during training (Table 12).

Table 15

Validation of >1 PEPI3+ threshold for predicting likely patients with CTL response in test/validation dataset Number of PEPI3+

1 2 3 4 5 6 7 8 9 10 AND 12 Sensitivity 0.75 0.52 0.26 0.23 0.15 0.13 0.08 0.05 0 0 0 0 1-specificity: 0.45 0.15 0.05 0 0 0 0 0 0 0 0 0

Example 4: Test >1 PEPI3+ predicts CD8+ CTL reactivity.

The >1 PEPI3+ test was compared with a previously described method to predict the CTL response of a particular human subject to peptide antigens.

HLA genotypes in 28 patients with cervical cancer and VIN-3 who received the synthetic long peptide vaccine (LPV) HPV-16 in two different clinical trials were determined based on DNA samples ^{8 8 9 10} . LPV consists of long peptides encompassing the E6 and E7 viral oncoproteins of HPV-16. The amino acid sequence of LPV was obtained from these publications. The publications also report the T cell responses of each vaccinated patient to pools of overlapping vaccine peptides.

For each patient, epitopes (9 mers) of LPV that are present in at least three of the patient's HLA class I (PEPI3+) were identified and their distribution among the peptide pools was determined. Peptides that contained at least one PEPI3+ (>1 PEPI3+) were predicted to induce a CTL response. Peptides that did not contain PEPI3+ were predicted not to induce a CTL response.

The >1 PEPI3+ test correctly predicted 489 of 512 negative CTL responses and 8 of 40 positive CTL responses measured after vaccination ( Fig. 3A ). Overall, agreement between the >1 PEPI3+ test and experimentally determined CD8+ T cell reactivity was 90% (p < 0.001).

For each patient, the distribution among the peptide pools of epitopes that are presented by at least one HLA class I (>I PEPI1+, HLA-restricted epitope prediction, prior art method) was also determined. The >1 PEPI1+ test correctly predicted 116 of 512 negative CTL responses and 37 of 40 positive CTL responses measured after vaccination ( Fig. 3B ). Overall, agreement between HLA-restricted epitope prediction (>1

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PEPI1+) and the reactivity of CD8+ T lymphocytes was 28% (not significant).

Example 5: Prediction of HLA class II-restricted epitopes of CD4+ T helper cells.

patients with cervical cancer and VTN-3 who received the synthetic long peptide vaccine (LPV) HPV-16 in two different clinical trials (as detailed in Example 4) were examined for CD4+ T helper cell responses following LPV vaccination (Fig. 4 ). The sensitivity for predicting HLA class II restricted epitopes was 78%, as the prior art tool predicted 84 positive responses (positive CD4+ T cell reactivity to the pool of peptides for human DP alleles) out of 107 (sensitivity=78%). The specificity was 22% because it could exclude 7 negative responses out of 31. Overall, the agreement between HLA class II restricted epitope prediction and CD4+ T cell reactivity was 66%, which was not statistically significant.

Example 6: Test >1 PEPI3+ predicts T-lymphocyte responses to full-length LPV polypeptides.

Using the same assay results as Examples 4 and 5, the >1 PEPI3+ test was used to predict patient CD8+ and CD4+ T cell responses to the full length E6 and E7 polypeptide antigens of the LPV vaccine. The results were compared with experimentally determined responses that were reported. The test correctly predicted CD8+ T cell reactivity (PEPI3+) in 11 of 15 patients with VIN-3 with positive CD8+ T cell reactivity test results (sensitivity 73%, PPV 85%) and in 2 of 5 patients with cervical cancer (sensitivity 40%, PPV 100%). CD4+ T cell reactivity (PEPI4+) was correctly predicted at 100% in both VIN-3 and cervical cancer patients (Figure 5).

It was also noted that the amount of HLA class I and class II-restricted PEPI3+ is consistent with the reported clinical efficacy for patients vaccinated with LPV. Patients with higher amounts of PEPI3+ had a complete or partial response after 3 months.

Example 7: Case Study.

pGX3001 is an HPV16-based DNA vaccine containing full-length E6 and E7 antigens with a linker between them. pGX3002 is an HPV18-based DNA vaccine containing full-length E6 and E7 antigens with a linker between them. A phase II clinical trial examined the T cell responses of 17 HPV-infected cervical cancer patients who were vaccinated with both pGX3001 and pGX3002 (VGX-3100 vaccination) ¹ .

Fig. 5-6 illustrate for two representative patients (patient 12-11 and patient 14-5) the position of each epitope (9-mer) presented by at least 1 (PEPI1+), at least 2 (PEPI2+), at least 3 ( PEPI3+), at least 4 (PEPI4+), at least 5 (PEPI5+) or all 6 (PEPI6) HLA class I of these patients in the full-length sequence of two HPV-16 and two HPV-18 antigens.

Patient 12-11 had a total PEPI1+ count of 54 for combination vaccines (54 epitopes presented by one or more HLA class I). Patient 14-5 had a PEPI1+ count of 91. Therefore, Patient 14-5 has a higher PEPI1+ count than Patient 12-11 for the four HPV antigens. PEPI1+ are sets of distinct vaccine antigen-specific HLA-restricted epitopes from patients 12-11 and 14-5. Only 27 PEPI1+ were common to these two patients.

For PEPI3+ counts (the number of epitopes presented by three or more HLA class I patients), the results for patients 12-11 and 14-5 were reversed. Patient 12-11 had 8 PEPI3+, including at least one PEPI3+ in each of the four HPV16/18 antigens. Patient 14-5 had 0 PEPI3+.

The recorded immune responses of these two patients corresponded to the amounts of PEPI3+, not to the amounts of PEPI1+. Patient 12-11 developed immune responses to each of the four antigens as measured by ELISpot following vaccination, while Patient 14-5 did not develop immune responses to any of the four vaccine antigens. A similar pattern was observed when comparing the PEPI1+ and PEPI3+ kits in all 17 patients in the trial. There was no agreement between the amount of PEPI1+ and experimentally determined T-lymphocyte responses obtained from the clinical trial. However, there was agreement between T-lymphocyte immunity predicted by the >1 PEPI3+ test and reported T-lymphocyte immunity. A >1 PEPI3+ test predicted patients to have an immune response to the HPV DNA vaccine.

In addition, the diversity of the patient's PEPI3+ repertoire resembled the diversity of T cell responses typically found in cancer vaccine trials. Patients 12-3 and 12-6, similar to patient 14-5, did not have PEPI3+ predictive of HPV vaccine failure to induce T-cell immunity. All other patients had at least one PEPI3 predictive of the likelihood that HPV vaccine would induce T-54 045699 lymphocyte immunity. Eleven patients had multiple PEPI3+ scores predicting that HPV vaccine is likely to induce polyclonal T-lymphocyte responses. Patients 15-2 and 15-3 could form a high level of T-lymphocyte immunity to E6 of both HPVs, but had poor immunity to E7. Other patients 15-1 and 12-11 had similar response rates to E7 for

HPV18 and HPV16, respectively.

Example 8: Development of a simulated population for in silico testing and identification of potential precision vaccine targets for a large population.

In silico test group of 433 human subjects with full 4-digit HLA class I genotype (2 x HLA-A*xx:xx; 2 x HLA-B *xx:xx; 2 x HLA-C*xx:xx) and demographic information. This simulated population includes subjects of mixed ethnicity having a total of 152 different HLA alleles, which are representative of more than 85% of currently known allelic G groups.

A large population database containing 7189 subjects characterized by 4-digit HLA genotype and demographic information was also created. The large population included 328 different HLA class I alleles. The distribution of HLA alleles in the simulated population was largely consistent with the large population (Table 16) (Pearson's goodness-of-fit test p < 0.001). Thus, the simulated population of 433 patients is representative of a population 16 times larger.

The simulated population is representative of 85% of the human race, as determined by HLA diversity and HLA frequency.

Table 16

Statistical analysis of HLA distribution in the Simulated population compared to the Large population

Group name 1 Group name 2 Pearson R value Correlation P value Simulated Large population 0.89 Strong P<0.001 population 433 7189

Example 9: In silico tests based on the identification of multiple HLA-binding epitopes predict reported frequencies of T-lymphocyte response in clinical trials.

The objective of this study was to determine whether a simulated population, such as that described in Example 8, could be used to predict vaccine CTL reactivity levels, i.e. used in in silico efficacy trials.

Twelve peptide vaccines derived from cancer antigens that induced T lymphocyte responses in a subpopulation of subjects were identified from expert publications. These peptides were studied in clinical trials involving 172 patients (4 ethnic groups). T-lymphocyte responses induced by vaccine peptides were determined from blood samples and recorded. The immune response rate was determined as the percentage of study subjects with positive T-lymphocyte responses measured in clinical trials (Fig. 7).

Table 17

Clinical trials conducted with peptide vaccines

Peptide vaccines Original antigen Peptide length Tlymphocyte Analysis Populations (n) Ethnicity Link MMNLMQPKTQQTYTYD JUP 16mer Multimer staining 18 Canadians 12 GRGSTTTNYLLDRDDYRNTSD ADA17 21mer Multimer staining 18 Canadians 12 LKKGAADGGKLDGNAKLNRSLK VAR31 22mer Multimer staining 18 Canadians 12 FPPKDDHTLKFLYDDNQRPYPP TOP2A 22mer Multimer staining 18 Canadians 12 RYRKPDYTLDDGHGLLRFKST AY-2 21mer Multimer staining 18 Canadians 12 QRPPFSQLHRFLADALNT DDR1 18mer Multimer staining 18 Canadians 12

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ALDQCKTSCALMQQHYDQTSCFSSP ITGB8 25step Multimer staining 18 Canadians 12 STAPP AHGVTSAPDTRPAPGST APP MUC-1 25step Proliferation 80 Canadians 13 YLEPGPVTA gplOO 9mer Tetramer 18 USA 14 MTPGTQSPFFLLLLLLTVLTW MUC-1 21mer Cytotoxicity 10 Israelis 15 SSKALQRPV Bcr-Abl 9mer ELISPOT (enzyme-linked immunospot method) 4 USA 16 RMFPNAPYL WT-1 9mer Multimer staining 24 USA 17 RMFPNAPYL (HLA-A*0201) WT-1 9mer Cytokine staining 18 CEU (Europeans) 18

peptides were screened using the >1 PEPI3+ test in each of the 433 subjects in the simulated population described in Example 8. The >1 PEPI3+ score for each peptide was calculated as the proportion of subjects in the simulated population having at least one vaccine-derived epitope that could bind with at least three HLA class I subjects (>1 PEPI3+). If the corresponding clinical trial stratified patients by the HLA allele of the selected population, the simulated population was also filtered for subjects with the corresponding allele(s) (example: WT1, HLA-A*0201).

Experimentally determined response rates obtained across trials were compared to >1 PEPI3+ scores. Overall percentage agreement (OPA) was calculated based on paired data (Table 18). A linear correlation was also found between PEPI3+ score >1 and response rate (R ² =0.77) (Figure 7). This result demonstrates that the identification of peptides predicted to bind to multiple HLAs in an individual is useful for predicting the results of clinical trials in silico.

Table 18

Comparison of >1 PEPI3+ and CTL response rates for 12 peptide vaccines

Peptide vaccine Original antigen Frequency answers (clinical trials) Indicator > 1 PEPI3* (simulated population) ORA MMNLMQPKTQQTYTYD JUP 0% 22% Not applicable GRGSTTTNYLLDRDDYRNTSD ADA 17 eleven % 18% 61% LKKGAADGGKLDGNAKLNRSLK BAP31 eleven % 7% 64% FPPKDDHTLKFLYDDNQRPYPP TOP2A eleven % 39% 28% RYRKPDYTLDDGHGLLRFKST Abl-2 17% 12% 71% QRPPFSQLHRFLADALNT DDR1 17% 5 % 29% ALDQCKTSCALMQQHYDQTSCFSSP ITGB8 28% 31% 90% STAPPAHGVTSAPDTRPAPGSTAPP MUC-1 20% 2% 10% YLEPGPVTA gplOO 28% 4% 14% MTPGTQSPFFLLLLLLTVLTW MUC-1 90% 95% 95% SSKALQRPV Bcr-Abl 0% 0% 100 % RMFPNAPYL WT-1 100 % 78% 78% RMFPNAPYL (HLA-A*0201) WT-1 81% 61% 75%

*% of subjects in the simulated population with >1 PEPI3+-derived vaccine.

Example 10: In silico tests based on identification of multiple HLA binding epitopes predict reported frequencies of T cell response in clinical trials II.

Nineteen clinical trials with published immune response rates (IRRs) using peptide or DNA vaccines were identified (Table 19). These studies included 604 patients (9 ethnic groups) and included 38 vaccines derived from tumor and viral antigens. Vaccine antigen-specific CTL responses were measured in each study patient, and response rates were calculated and recorded in the clinical study populations.

Each vaccine peptide from the 19 clinical trials was tested using a >1 PEPI3+ test in each subject of the simulated population. A score of >1 PEPI3+ for each peptide was calculated as the proportion of subjects in the modeled population having at least one vaccine-derived PEPI3+. Experimentally determined response frequencies obtained during the tests were compared with PEPI indicators, as in example 9 (Table 20). There was a linear correlation between response rate and PEPI3+ score >1 (R2=0.70) (Fig. 8). This result confirms that identifying peptides predicted to bind to multiple HLAs in an individual can predict T cell responses in subjects, and in silico testing can predict clinical trial outcomes.

Table 19

Response rates published in clinical trials

Immunotherapy Type CTL analysis Population (P) Race/Ethnicity Link StimuVax peptide Proliferation 80 Canadians 13 Vaccine gp 100 DNA Tetramer 18 USA 14 IMA901 phase! peptide ELISPOT (enzymelinked immunospot method) 64 CEU (Europeans) 19 IMA901 phase II peptide Multimer staining 27 CEU (Europeans) ICT107 peptide ICC 15 USA 20 ProstVac DNA ELISPOT (enzymelinked immunospot method) 32 CEU 87%, Africa. Amer, 12% Spanish 1 % 21 Synchrotope TA2M DNA Tetramer 26 USA 22 MELITAC 12.1 peptide ELISPOT (enzymelinked immunospot method) 167 USA 23 WT1 vaccine peptide Tetramer 22 Japanese 24 Ipilimumab (NY-ESO-1) checkpoint inhibitor** ICC 19 USA 5 VGX-3100 DNA ELISPOT (enzymelinked immunospot method) 17 USA 1 HIVIS-1 DNA ELISPOT (enzymelinked immunospot method) 12 CEU98%,. Asians 1%. Spanish 1 % 2 ImMucin peptide Cytotoxicity 10 Israelis 15 NY-ESO-1 OLP peptide IFN-gamma 7 Japanese 7 GVX301 peptide Proliferation 14 CEU (Europeans) 25 WT1 vaccine peptide ELISPOT (enzymelinked immunospot method) 12 USA 26 WT1 vaccine peptide ICC 18 CEU (Europeans) 18 DPX-0907* peptide Multimer staining 18 Canadians 12 Peptide vaccine against melanoma peptide ELISPOT (enzymelinked immunospot method) 26 White 27

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Table 20

Linear correlation between PEPI score and response rate (R ² =0.7)

Immunotherapy Clinical trial response rate Indicator >1 PEPI3+* ORA StimuVax (failed to show 20 % 2% 10 % effectiveness in phase III) Vaccine gp 100 28% 4% 14% IMA901 phase! 74% 48% 65% IMA901 phase II 64% 48% 75% ICT107 33% 52% 63% ProstVac 45% 56% 80% Synchrotope TA2M 46% 24% 52% MELITAC 12.1 49% 47% 96% WT1 vaccine 59% 78% 76% Ipilimumab (NY-ESO-1*) 72% 84% 86% VGX-3100 78% 87% 90% HIVIS-1 80% 93% 86% ImMucin 90% 95% 95% NY-ESO-1 OLP 100 % 84% 84% GVX301 64% 65% 98% WT1 vaccine 83% 80% 96% WT1 vaccine 81% 61% 75% DPX-0907 61% 58% 95% Peptide vaccine against melanoma 52% 42% 81%

* % of subjects in the simulated population with > 1 PEPI3+-derived vaccine.

Example 11 An in silico test based on the identification of multiple HLA binding epitopes in a multipeptide vaccine predicts the incidence of immune response in clinical trials.

IMA901 is a therapeutic vaccine against renal cell cancer (RCC) containing 9 peptides derived from tumor-associated peptides (TUMAPs), which are naturally present in human cancer tissue. A total of 96 HLA-A*O2+ patients with advanced RCC were treated with IMA901 in two independent clinical trials (Phase I and Phase II). Each of the 9 IMA901 peptides have been identified in the prior art as HLA-A2 restricted epitopes. Based on currently accepted standards, all of these are stable potential peptides for enhancing T lymphocyte responses against kidney cancer in test subjects, since their presence was found in patients with kidney cancer, and also because test patients were specifically selected to have at least one an HLA molecule capable of presenting each of the peptides.

For each subject in the simulated population, it was determined how many of the nine IMA901 vaccine peptides were able to bind to three or more HLAs). Since each peptide in the IMA901 vaccine represents 9 mer, this corresponds to the number of PEPI3+. The results were compared with the immune response rates reported for phase I and phase II clinical trials (Table 21).

Table 21

Immune response rates in a simulated population and two clinical trials IMA9 01

Immune responses to TUMAP Simulated population (HLA-A2+) (n=180) Phase I (n=27)* Phase II (n=64)* No peptides 39% 25% 36% 1 peptide 34% 44% 38% = 2 peptides 27% (MultiPEPI indicator) 29% 26% = 3 peptides 3% Not detected 3%

*No patients assessed for immune responses.

Results from phase I and phase II studies show variability in immune responses to the same vaccine across trial arms. However, overall there was good agreement between response rates predicted by the >2 PEPI3+ test and reported clinical response rates.

In a retrospective analysis, clinical investigators of the trials described above found that subjects who responded to multiple IMA901 vaccine peptides had significantly greater (p=0.019) disease control (stable disease, partial response) than subjects who responded to multiple peptides in the IMA901 vaccine.

- 58 045699 which had a response to only one peptide, or for which there was no response. 6 of 8 subjects (75%) with response to multiple peptides experienced clinical efficacy in the trial, in contrast

14% and 33% with a response to 0 and 1 peptide, respectively. Randomized phase II trials confirmed that immune responses to multiple TUMAPs were associated with longer overall survival.

Because the presence of PEPI accurately predicted patients with a response to TUMAP, clinical patients with a response to IMA901 are likely patients to present >2 PEPI from TUMAP. This subpopulation represents only 27% of selected HLA-A*02 patients, and based on clinical trial results, 75% of this subpopulation is expected to experience clinical efficacy. The same clinical results suggest that 100% of patients would experience clinical benefit if patient selection was based on >3 PEPIs from TUMAP, although this population would represent only 3% of the selected HLA-A*02 patient population. These results suggest disease control rates (stable disease or partial response) of 3% to 27% in the patient population studied in the IMA901 clinical trial. In the absence of a complete response, only a subset of these patients may experience a survival benefit.

These data explain the lack of survival benefit in the phase III IMA901 clinical trial. These results also demonstrated that the HLA-A*02 expansion of the study population was insufficient to achieve the primary overall survival endpoint in the phase III IMA901 trial. As noted by the IMA901 trial investigators, there is a need to develop a companion diagnostic (CDx) to select patients likely to respond to peptide vaccines. These data also suggest that selecting patients with >2 TUMAP-specific PEPIs may provide sufficient expansion to demonstrate significant clinical benefit from IMA901.

Example 12 An in silico test based on the identification of multiple vaccine-derived HLA-binding epitopes predicts reported experimental clinical response rates.

Agreement was determined between the >2 PEPI3+ score for immunotherapy vaccines determined in the model population described in Example 8 and the reported Disease Control Rate (DCR, the proportion of patients with complete responses and partial responses and stable disease) determined in clinical trials. tests.

Seventeen clinical trials using peptide and DNA vaccines for cancer immunotherapy that had published disease control rates (DCR) or objective response rate (ORR) were identified from peer-reviewed scientific journals (Table 22). These trials included 594 patients (5 ethnic groups) and covered 29 tumor and viral antigens. DCR was determined according to the Response Evaluation Criteria in Solid Tumors (RECIST), which is the current standard for clinical trials in which clinical responses are based on changes in maximum cross-sectional ^{dimensions42,43,44} . If DCR data were not available, objective response rate (ORR) data were used, also determined according to RECIST guidelines.

In table 23 compares >2 PEPI3+ for each vaccine in the modeled population and the published DCR or ORR. Agreement was observed between predicted and measured DCR, providing further evidence that not only the immunogenicity but also the efficacy of cancer vaccines depends on multiple individuals' HLA sequences (R2=0.76) (Fig. 9).

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Table 22

Clinical trials selected to predict disease control rates (DCR)

Immunotherapia Antigen Sponsor Disease Population (n) Study population/ethnicity HLA restriction Admission form Dose (mg) Reception scheme Research Institute assessment time (weeks) Link IMA901 phase I 9TAA Immatics Renal cell carcinoma 28 CEU (Europeans) A02 internally 0.4 8x at 10 weeks 12 19 IMA901 phase II 9TAA Immatics Renal cell carcinoma 68 CEU (Europeans) A02 internally 0.4 7x at 5 weeks, then yuh 3 weeks 24 19 Ipilimuma b NY-ESO1 MSKCC Melanoma 19 USA No intravenously o,z 3 10 4 x every 3 weeks 24 5 HPV-SLP* HPV-16 E6, E7 Leiden University VIN 20 CEU (Europeans) No subcutaneously 0.3 3 x every 3 weeks 12 9 HPV-SLP* Leiden University Cervical cancer caused by human papillomavirus 5 CEU (Europeans) No subcutaneously 0.3 3 x every 3 weeks 12 (OR) 10 gplOO-2* peptides gplOO BMS Melanoma 136 USA A*020 1 subcutaneously 1 4 x every 3 weeks 12 28 Immucin Muc-1 VaxilBio Myeloma 15 Israelis No subcutaneously 0.1 6 x every 2 weeks 12** 29 StimuVax Muc-1 Merck NSCLC (Nonsmall Cell Lung Cancer, non-small cell lung cancer) 80 Canadians No subcutaneously 1 8x per week, then every 6 weeks 12 13, 30 VGX-3100 HPV-16 and 18 Inovio Cervical cancer caused by 125 USA No intramuscular 6 0, 4, 12 weeks 36 31 human papillomavirus TSPP peptide vaccine Timidyl atsynthase University of Siena Colorectal carcinoma, non-small cell lung cancer, gallbladder carcinoma, breast cancer, stomach cancer 21 CEU (Europeans) No subcutaneously 0.1 0.2 0.3 3x3 weeks 12 32 Peptide vaccine KIF20A66* KIF20A Chibu Tokushuka Hospital Metastatic pancreatic cancer 29 Japanese A*240 2 subcutaneously 1 3 2 cycles 1, 8, 15, 22 days, then every 2 weeks 12 (OR) 33 Peptide vaccine* ZTAA Kumamoto University HNSCC (squamous cell carcinoma of the head and neck organs, squamous cell carcinoma of the head and neck organs) 37 Japanese A*240 2 subcutaneously 1 8x weekly, then every 4 weeks 12 34 Serum-based vaccine made from 7 peptides* 7TAA Kinki University Metastatic colorectal cancer thirty Japanese A*240 2 subcutaneously 1 Cycles: 5 x weekly, then every 1 week break 10 (OR) 35 GVX301* hTERT University of Genoa Prostate and kidney cancer 14 Japanese A02 Internal O 0.5 1, 3, 5, 7, 14, 21,35,63 days 12 25 MAGE-A3 MAGE- Cancerous Plural 26 USA No subcutaneously 0.3 14, 42, 90, 24 36 Trojan* AZ Abramson center myeloma But 120, 150 days RerSap HPV-16 E6 Arkansas Cue University Cervical dysplasia, 2/3 23 USA No intramuscular 0.05 0.1 0.25 0.5 4x3 weeks 24 37 Peptide vaccine against melanoma* Tyrosine for, gplOO Vir Jeans University Melanoma 26 USA A1, A2 or AZ subcutaneously 0.1 6 cycles: 0, 7, 14, 28, 35, 42 days 6 27

*Montanide ISA51 VG as an adjuvant.

**Disease response was assessed according to the International Myeloma Working Group response criteria ⁴⁵ .

-60045699

Table 23

Disease control rates (DCR) and MultiPEPI scores (predicted DCR) in 17 clinical trials Immunotherapy DCR MultiPEPI (Predicted DCR) Indicator Overall percentage of agreement IMA901 phase! 43% 27% 61% IMA901 phase II 22% 27% 81% Ipilimumab 60% 65% 92% HPV-SLP 60% 70% 86% HPV-SLP 62% 70% 89% gplOO - 2 peptides 15 % eleven % 73% Immucin 73% 59% 81% StimuVax 0% 0% 100% VGX-3100 50% 56% 89% TSPP peptide vaccine 48% 31% 65% Peptide vaccine KIF20A-66 26% 7% 27% Peptide vaccine 27% 10% 37% 7-peptide serum vaccine 10% 9% 90% GVX301 29% 7% 24%> MAGE-A3 Trojan 35% 10% 29%> RerSap 52% 26% 50%> Peptide vaccine against melanoma 12% 6% 50%>

Example 13: A panel of multiple HLA-binding peptides from tumor antigens predicts patients' response to immunotherapy with the checkpoint inhibitor ipilimumab.

It was determined whether the survival benefit of melanoma patients treated with the checkpoint inhibitor ipilimumab could be predicted by the amount of melanoma-specific PEPI3+ potentially expressed in the patient's tumor.

Eighty melanoma-associated antigens (MAAs) were identified, from which a PEPI3+ panel was selected (IPI-PEPI panel: 627 PEPI), that are common to ipilimumab-treated melanoma patients with prolonged clinical efficacy and absent in patients without prolonged efficacy . These PEPI3+ identify specific T lymphocytes that are reactivated by ipilimumab to target the patient's tumor cells. Patients with certain HLA sequences that can present more melanoma-specific PEPIs have more T cells reactivated by ipilimumab and are more likely to benefit from ipilimumab immunotherapy.

The clinical efficacy of ipilimumab treatment was determined in 160 patients from four independent clinical trial arms. Two arms were drawn from the CA184007 (10 mg/kg ipilimumab) and CA184-002 (3 mg/kg ipilimumab) trials and two arms from the published clinical trial data sets of 10 mg/kg and 3/mg/kg ipilimumab ^5,38,39 .

Epitopes from 80 melanoma antigens restricted to all 6 HLA class I of each patient were predicted, and the number of melanoma-specific PEPI3+ restricted to at least 3 HLA class I of each patient was then calculated (4668 PEPI). Each patient with at least one of the 627 PEPIs qualified as a responder. The LPI-PEPI panel predicts overall survival when receiving both 10 mg/kg and 3 mg/kg ipilimumab doses. The results were highly significant and consistent across four independent groups (Figure 10).

Example 14: Multiple HLA-binding epitopes define a patient's mutational neoantigens.

The ability of PEPI3+ to identify neoantigens from mutations was determined. PEPI3+ from 110 melanoma patients treated with ipilimumab was determined using published exome mutation data ³⁹ . According to the exome mutation data, 9502 antigens from 110 patients were mutated (Fig. 11A). The mean nonsynonymous mutational load per sample was highly variable: 309 (294,738) in the clinical efficacy group and 147 (7-5854) in the minimal or no clinical efficacy group. Due to the epitope prediction results, there were 211 (8–1950) and 56 (2–3444) neoepitopes in these mutations in the clinical efficacy group and the minimal or no clinical efficacy group, respectively.

PEPI3+ mutational neoepitopes from published mutations were determined (Fig. 11B and Table 24). These mutations yielded an average of 16 PEPI and 6 PEPI neoepitopes in the clinically effective group and in the minimal or no clinically effective group, respectively.

The results indicate that PEPIs detect mutational neoantigens derived from genetic

- 61 045699 skiy altered proteins expressed in an individual. Such neoantigens are PEPI3+ peptides, which are capable of activating T-lymphocytes in the patient’s body. If a genetic change occurs in an individual's tumor cell that creates PEPI3+, then that PEPI3+ can induce T-lymphocyte responses. These PEPI3+ containing peptides can be included in a medicinal product (eg, vaccine, T-lymphocyte therapy) to induce an immune response against a specific tumor.

Table 24

Prediction of mutational neoantigen using the PEPI test: results from Van Allen et al. and the PEPI test in 110 melanoma patients

Options Results published by Van Allen et al. Result obtained from PEPI test analyzes (confirmed epitopes and PEPI) Patients Clinical effectiveness (n=27) Minimal or no clinical efficacy (n=73) Clinical effectiveness (n=27) Minimal or no clinical efficacy (n=73) Medium Mutations 555 281 - - Average non-synonymous mutations 309 147 - - Average expressed mutational antigens 198 - - Medium neoepitopes (9mer only) 211 56 130 50 Recurrent neoepitopes 28 Not provided 10 76 Average neoepitopes of PEPI - - 16 6 Recurrent neoepitopes of PEPI - - 1 5

Example 15 In silico tests based on the identification of multiple HLA binding epitopes predict reported frequencies of cellular immune response to a vaccine targeting a mutational antigen.

Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioblastoma multiforme (GBM) and other neoplasms. The mutation involves an 801 bp in-frame deletion from the extracellular domain of EGFR, which cleaves the codon and produces a new glycine at the ^1,2 fusion junction. This mutation encodes a constitutively active tyrosine kinase that increases tumor formation and migration of tumor cells and increases resistance to radiation and chemotherapy ^{3,4,5,6,7, 8,9} . This insertion produces a tumor-specific epitope that is not found in normal adult tissues ^10. Rindopepimut is a 13-amino acid peptide vaccine (LEEKKGNYVVTDHC) covering the EGFRvIII mutation with an additional C-terminal cysteine residue ¹¹ .

In a phase II clinical trial, keyhole limpet hemocyanin (KLH)-conjugated peptide was administered to newly diagnosed patients with GBM expressing EGFRvIII. The first three vaccinations were given every two weeks, starting 4 weeks after completion of irradiation. Subsequent vaccines were administered monthly until tumor progression or death was radiologically confirmed. All vaccines were administered intradermally in the groin area. Immunological evaluation showed that only 3 of 18 patients developed a cellular immune response, assessed using the delayed-type hypersensitivity test (DTH).

An in silico trial was conducted with a simulated population of 433 subjects with the Rindopepimut sequence. 4 of 433 subjects were PEPI3+, confirming the low immunogenicity found in the phase II study (Table 25).

Table 25

Clinical trial and in silico study results

Patients with response Response rate Clinical Trial (Phase II) 3/18 16.6% In silico study (PEPI3+ test) 4/433 1 %

HLA map for Rindopepimut on the HLA alleles of subjects in the simulated population (Figure 12)

- 62 045699 shows that very few HLA-A and HLA-C alleles can bind to vaccine epitopes, which explains the absence of PEPI3+ in the in silico group.

In a recent phase III clinical trial, failure was further demonstrated when 745 patients were enrolled and randomly assigned to the Rindopepimut and temozolomide group (n=371) or the control and temozolomide group (n=374). The trial was stopped due to ineffectiveness after an interim analysis. The analysis revealed no significant differences in overall survival: median survival was 20.1 months (95% CI 18.5-22.1) in the Rindopepimut group versus 20.0 months (18.1-21.9) in the control group (HR 1.01, 95% CI 0.791.30; p=0.93).

Links for example 15.

Bigner et al, Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts, Cancer Res 1990;50: 8017-22.

Libermann et al, Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumors of glial origin, Nature 1985;313: 144-7.

Chu et al, Receptor dimerization is not a factor in the signaling activity of a transforming variant epidermal growth factor receptor (EGFRvIII), Biochem J 1997; 324:855-61.

Batra et al, Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene, Cell Growth Differ 1995;6: 1251-9.

Nishikawa et al, A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity, PNAS 1994; 91: 7727-31.

Lammering et al, Inhibition of the type III epidermal growth factor receptor variant mutant receptor by dominant-negative EGFR-CD533 enhances malignant glioma cell radio sensitivity, Clin Cancer Res 2004; 10: 6732-43.

Nagane et al, A common mutant epidermal growth factor receptor confers enhanced igenicity on human tumor glioblastoma cells by increasing proliferation and reducing apoptosis, Cancer Res 1996; 56:5079-86.

Lammering et al, Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance, Radiother Oncol 2004; 72: 267-73.

Montgomery et al, Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters β-tubulin isotype expression, J Biol Chem 2000; 275:17358-63.

Humphrey et al, Anti-synthetic peptide antibody reacting at the fusion junction of deletionmutant epidermal growth factor receptors in human glioblastoma, PNAS 1990; 87:4207-11.

Sampson et al, Immunologic Escape After Prolonged Progression-Free Survival With Epidermal Growth Factor Receptor Variant III Peptide Vaccination in Patients With Newly Diagnosed Glioblastoma, J Clin Oncol 28:4722–4729.

Weller at al, Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII expressing glioblastoma (ACT IV): a randomized, double-blind, international phase 3 trial, Lancet Oncol 2017; 18(10): 1373-1385.

Example 16: Multiple HLA peptide-binding individuals predict immunotoxicity.

Thrombopoietin (TPO) is a highly immunogenic protein drug that causes toxicity in many patients. EpiVax/Genentech used an existing method to identify HLA class II-restricted epitopes and found that the most immunogenic region of TPO was at the C terminus of TPO (US20040209324 A1).

In accordance with the present invention, we identified multiple HLA class II binding epitopes (PEPI3+) from TPO in 400 HLA class II genotyped US subjects. Most of the PEPI3+ peptides from these individuals are located at the N-terminus of TPO between amino acids 1–165. PEPI3+ were sporadically identified in some subjects also in the C-terminus. However, these results differed from the state of the art.

Published literature confirms the disclosed results, demonstrating experimental evidence that the immunotoxic region is located at the N‐terminal region of TPO ^{40 , 41} . Most people treated with the drug TPO have formed anti-drug antibodies (ADA) to this region of the drug. These antibodies not only negated the therapeutic efficacy of the drug, but also caused systemic side effects, that is, immunotoxicity such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity associated with thrombocytopenia, neutropenia and anemia. These data demonstrate that identification of multiple individuals' HLA-binding peptides predicts TPO immunotoxicity. Thus, the invention is useful for identifying the toxic immunogenic region of drugs, for identifying subjects who are likely to experience immunotoxicity from drugs, for determining

- 63 045699 regions of a polypeptide drug that may be targets of ADA, and to identify subjects who are likely to experience the formation of ADA.

Example 17. Personalized immunotherapeutic composition for the treatment of ovarian cancer.

This example describes the treatment of a patient with ovarian cancer with a personalized immunotherapy composition, wherein the composition was specifically designed for the patient based on the patient's HLA genotype based on the invention described herein. This example and Example 19 below provide clinical data to support the principles regarding the binding of epitopes to multiple HLAs of a subject to induce a cytotoxic T lymphocyte response on which the present invention is based.

The HLA class I and class II genotype of metastatic ovarian cancer in a patient with XYZ adenocarcinoma was determined from a saliva sample.

Thirteen peptides were selected to create a personalized pharmaceutical composition for patient XYZ, each of which met the following two criteria: (i) derived from an antigen that is expressed in ovarian cancer as reported in peer-reviewed scientific publications; and (ii) contains a fragment that is a T-lymphocyte epitope capable of binding to at least three HLA class I of patient XYZ (Table 26). Additionally, each peptide is optimized to bind to as many of the patient's HLA class II as possible.

Table 26

Personalized vaccine for patient XYZ with ovarian cancer

Vaccine for patient XYZ Antigentarget Antigen expression Peptides 20mer Max. HLA class I Max. HLA class II ROS01 R1 AKAR4 89% NSLQKQLQAVLQWIAASQFN 3 5 ROS01 R2 BORIS 82% SGDERSDEIVLTVSNSNVEE 4 2 ROS01 RZ SPAG9 76% VQKEDGRVQAFGWSLPQKYK 3 3 ROS01 P4 OY-TES-1 75% EVESTPMIMENIQELIRSAQ 3 4 ROS01 P5 SP17 69% AYFESLLEKREKTNFDPAEW 3 1 ROS01 P6 WT1 63% PSQASSGQARMFPNAPYLPS 4 1 ROS01 P7 HIWI 63% RRSIAGFVASINEGMTRWFS 3 4 ROS01 P8 PRAME 60% MQDIKMILKMVQLDSIEDLE 3 4 ROS01 P9 AKAR-3 58% ANSVVSDMMVSIMKTLKIQV 3 4 ROS01 RYU MAGE-A4 37% REALSNKVDELAHFLLRKYR 3 2 ROS01 P11 MAGE-A9 37% ETSYEKVINYLVMLNAREPI 3 4 ROS01 R12a MAGE-A10 52% DVKEVDPTGHSFVLVTSLGL 3 4 ROS01 R12b BAGE thirty% SAQLLQARLMKEESPWSWR 3 2

The eleven PEPI3 peptides in this immunotherapy composition can induce T-lymphocyte responses in patient XYZ with an 84% probability, and the two PEPI4 peptides (POC01-P2 and POC01-P5) with a 98% probability, consistent with the validation for the PEPI test shown in table 10. T-lymphocyte responses are directed to 13 antigens expressed in ovarian cancer. The expression of these cancer antigens has not been tested in patient XYZ. Instead, the likelihood of successful cancer cell killing was determined based on the likelihood of antigen expression in the patient's cancer cells and the positive predictive value of the test >1 PEPI3+ (AGP count). The AGP count predicts vaccine efficacy in a subject: the number of antigens used to prepare the vaccine that are expressed in the patient's tumor (ovarian adenocarcinoma) with PEPI. The amount of AGP indicates the number of tumor antigens that the vaccine recognizes and induces a T cell response to the patient's tumor (target attack). The amount of AGP depends on the frequency of expression of the antigen used to prepare the vaccine in the subject's tumor and the HLA genotype of the subject. The correct value should be between 0 (no PEPI presented by the expressed antigen) and the maximum number of antigens (all antigens expressed and presented by PEPI).

The probability that patient XYZ will express one or more of the 12 antigens is shown in FIG. 13. AGP95=5, AGP50=7.9, mAGP=100%, AP=13.

The pharmaceutical composition for patient XYZ may contain at least 2 of the 13 peptides (Table 26), since the presence in the vaccine or immunotherapeutic composition of at least two polypeptide fragments (epitopes) that can bind to at least three HLAs of the individual (> 2 PEPI3+) was identified as predictive of clinical response. Before injection, the peptides are synthesized, dissolved in a pharmaceutically acceptable solvent and mixed with an adjuvant. It is advisable for the patient to receive personalized immunotherapy with at least two peptide vaccines, but preferably more, to increase the likelihood of killing cancer cells and reduce the likelihood of relapse.

For the treatment of patient XYZ, 12 peptides were formulated as 4 x 3/4 peptides (POC01/1, POC01/2, POC01/3, POC01/4). One treatment cycle is defined as administration of all 13 peptides over 30 days.

- 64 045699

Disease history.

Diagnosis: metastatic ovarian adenocarcinoma.

Age: 51 years old.

Family history: colon and ovarian cancer (mother), breast cancer (grandmother).

Tumor pathology:

BRCAl-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCH1-S2450N.

2011: primary diagnosis of ovarian adenocarcinoma; Wertheim's operation and chemotherapy; removal of a lymph node.

2015: metastasis to pericardial adipose tissue, excision.

2016: liver metastases.

2017: retroperitoneal and mesenteric lymph nodes progressed; incipient peritoneal carcinosis with slight accompanying ascites.

Previous therapy:

2012: paclitaxel-carboplatin (6x) 2014: kelix-carboplatin (1x),

2016-2017 (9 months): limparza (Olaparib) 2x400 mg/day, orally,

2017: Hycamtin for infusion 5x2.5 mg (3x one series/month) Treatment with PIT vaccine began on April 21, 2017.

Table 27

Peptide treatment regimen for patient XYZ

Lot No. Vaccinations ^1st cycle OH λ cycle OH 3 cycle L Y 4 cycle ROS01/1 N1727 04/21/2017 06/16/2017 08/30/2017 10/19/2017 ROS01/2 N1728 04/28/2017 05/31/2017 ROS01/3 N1732 06/16/2017 08/02/2017 09.20.2017 ROS01/4 N1736 05/15/2017 07/06/2017

Patient's tumor CT scan results (baseline April 15, 2016)

The disease was limited primarily to the liver and lymph nodes. The use of computed tomography limits the detection of pulmonary (pulmonary) metastases.

May 2016-January 2017: treatment with Olaparib.

December 2016 (before treatment with PIT vaccine) There was a dramatic decrease in tumor weight with confirmation of the response obtained to FU2.

January-March 2017 - TORO protocol (topoisomerase).

April 2017 FU3 demonstrated an increase in existing pathological changes and the emergence of new pathological changes leading to disease progression.

April 2017 the beginning of the use of PIT.

July 2017 (after 2nd cycle of PIT) FU4 demonstrated continued increase in pathological changes and overall enlargement of the pancreas and abnormal peripancreatic signal along with increasing ascites.

July 2017 - CBP+Gem+Avastin.

September 2017 (after 3 cycles of PIT) FU5 demonstrated reversal of lesion growth and improved pancreatic/parapancreatic signal. The data obtained indicate pseudo progression.

November 2017 (after 4 cycles of PIT) FU6 demonstrated the best response with resolution of non-target lesions.

The CT scan data for patient XYZ is shown in Table. 28 and FIG. 14.

-65 045699

Table 28

Summary table of responses to pathological changes

Pathological changed not / point in time Base level (%D from BL (Baseline, basic level) FU1 (%A of BL) FU2 (%A of BL) FU3 (%A of BL) FU4 (%A of BL) FU5 (%A of BL) FU6 (%A of BL) Best answer cycle Moment of time and disease progression (PD, progressive disease) TL1 Not applicable -56.1 -44.4 -44.8 +109.3 -47.8 -67.3 FU6 FU4 TL2 Not applicable -100.0 -100.0 -47.1 -13.1 -100.0 -100.0 FU1 FU3 TL3 Not applicable -59.4 -62.3 -62.0 -30.9 -66.7 -75.9 FU6 FU4 TL4 Not applicable -65.8 -100.0 -100.0 -100.0 -100.0 -100.0 FU2 Not applicable Bottom line Not applicable -66.3 -76.0 -68.9 -23.5 -78.2 -85.2 FU6 FU4

Example 18. Development of a personalized immunotherapeutic composition for the treatment of breast cancer.

The HLA class I and II genotype of patient ABC's metastatic breast cancer was determined from a saliva sample. To create a personalized pharmaceutical composition for patient ABC, twelve peptides were selected, each of which met the following two criteria: (i) derived from an antigen that is expressed in breast cancer, as reported in peer-reviewed scientific publications; and (ii) contains a fragment that is a T-lymphocyte epitope capable of binding to at least three HLA class I ABC patients (Table 29). Additionally, each peptide is optimized to bind to as many of the patient's HLA class II as possible. Twelve peptides target twelve breast cancer antigens. The probability that patient ABC will express one or more of the 12 antigens is shown in FIG. 15.

Table of 29 peptides for patient ABC with breast cancer

BRC09 vaccine peptides Antigentarget Express nya antigen Peptide 20 tag Max. HLA class I Max. HLA class II PBRC01 сР1 FSIP1 49% ISDTKDYFMSKTLGIGRLKR 3 6 PBRC01 сР2 SPAG9 88% FDRNTESLFEELSSAGSGLI 3 2 PBRC01 sRZ AKAR4 85% SQKMDMSNIVLMLIQKLLNE 3 6 PBRC01 сР4 BORIS 71% SAVFHERYALIQHQKTHKNE 3 6 PBRC01 сР5 MAGE-A11 59% DVKEVDPTSHSYVLVTSLNL 3 4 PBRC01 сР6 NY-SAR-35 49% ENAHGQSLEEDSALEALLNF 3 2 PBRC01 сР7 HOM-TES-85 47% MASFRKLTLSEKVPPNHPSR 3 5 PBRC01 сР8 NY-BR-1 47% KRASQYSGQLKVLIAENTML 3 6 PBRC01 сР9 MAGE-A9 44% VDPAQLEFMFQEALKLKVAE 3 8 PBRC01 сР1О SCP-1 38% EYEREETRQVYMDLNNNIEK 3 3 PBRC01 sRP MAGE-A1 37% PEIFGKASESLQLVFGIDVK 3 3 PBRC01_cP12 MAGE-C2 21% DSES SFTYTLDEKVAELVEF 4 2

Predicted efficiency: AGP95=4; There is a 95% chance that the PIT vaccine induces CTL responses to 4 CTAs expressed in BRC09 breast cancer cells. Additional efficiency parameters: AGP50=6.3, mAGP=100%, AP=12. Detected effectiveness after the 1st vaccination with all 12 peptides: a decrease in the metabolic activity of the tumor by 83% (data from PET CT (Positional Emission Tomography-Computed Tomography)).

For patient ABC treatment, 12 peptides were formulated as 4x3 peptides (PBR01/1, PBR01/2, PBR01/3, PBR01/4). One treatment cycle is defined as administration of all 12 different vaccine peptides over a 30-day period.

Disease history.

Diagnosis: bilateral metastatic breast carcinoma: right breast is ER-positive, PR-negative, Her2-negative; left mammary gland - ER, PR and Her2 negative.

Primary diagnosis: 2013 (4 years before treatment with the PIT vaccine).

2016 extensive metastatic disease involving nodes both above and below the diaphragm.

- 66 045699

Multiple metastases in the liver and lungs.

Treatments 2016-2017: Etrozol, Ibrance (palbociclib) and Zoladex.

Results.

March 2017: before treatment with the PIT vaccine.

Hepatic multimetastatic process with truly external compression of the beginning of the bile duct and massive dilatation of the entire intrahepatic bile tract. Celiac disease, intraductal hepatic and retroperitoneal adenopathy.

May 2017: after 1 cycle of PIT.

Detected effectiveness: 83% reduction in tumor metabolic activity (PET CT) of the liver, lung lymph nodes and other metastases. Detected safety: skin reactions.

Local inflammation at the injection site within 48 hours after vaccine administration.

Follow-up activities.

BRC-09 was treated with 5 cycles of PIT vaccine. She felt very well, but in September 2017 she refused PET ST. She became symptomatic in November and a PET CT scan showed progressive disease, but she refused all treatments. Additionally, her oncologist learned that she had not taken palbociclib since the spring/summer. Patient ABC died in January 2018.

The combination of pablociclib and the personalized vaccine was likely responsible for the remarkable early response observed after vaccine administration. Palbociclib has been shown to improve immunotherapy activity by increasing HLA CTA presentation and decreasing regulatory T cell proliferation: (Goel et al. Nature. 2017:471475). The PIT vaccine can be used as an adjunct to current therapy to achieve maximum effectiveness.

Example 19. Personalized immunotherapeutic composition for the treatment of a patient with advanced metastatic breast cancer.

Patient BRC05 was diagnosed with inflammatory breast cancer on the right with extensive lymphogenous carcinomatosis. Inflammatory breast cancer (IBC) is a rare but aggressive form of locally advanced breast cancer. It is called inflammatory breast cancer because its main symptoms are swelling and redness (the breasts often look inflamed). Most inflammatory forms of breast cancer are invasive ductal carcinomas (starting in the milk ducts). This type of breast cancer is associated with a high risk of expressing human papillomavirus oncoproteins ¹ . Indeed, HPVI6 DNA was diagnosed in this patient's tumor.

Patient stage in 2011 (6 years before PIT vaccine treatment).

T4: Tumor of some size with direct extension to the chest wall and/or skin (ulceration or skin nodules).

pN3a: metastases in > 10 axillary lymph nodes (at least 1 tumor metastasis > 2.0 mm); or metastases in subclavian (level III axillary lymph nodes) nodes.

For the BRC05 vaccine, 14 vaccine peptides were developed and obtained (Table 30). For this patient, peptides PBRC05-P01-P10 were designed based on population expression data. The last 3 peptides in the table. 29 (SSX-2, MORC, MAGE-B1) were composed of antigens, the expression of which was measured directly in the patient’s tumor.

Table 30

Vaccine peptides for patient BRC05

Vaccine peptides for BRC05 Antigentarget Antigen expression Peptide 20 tag Max. HLA class I Max. HLA class II PBRC05 P1 SPAG9 88% xxxxxxxxxxxxxxxxxxxxxxxxx 3 4 PBRC05 P2 AKAR4 85% xxxxxxxxxxxxxxxxxxxxxxxxx 3 4 PBRC05 RZ MAGE-A11 59% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 P4 NY-SAR-35 49% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 P5 FSIP1 49% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 P6 NY-BR-1 47% xxxxxxxxxxxxxxxxxxxxxxxxx 3 4 PBRC05 P7 MAGE-A9 44% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 P8 SCP-1 38% xxxxxxxxxxxxxxxxxxxxxxxxx 3 6 PBRC05 P9 MAGE-A1 37% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 RYU MAGE-C2 21% xxxxxxxxxxxxxxxxxxxxxxxxx 3 3 PBRC05 P11 MAGE-A12 13% xxxxxxxxxxxxxxxxxxxxxxxxx 3 4 PBRC05 P12 SSX-2 6% xxxxxxxxxxxxxxxxxxxxxxxxx 3 1 PBRC05P13 MORC Not detected xxxxxxxxxxxxxxxxxxxxxxxxx 3 4 PBRC05P14 MAGE-B1 Not detected xxxxxxxxxxxxxxxxxxxxxxxxx 3 3

Note: Bold red indicates PEPI CD8, underline indicates best binding CD4 allele.

T-lymphocyte responses were measured in peripheral blood mononuclear cells 2 weeks after the 1st vaccination with a mixture of peptides PBRC05P1, PBRC05P2, PBRC05P3, PBRC05P4, PBRC05P5, PBRC05P6, PBRC05P7.

- 67 045699

Table 31

Antigen-specific T-lymphocyte responses: number of spots/300,000 PBMCs

Antigen Stimulant Example 1 Example 2 Average SPAG9 PBRC05 P1 2 1 1.5 AKAR4 PBRC05 P2 eleven 4 7.5 MAGE-A11 PBRC05 RZ 26 32 29 NY-SAR-35 PBRC05 P4 472 497 484.5 FSIP1 PBRC05 P5 317 321 319 NY-BR-1 PBRC05 P6 8 12 10 MAGE-A9 PBRC05 P7 23 27 25 No Negative control (DMSO, dimethyl sulphoxide, dimethyl sulfoxide) 0 3 1.5

The results show that a single immunization with 7 peptides induced strong T-lymphocyte responses to 3 of the 7 peptides, demonstrating strong MAGE-A11, NYSAR-35, FSIP1 and MAGE-A9-specific T-lymphocyte responses. There were weak responses against AKAP4 and NY-BR1 and no response against SPAG9.

Example 20. Personalized immunotherapeutic composition for the treatment of a patient with early stage metastatic breast cancer.

Medical history: in 2011, the left sector of the breast was removed due to a tumor. Treatment: aromatase inhibitor and irradiation of the lumbar spine (bone tissue).

In 2017, before starting treatment with the PIT vaccine, metastatic lesions were observed in the ventral floor of the right 5th rib and the right 3rd rib. Recurrence of a malignant neoplasm in the left breast should be excluded. A malignant tumor may exist in the right breast with a metastatic right axillary lymph node.

Table 32

Vaccine peptides for the patient of Example 20

Vaccine peptides for the patient Antigentarget Antigen expression Peptide 20 mer Max. HLA CD8 Max. HLA CD4 PBRC04 P1 SPAG9 88% xxxxxxxxxxxxxxxxxxx 3 1 PBRC04 P2 AKAR4 85% xxxxxxxxxxxxxxxxxxx 4 4 PBRC04 RZ BORIS 71% xxxxxxxxxxxxxxxxxxx 3 2 PBRC04 P4 MAGE-A11 59% XXXXXXXXXXXXXXXXXXXX 3 1 BRC04 P6 NY-SAR-35 49% XXXXXXXXXXXXXXXXXXXX 3 5 PBRC04 P7 FSIP1 49% XXXXXXXXXXXXXXXXXXXX 3 6 PBRC04 P8 NY-BR-1 47% XXXXXXXXXXXXXXXXXXXX 3 1 PBRC04 RYU LDHC 35% XXXXXXXXXXXXXXXXXXXX 3 5 PBRC04 RN GATA-3 31% xxxxxxxxxxxxxxxxxxx 3 1 PBRC04 P13 Survivin 71% xxxxxxxxxxxxxxxxxxx 3 2 PBRC04 P14 MAGE-CI 12% xxxxxxxxxxxxxxxxxxx 3 8 PBRC04P15 PRAME 55% xxxxxxxxxxxxxxxxxxx 3 5

The patient received 2 cycles of PIT vaccine - immuno-BLAST.

A method has been developed to target an antigen to determine its ability to cause a toxic immune response, such as autoimmunity. The method is referred to herein as immuno-BLAST.

PolyPEPI1018 contains six 30-mer polypeptides. Each polypeptide consists of two 15-mer peptide fragments derived from antigens expressed in the CRC) Neoepitopes can be generated at the junction of two 15-mer peptides and can cause unwanted T-lymphocyte responses to healthy cells (autoimmunity). This was assessed using the immunoBLAST technique.

A 16-mer peptide was designed for each of the 30-mer components of PolyPEP1018. Each 16-mer contains 8 amino acids from the end of the first 15 residues of the 30-mer and 8 amino acids from the beginning of the second 15 residues of the 30-mer - thus precisely spanning the region where two 15-mers meet. These 16-mers were then analyzed to identify cross-reactive regions of local similarity to human sequences using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), which compares protein sequences to sequence databases and calculates statistical significance of the matches. The 8-mer out of the 16-mer was chosen as the length tested because this length represents the minimum length required for the peptide to form an epitope and represents the distance between anchor points during HLA binding.

As shown in FIG. 16, the amino acid positions in the polypeptide are numbered. The starting positions of potential 9-mer peptides that can bind to HLA and form neoepitopes are 8 amino acids at positions 8-15. Starting positions of peptides obtained from

- 68 045699 tumor antigen contained in 15 -mers that can form pharmaceutically active epitopes are 7 + 7 = 14 amino acids at positions 1-7 and 16-22. The ratio of possible neoepitope-generating peptides is 36.4% (8/22).

The PEPI3+ test was used to identify neoepitopes and neoPEP1 among the 9-mer epitopes in the junction region. The risk that PolyPEPI1018 induces unwanted T cell responses was assessed in 433 subjects in a simulated population by determining the proportion of subjects with PEPI3+ among the 9-mers at the junction. The results of the neoepitope/neo-PEPI analysis are shown in Table. 33. In the 433 subjects in the simulated population, the average predicted number of epitopes that could be produced by intracellular processing was 40.12. Neoepitopes were frequently generated; 11.61 of 40.12 (28.9%) epitopes are neoepitopes. Most of the peptides could be identified as neoepitopes, but the number of subjects presenting neoepitopes varied.

The epitopes contained in PolyPEPI1018 generate an average of 5.21 PEPI3+. Said PEPIs may activate T lymphocytes in a subject. The number of potential neo-PEPIs was much lower than neo-epitopes (3.7%). There is a small possibility that these neo-PEPIs compete for T cell activation with PEPIs in some subjects. It is important to note that the activated neo-PEPI-specific T cells were not targeted on healthy tissues.

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Table 33

Identification of potential neoepitopes of PolyPEPI1018

Peptide identifier Potential neoepitope Epitope and PEPI3+ binding in 433 subjects in a simulated population Epitope binding (1 x HLA) PEPI3+ binding (3 x HLA) Col. % NeoER! Col. Col. % Col. PolyPEPIl 018: subjects subjects neoER! subjects subjects neoPER! CRC-P1 QFPVSEGKS 0 0.0% 7 0 0.0% FPVSEGKSR 160 37.0% X 1 0.2% X PVSEGKSRY 150 34.6% X 0 0.0% VSEGKSRYR 194 44.8% X 1 0.2% X SEGKSRYRA 113 26.1% X 0 0.0% EGKSRYRAQ 77 17.8% X 0 0.0% GKSRYRAQR 37 8.5% X 0 0.0% KSRYRAQRF 337 77.8% X 33 7.6% X CRC-P2 IELKHKART 32 7.4% X 7 0 0.0% ELKHKARTA 63 14.5% X 0 0.0% LKHKARTAK 59 13.6% X 0 0.0% KHKARTAKK 166 38.3% X 1 0.2% X HKARTAKKV 0 0.0% 0 0.0% KARTAKKVR 70 16.2% X 0 0.0% ARTAKKVRR 134 30.9% X 0 0.0% RTAKKVRRA 41 9.5% X 0 0.0% CRC-P3 EFSMQGLKD 0 0.0% 5 0 0.0% FSMQGLKDE 188 43.4% X 0 0.0% SMQGLKDEK 138 31.9% X 0 0.0% MQGLKDEKV 16 3.7% X 0 0.0% QGLKDEKVA 0 0.0% 0 0.0% GLKDEKVAE 0 0.0% 0 0.0% LKDEKVAEL 186 43.0% X 3 0.7% X KDEKVAELV 51 11.8% X 0 0.0% CRC-P6 LLALMVGLK 252 58.2% X 7 0 0.0% LALMVGLKD 86 19.9% X 0 0.0% ALMVGLKDH 65 15.0% X 0 0.0% LMVGLKDHR 97 22.4% X 0 0.0% CRC-P7 MVGLKDHRI 67 15.5% X 0 0.0% VGLKDHRIS 0 0.0% 0 0.0% GLKDHRIST 4 0.9% X 0 0.0% LKDHRISTF 195 45.0% X 5 1.2% X PALFKENRS 0 0.0% 5 0 0.0% ALFKENRSG 0 0.0% 0 0.0% LFKENRSGA 41 9.5% X 0 0.0% FKENRSGAV 114 26.3% X 0 0.0% KENRSGAVM 261 60.3% X 0 0.0% ENRSGAVMS 0 0.0% 0 0.0% NRSGAVMSE 227 52.4% X 0 0.0% RSGAVMSER 197 45.5% X 2 0.5% X CRC-P8 AVLTKKFQK 181 41.8% X 7 0 0.0% VLTKKFQKV 208 48.0% X 2 0.5% X LTKKFQKVN 0 0.0% 0 0.0% TKKFQKVNF 25 5.8% X 0 0.0% KKFQKVNFF 250 57.7% X 12 2.8% X KFQKVNFFF 273 63.0% X 23 5.3% X FQKVNFFFE 163 37.6% X 0 0.0% QKVNFFFER By 25.4% X 0 0.0%

Abbreviations: CRC=colorectal cancer; HLA=human leukocyte antigen; PEPI=personal epitope.

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Claims (24)

1. A method of identifying a polypeptide fragment as immunogenic for a particular human subject or predicting whether a polypeptide or polypeptide fragment is immunogenic for a particular human subject, comprising the steps of: (i) determining whether the polypeptide contains: (a) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, or (b) an amino acid sequence that is a T lymphocyte epitope capable of binding to at least , with three HLA class II molecules of the subject, and (ii) (I) identifying said sequence as a polypeptide fragment that is immunogenic for that subject; - 72 045699 (II) forecasting that: A) the polypeptide is immunogenic in a subject if the polypeptide contains at least one sequence that meets the requirements of step (i), or B) the polypeptide is not immunogenic for the subject if the polypeptide does not contain at least one sequence that meets the requirements of step (i). 2. The method according to claim 1, in which: (a) The T-lymphocyte epitope is capable of binding to at least three HLA class I molecules of the subject and consists of 9 consecutive amino acids of a polypeptide or in which the T-lymphocyte epitope is capable of binding to at least three HLA class II molecules of the subject and consists of 15 consecutive amino acid polypeptides; (b) step (i) includes determining whether the polypeptide contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject; (c) step (i) includes determining whether the polypeptide contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class II molecules of the subject; and/or (d) step (i) includes determining that the polypeptide contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject and identifying a fragment of the polypeptide that is T -a lymphocyte epitope capable of binding to at least one HLA class II molecule of a subject, wherein the HLA class II binding epitope contains the amino acid sequence of a T lymphocyte epitope that binds to HLA class I. 3. The method according to claim 1 or 2, in which the polypeptide: (a) is expressed by a pathogenic organism, virus or cancer cell, is associated with an autoimmune disorder, or is an allergen or ingredient in a pharmaceutical composition; (b) selected from the antigens listed in table. 2-6; (c) is an antigen or neoantigen expressed by a cancer cell, or is an antigen or neoantigen expressed by a cancer cell, wherein the cancer cell, antigen or neoantigen is present in a sample obtained from the subject; (d) is a mutational neoantigen or is a mutational neoantigen, where: A) the neoantigen is present in the sample obtained from the subject, and/or B) the immunogenic fragment contains a neoantigen-specific mutation; or (e) is associated or is believed to be associated with an autoimmune disorder or autoimmune response in a subject, wherein determining that the polypeptide contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules subject, identifies the polypeptide and/or fragment as immunogenic or associated with an autoimmune disorder or autoimmune response in the subject. 4. The method according to any one of claims 1-3, in which all polypeptide fragments representing a T-lymphocyte epitope capable of binding to at least three HLA class I molecules are identified, and/or all polypeptide fragments representing T -a lymphocyte epitope capable of binding to at least three HLA class II molecules of a subject. 5. The method of any one of the preceding claims, further comprising predicting whether the subject will have a cytotoxic T lymphocyte response or a helper T cell response upon administration of one or more polypeptides or a pharmaceutical composition or kit containing one or more polypeptides as active ingredients , wherein: A) a cytotoxic T lymphocyte response is predicted if the polypeptide(s) contains at least one amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject; B) a T helper response is predicted if the polypeptide(s) contains at least one amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class II molecules of the subject; C) predicting the absence of a cytotoxic T lymphocyte response if the polypeptide(s) does not contain any amino acid sequence that represents a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject; or D) predicting absence of a T helper cell response if the polypeptide(s) do not contain any amino acid sequence that represents a T lymphocyte epitope capable of binding to at least three HLA class II molecules of the subject. - 73 045699 6. Method according to claim 5, where: (I) predicting that the subject will have a cytotoxic T lymphocyte response and/or a T helper cell response, and the method further includes determining the probability that the subject will have a cytotoxic T lymphocyte response and/or a T helper cell response directed to the polypeptide antigen expressed in a subject, wherein the method includes: (i) identifying one or more polypeptide antigens containing an amino acid sequence that: (a) is a T lymphocyte epitope capable of binding to at least three HLA class I molecules or at least three HLA class II molecules of the subject, and (b) is contained in the amino acid sequence of the polypeptide(s), ( ii) using population expression frequency data for one or more of the peptide antigens identified in step (i) to determine the likelihood that a subject will have a cytotoxic T lymphocyte response and/or a helper T cell response directed to the polypeptide antigen, expressed in a subject; or (II) the polypeptide is a component of a pharmaceutical composition, and the method includes determining the probability that the subject will form anti-drug antibodies (ADA) after administration of the polypeptide, wherein the predicted T helper cell response corresponds to a higher probability of forming ADA, and the absence predicted T helper response corresponds to a lower probability of ADA formation. 7. The method according to any one of claims 1-6, additionally including: (I) predicting whether a subject will have a clinical response to administration of a pharmaceutical composition or pharmaceutical kit containing one or more polypeptides as active ingredients, the method comprising determining whether one or more active ingredient polypeptides contain together at least two different amino acid sequences, each of which is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, and the prediction that: A) a subject will have a clinical response to administration of a pharmaceutical composition or kit if one or more polypeptides of the active ingredient together contain at least two different sequences, each of which is a T-lymphocyte epitope capable of binding to at least three molecules subject's HLA class I, or B) a subject will not have a clinical response to administration of a pharmaceutical composition or kit if one or more polypeptides of the active ingredient together contain no more than one sequence that is a T-lymphocyte epitope capable of binding to at least three HLA class molecules I subject; and/or (II) determining the likelihood that a particular human subject will have a clinical response to administration of a pharmaceutical composition or kit containing one or more polypeptides as an active ingredient, wherein: (1) one or more of the following factors corresponds to a higher likelihood of clinical response: (a) the presence in the polypeptide(s) of the active ingredient of more amino acid sequences and/or different amino acid sequences, each of which is a T-lymphocyte epitope capable of binding to at least three HLA class I of the subject; (b) a larger number of target polypeptide antigens containing at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T cell epitope capable of binding to at least three HLA class I of the subject; or a T cell epitope capable of binding to at least three HLA class I of a subject, wherein the target polypeptide antigens are expressed in the subject or present in one or more samples obtained from the subject; (c) a higher probability that the subject expresses target polypeptide antigens, or a threshold number of target polypeptide antigens and/or target polypeptide antigens that have been determined to contain at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class I of the subject, and/or - 74 045699 (d) a greater number of target polypeptide antigens that a subject is predicted to express, or a greater number of target polypeptide antigens that a subject has a threshold probability of expressing, and/or a greater number of target polypeptide antigens that are predicted to express determined to contain at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class I of the subject; and/or (2) the method includes: (i) identifying which polypeptide antigens targeted by the active ingredient polypeptide(s) contain an amino acid sequence that both: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class I of the subject; (ii) using population expression data for each antigen identified in step (i) to determine the likelihood that a subject expresses one or more antigens identified in step (i) that together contain at least two different amino acid sequences step (i), or using population expression data for each antigen identified in step (i), to determine the likelihood that a subject expresses one or more antigens identified in step (i) that together contain at least two different amino acid sequences of step (i); and (iii) determining the probability that the subject will have a clinical response to administration of the pharmaceutical composition or kit, with a higher probability determined in step (ii) corresponding to a more likely clinical response; and/or (3) one or more of the following factors additionally corresponds to a higher likelihood of clinical response: (a) the presence in the polypeptide(s) of the active ingredient of more amino acid sequences and/or different amino acid sequences, each of which is a T-lymphocyte epitope capable of binding to at least three HLA class II of the subject; (b) a larger number of target polypeptide antigens containing at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T lymphocyte epitope capable of binding to at least three HLA class II of the subject, or a T lymphocyte epitope capable of binding to at least three HLA class II of the subject, where the target polypeptide antigens are expressed in the subject or are present in one or more samples obtained from the subject; (c) a larger number of polypeptide target antigens containing: i) at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T lymphocyte epitope capable of binding to at least three HLA class I of the subject, and ii) at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class II of the subject; (d) a higher probability that the subject expresses target polypeptide antigens, or a threshold number of target polypeptide antigens that have been determined to contain at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class II of the subject; (f) a higher probability that the subject expresses target polypeptide antigens or a threshold amount of target polypeptide antigens that have been determined to contain: i) at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T lymphocyte epitope capable of binding to at least three HLA class I of the subject, and ii) at least one amino acid sequence that simultaneously: A) contained in the polypeptide of the active ingredient and - 75 045699 B) is a T-lymphocyte epitope capable of binding to at least three HLA class II of the subject; (f) a greater number of target polypeptide antigens that the subject is predicted to express, or a greater number of target polypeptide antigens that the subject expresses with a threshold probability, and which have been determined to contain at least one amino acid sequence that simultaneously : A) contained in the active ingredient polypeptide and B) is a T lymphocyte epitope capable of binding to at least three HLA class II of the subject, and/or (g) a greater number of target polypeptide antigens that the subject is predicted to express, or a greater number of polypeptide antigens - targets that the subject expresses with a threshold probability and which have been determined to contain: i) at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T lymphocyte epitope capable of binding to at least three HLA class I of the subject, and ii) at least one amino acid sequence that simultaneously: A) contained in the active ingredient polypeptide and B) is a T-lymphocyte epitope capable of binding to at least three HLA class II of the subject. 8. The method according to claim 7, additionally including: (I) repeating the method for one or more additional pharmaceutical compositions or kits and ranking the compositions or kits by their likelihood of inducing a clinical response in the subject; and/or (II) predicting whether administration of the polypeptide, pharmaceutical composition or kit will induce a toxic immune response in a subject, wherein: (a) the polypeptide(s) contains at least one amino acid sequence that: i) is capable of binding to at least three HLA class I of the subject, and ii) corresponds to a human polypeptide fragment expressed in healthy cells; and a toxic immune response is predicted, or (b) the polypeptide(s) does not contain any amino acid sequence that: A) capable of binding to at least three HLA class I of the subject, and B) corresponds to a human polypeptide fragment expressed in healthy cells; and predict the absence of a toxic immune response. 9. The method of any of the preceding claims, further comprising administering to the specified subject or selecting or recommending for treatment of the specified human subject by administering to the subject a polypeptide containing a fragment of a polypeptide identified as immunogenic for the subject, or a polypeptide that is predicted to be immunogenic or to induce a response a cytotoxic T lymphocyte or T helper cell response, or a pharmaceutical composition or kit that is predicted to induce a clinical response, or a polypeptide or pharmaceutical composition that is not predicted to induce a toxic immune response or induce the formation of ADA in a subject. 10. The method of any one of claims 1 to 4, further comprising predicting whether the subject will have a clinical response to administration of a checkpoint inhibitor for the treatment of cancer, comprising determining whether one or more cancer-associated antigens contains at least two different amino acid sequences, each of which is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, and the prediction that: A) a subject will have a clinical response to checkpoint inhibitor administration if one or more cancer-associated antigens together contain at least two different sequences, each of which is a T-lymphocyte epitope capable of binding to at least three molecules subject's HLA class I, or B) the subject will not have a clinical response to administration of a checkpoint inhibitor if one or more cancer-associated antigens together contain no more than one sequence that is a T-lymphocyte epitope capable of binding to at least three HLA class molecules I subject. 11. The method according to claim 10, where said method further includes: (I) determining the likelihood that the subject will have a clinical response to administration of a checkpoint inhibitor for the treatment of cancer, the method comprising: (i) selecting a plurality of polypeptide antigens that are associated with the subject's type of cancer; - 76 045699 (ii) determining which of the specified cancer-associated antigens contains an amino acid sequence representing a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, and (iii) using expression data in the population for each cancer-associated antigen identified in the step to determine the probability that a subject will have a clinical response to administration of a checkpoint inhibitor for the treatment of cancer, wherein the greater probability is that the subject expresses one or more of the cancer-associated antigens, identified in step (ii), which together contain at least two amino acid sequences, each of which is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, corresponds to a more likely clinical response; and/or (II) selecting or recommending administration of a checkpoint inhibitor to treat the subject and/or administration of a checkpoint inhibitor to the subject. 12. The method according to any one of claims 5-7, 10 and 11, wherein the subject is predicted to have a toxic immune response or ADA formation, or to have no cytotoxic T lymphocyte or T helper cell response, or a clinical response, or to not respond for treatment with a checkpoint inhibitor, the method further comprising selecting or recommending another treatment for the subject. 13. A method of formulating or producing a human subject-specific pharmaceutical composition or pharmaceutical kit for use in a method of treating a specific human subject, comprising: (i) selecting a polypeptide fragment, wherein said fragment has been identified as immunogenic for the subject by the method of any one of claims 1 to 12; (iii) selecting a first sequence of up to 50 consecutive amino acids of the polypeptides, wherein the consecutive amino acids comprise the amino acid sequence of the fragment selected in step (i); (iv) repeating steps (i)-(iii) to select a second amino acid sequence from up to 50 consecutive amino acids of the same or a different polypeptide relative to the first amino acid sequence; and (vi) formulating or preparing a subject-specific pharmaceutical composition or kit having as active ingredients one or more polypeptides that together contain all of the amino acid sequences selected in the previous steps. 14. The method according to claim 13, additionally containing: (ii) if the fragment selected in step (i) is an HLA class I binding epitope, selecting a polypeptide fragment that: a) contains the fragment selected in step (i), and b) is a T-lymphocyte epitope capable of binding to at least three or the most possible HLA class II molecules of the subject, and in said (iii) said consecutive amino acids contain the amino acid sequence of the fragment selected in step (i), or fragment selected in step (ii). 15. The method according to claim 13 or 14, in which: (a) each polypeptide either consists of one of the selected amino acid sequences or contains or consists of: (I) two or more selected amino acid sequences located end to end or overlapping in a single polypeptide; or (II) two or more of the selected amino acid sequences located end to end or overlapping in the same polypeptide, wherein any neo-epitopes formed at the junction between any two of the selected amino acid sequences located end to end in the same polypeptide were subjected to screening to eliminate polypeptides containing a neoepitope amino acid sequence that: (i) corresponds to a human polypeptide fragment expressed in healthy cells; (ii) is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, or (iii) meets the requirements of both (i) and (ii); and/or (b) one or more polypeptides have been screened to eliminate polypeptides containing an amino acid sequence that: (i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject. 16. Use of a human subject-specific pharmaceutical composition or pharmaceutical kit in a method of inducing an immune response in a specific human subject, wherein said composition or kit is formulated or obtained for the subject in accordance with the method of any one of claims 13 to 15. - 77 045699 17. The use of a human subject-specific pharmaceutical composition or pharmaceutical kit in a method of treating a specific human subject in need thereof, wherein the composition contains as active ingredients: (a) a first and second peptide, each peptide comprising an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules and/or at least three HLA class II molecules of the subject , wherein the amino acid sequence of the T-lymphocyte epitope of the first and second peptides differ from one another; or (b) a first region and a second region, each of which contains an amino acid sequence representing a T lymphocyte epitope capable of binding to at least three HLA class I molecules and/or at least three HLA class II molecules subject, and the amino acid sequence of the T-lymphocyte epitope of the first and second sections differ from one another. 18. Application according to claim 17, where: (a) one or more or each of the peptides or regions comprises an amino acid sequence that is a T lymphocyte epitope capable of binding to at least one HLA class II molecule of a subject, wherein the HLA class II binding T lymphocyte epitope comprises the amino acid sequence , which is a T-lymphocyte epitope capable of binding to at least three HLA class I molecules of a subject; (b) one or more polypeptides do not contain any amino acid sequence that: (i) corresponds to a human polypeptide fragment expressed in healthy cells, or (ii) corresponds to a human polypeptide fragment expressed in healthy cells and is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject; (c) one or more or each of the peptides or regions contains a sequence of up to 50 consecutive amino acids of a polypeptide that: A) is expressed by a pathogenic organism, virus or cancer cell, is associated with an autoimmune disorder or is an allergen, wherein the sequence contains a T-lymphocyte epitope of a peptide or region capable of binding to at least three HLA class I or class II molecules of the subject; B) selected from the antigens listed in table. 2-6; C) is an antigen or neoantigen expressed by a cancer cell, or a cancer cell in a sample obtained from the subject; D) is a mutational neoantigen or neoantigen that is present in a sample obtained from the subject; and/or each T-lymphocyte epitope(s) contains a neoantigen-specific mutation. 19. Application according to claim 17 or 18, where: (I) one or more or each of the polypeptide sequences is flanked at the N- and/or C-terminus by additional amino acids that are not part of the amino acid sequence of the polypeptide(s); (ii) two or more, or each of the polypeptide sequences containing up to 50 consecutive amino acids, are derived from different polypeptides; (III) one or more or each of up to 50 consecutive amino acid sequences comprises an amino acid sequence that (i) contains an amino acid sequence that is a T lymphocyte epitope capable of binding to at least three HLA class I molecules subject, and (ii) is a T lymphocyte epitope capable of binding to at least three HLA class II molecules in the subject or to the most possible HLA class II molecules in the subject for a sequence containing an HLA class I binding epitope (i); (IV) one or more or each active ingredient of a peptide or polypeptide of a pharmaceutical composition or kit; or: (i) consists of one of the specified sequences of up to 50 consecutive amino acids from a polypeptide expressed by a pathogenic organism, virus or cancer cell, associated with an autoimmune disease, or representing an allergen, or (ii) contains or consists of: (I) two or more of the above sequences of up to 50 consecutive amino acids located end to end or overlapping in a single peptide; or - 78 045699 (II) two or more of the specified sequences of up to 50 consecutive amino acids located end to end or overlapping in a single peptide, where one or more peptides or polypeptides do not contain a single neoepitope spanning the junction between any two of the specified amino acids sequences located end to end in a single peptide, and which: i) corresponds to a human polypeptide fragment expressed in healthy cells; ii) is a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject; or (iii) meets both (i) and (ii). 20. A method of treating a human subject in need, comprising: (A) administering to a subject a polypeptide containing a polypeptide fragment that has been identified as immunogenic, or a polypeptide that has been predicted to be immunogenic, or a polypeptide or pharmaceutical composition that has been predicted to induce a cytotoxic T lymphocyte response or a T response -helpers, or a pharmaceutical composition or pharmaceutical kit that has been predicted to induce a clinical response, or a pharmaceutical composition or pharmaceutical kit that has been determined to have a threshold minimum likelihood of inducing a clinical response, or a polypeptide or pharmaceutical composition that is is predicted not to induce a toxic immune response or the formation of ADA in the subject, using the method according to one of claims. 1-7, or one or more polypeptides or pharmaceutical compositions that have been selected or recommended for treating a subject using the method of claim 9; (B) administering to the subject a checkpoint inhibitor, wherein the subject is predicted to have a response or is likely to have a response to administration of the checkpoint inhibitor by the method of claim 11; (C) administering to the subject a human subject-specific pharmaceutical composition or polypeptides from a kit according to any one of claims 17 to 19, wherein the pharmaceutical composition or kit is specific to the subject. 21. Method according to claim 20, where: (a) the method is intended to treat cancer and/or (b) the treatment consists of the administration, in combination with chemotherapy, of a targeted therapy or a checkpoint inhibitor. 22. A method of designing or producing a polypeptide for inducing an immune response in a particular human subject, comprising selecting an amino acid sequence that represents a T-lymphocyte epitope capable of binding to at least three HLA class I molecules or at least three HLA class II molecules of the subject, and forming or producing a polypeptide containing the selected amino acid sequence. 23. The method according to claim 22, which is: (a) a method of designing or producing a polypeptide for inducing a cytotoxic T lymphocyte response in a particular human subject, comprising selecting an amino acid sequence that represents a T lymphocyte epitope capable of binding to at least three HLA class I molecules of the subject, and forming or producing a polypeptide containing a selected amino acid sequence, or (b) a method of forming or producing a polypeptide for inducing a T helper cell response, comprising selecting an amino acid sequence that represents a T lymphocyte epitope capable of binding to at least three HLA molecules a class II subject, and forming or producing a polypeptide containing the selected amino acid sequence; and where the method optionally further includes administering the polypeptide to the subject. 24. A method of inducing an immune response in a subject, comprising administering to the subject a polypeptide formulated in accordance with the method of claim 22 or 23.