Vaccines that target neoepitopes

Abstract

The present invention provides vaccination methods utilizing at least one neoepitope and a cationic liposome adjuvant mixed with a solvent. Also provided are unit doses and compositions for use in the methods.

Description

Technical Field

[0001] Field of the Invention The present invention relates to the field of immunotherapy. In particular, the present invention relates to therapeutic immunotechnology for the treatment of neoplastic diseases.

Background Art

[0002] Background of the Invention The treatment of malignant tumors in patients has traditionally focused on eradicating / removing malignant tissue by surgery, radiotherapy, and / or chemotherapy with cytotoxic agents in an administration regimen aimed at preferentially killing malignant cells over non-malignant cells.

[0003] In addition to the use of cytotoxic agents, more recent approaches have focused on targeting specific biological markers in cancer cells in order to reduce the systemic side effects that occur with conventional chemotherapy. Monoclonal antibody therapy targeting cancer-related antigens has been shown to be very effective in prolonging median survival in many malignant tumors. Although a successful drug, monoclonal antibodies targeting cancer-related antigens, by their nature, can only be developed to target expression products seen in multiple patients, i.e., as will be seen below, since many cancer-specific antigens are only seen in tumors from a single patient, the majority of cancer-specific antigens cannot be addressed by this type of therapy.

[0004] As early as the late 1950s, the immunosurveillance theory proposed by Burnet and Thomas suggested that lymphocytes recognize and eliminate self-cells - including cancer cells - that exhibit altered antigenic determinants, and today it is generally accepted that the immune system highly inhibits carcinogenesis. However, immunosurveillance is not 100% effective, and devising cancer therapies that improve / stimulate the immune system's ability to eliminate cancer cells remains an ongoing challenge.

[0005] One approach is to introduce immunity against cancer-related antigens. While this approach may be effective, it suffers from the same drawback as antibody therapy: it can only address a limited number of antigens.

[0006] Many tumors, though not all, express mutations. These mutations can potentially create novel target antigens (neoantigens), and if it is possible to identify these neoantigens and their antigenic determinants within a clinically appropriate timeframe, they may be effective in specific T-cell immunotherapy. Modern technology allows for the sequencing of entire cell genomes and the analysis of altered or novel expression products, making it possible to design personalized vaccines based on neoantigens. However, attempts to provide satisfactory clinical endpoints have so far failed. [Overview of the project] [Problems that the invention aims to solve]

[0007] Therefore, there is a need to provide an anti-cancer vaccine that can effectively target neoantigens in vaccinated individuals and induce a clinically significant immune response.

[0008] Purpose of the invention An objective of the embodiments of the present invention is to provide a method for inducing a therapeutically effective immune response to neoantigens, including neoepitopes. Furthermore, another objective is to provide a composition containing neoepitope material that can be used in cancer immunotherapy. [Means for solving the problem]

[0009] Summary of the Invention The inventors have found that formulating a neopeptide together with a specific cationic liposomal adjuvant, and administering such a formulation using a relatively high dose of the neopeptide, improves the immune response in vaccinated individuals. This is expected to lead to improvements in cancer immunotherapy for cancers characterized by the expression of neoepitopes, including the expression product.

[0010] Therefore, in a first embodiment, the present invention relates to a method for treating tumors, for example, malignant tumors, in mammalian patients, wherein the tumors exhibit T-cell epitopes (neoepitopes) not shown in non-tumor cells in the patient, and wherein the method comprises administering an immunologically effective amount of a liposome composition comprising: 1) At least one peptide (neopeptide) containing the amino acid sequence of a neoepitope in the patient's tumor cells, 2) Solvent, and 3) Cationic liposome adjuvants.

[0011] A second aspect of the present invention relates to a unit dose of an immunogenic composition, the unit dose comprising an immunologically effective amount of at least one peptide (neopeptide) containing the amino acid sequence of a neoepitope of a patient's tumor cells, a cationic liposome adjuvant, and a solvent.

[0012] A third aspect of the present invention relates to a liposome composition having the characteristics of a liposome composition administered as part of the first aspect of the present invention.

[0013] Related embodiments relate to unit doses of the second embodiment and compositions of the third embodiment for use in therapeutic and prophylactic therapies, particularly for use in the method of the first embodiment of the present invention. [Brief explanation of the drawing]

[0014] Legend of the drawing [Figure 1]Figure 1: Experimental design for the immunotherapy test in Example 1. Mice underwent cluster priming, including immunization on consecutive days, or were immunized according to a conventional prim-boost regimen. The dose of C22 and the number of immunization days are shown in the figure. Ip: Intraperitoneal administration

[0015] [Figure 2] Figure 2: Overview of the MHCI multimer assay. A stabilized peptide was loaded onto an MHC class I molecule, which was then exchanged for the C22 minimal epitope KFKASRASI by exposure to UV light. The MHCI molecule was conjugated to fluorescently labeled streptavidin and multimerized. To identify neopeptide-positive CD8+ T cells, blood cells were co-stained with the multimer and with fluorescently conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies. The samples were then analyzed by flow cytometry, and the fraction of MHC:C22-positive CD8+ was calculated.

[0016] [Figure 3-1] Figure 3: Detection of neopeptide-specific CD8+ T cells in whole blood at day 21. Figure 3A shows the percentage of multimer-positive CD8+ cells in the blood of two mice from each group 21 days after initial immunization. Circulating C22-specific CD8+ T cells were detected in mice immunized with 10.0 and 50.0 μg of C22 combined with CAF09b, regardless of the immunization schedule. Only blood from two mice from each group was stained. Figures 3B and 3C show representative plots from flow cytometry analysis, with Figure 3B showing the control. [Figure 3-2] Same as above.

[0017] [Figure 4-1]Figure 4: Detection of neo-peptide specific CD8+ T cells in whole blood on day 28, which is the endpoint. Figure 4A shows the percentage of multimer positive CD8+ in the blood from two mice in each group 28 days after the first immunization. Circulating C22-specific CD8+ T cells were detected in mice immunized with 10.0 and 50.0 μg of C22 formulated with CAF09b, regardless of the immunization schedule. Also, a slight increase was seen in the fraction of mice cluster-primed with 2.0 μg of C22. Figures 4B and C show representative plots from flow cytometry analysis, with Figure 4B showing the control. [Figure 4-2] The same as above.

[0018] [Figure 5-1] Figure 5: Detection of neo-peptide specific splenic CD8+ T cells. Figure 5A shows the percentage of multimer positive splenic CD8+ T cells at the endpoint (28 days after the first immunization) of cluster-primed mice. C22-specific CD8+ T cells were detected in mice immunized with 10.0 and 50.0 μg of C22 formulated with CAF09b. Figures 5B and C show representative plots from flow cytometry analysis, with Figure 5B showing the negative control. [Figure 5-2] The same as above.

[0019] [Figure 6-1] Figure 6: Immunization data in "Patient 1". A: ELISPOT data showing IFN-γ release from PBMC after immunization, stimulated with 9 peptides of the vaccine containing neo-epitopes. The healthy control group is the data from a parallel experiment using PBMC from control donors. CA019 was administered at twice the dose. [Figure 6-2]B: Flow cytometry data of PBMC obtained after intraperitoneal immunization. The left panel shows TNF-α and IFN-γ release from unstimulated PBMC, the middle panel shows TNF-α and IFN-γ release from PBMC stimulated with irrelevant peptides, and the right panel shows TNF-α and IFN-γ release from PBMC stimulated with nine peptides of the vaccine.

[0020] [Figure 7-1] Figure 7: Immune data in "Patient 2". A: ELISPOT data showing IFN-γ release when PBMC obtained before immunization were stimulated with five peptides of the neoepitope-containing vaccine. The healthy control is the data from a parallel experiment using PBMC of control donors. B: ELISPOT data showing IFN-γ release from PBMC obtained after intraperitoneal immunization and after stimulation with five peptides of the neoepitope-containing vaccine. The healthy control group is the data from a parallel experiment using PBMC of control donors. [Figure 7-2] C: Flow cytometry data of TNF-α and IFN-γ release from PBMC obtained before immunization. The left panel shows the release from unstimulated PBMC, the middle panel shows the release from PBMC stimulated with irrelevant peptides, and the right panel shows the release from PBMC stimulated with five peptides of the vaccine. D: Flow cytometry data of TNF-α and IFN-γ release from PBMC obtained after intraperitoneal immunization. The left panel shows the release from unstimulated PBMC, the middle panel shows the release from PBMC stimulated with irrelevant peptides, and the right panel shows the release from PBMC stimulated with five peptides of the vaccine. q

[0021] Detailed Disclosure of the Invention Definitions "Cancer-specific" antigens are antigens that are not expressed as products in the non-tumor somatic cells of an individual, but are expressed as products in the cancer cells of that individual. This is in contrast to "cancer-related" antigens, which are expressed at low concentrations in normal somatic cells but are found at high concentrations in at least some tumor cells.

[0022] The term "adjuvant" has its usual meaning in vaccine technology, namely, a substance or composition of substances that 1) cannot induce a specific immune response to the vaccine's immunogen on its own, but 2) can nevertheless enhance the immune response to the immunogen. In other words, vaccination with an adjuvant alone does not evoke an immune response to the immunogen, vaccination with an immunogen may or may not enhance the immune response to the immunogen, but vaccination with both an immunogen and an adjuvant induces a stronger immune response to the immunogen than that induced by the immunogen alone.

[0023] "CAF09" (Cationic Adjuvant Formulation 09) is an immunological adjuvant liposome formulation comprising N,N-dimethyl-N,N-dioctadecylammonium (DDA), a quaternary ammonium surfactant; synthetic 3-hydroxy-2-tetradecyl-octadecanoic acid-2,3-dihydroxypropyl ester (monomicholylglycerol, "MMG"), which acts as a ligand for C-type lectin receptors (CLRs); and polyinosinic acid-polycytidylic acid (sodium salt) ("polyIC" or "poly(I:C)"), which acts as a ligand for Toll-like receptors (TLRs). Numerous CAF family adjuvants, including CAF09, are disclosed in detail in US2014 / 0112979 and US2016 / 0228528. The relative amounts (w:w:w) of DDA:MMG:Poly(I:C) are 5:1:1.

[0024] "CAF09b" is a version of CAF09 in which the relative amount of poly(I:C) has been reduced to about one-quarter of the amount in CAF09 disclosed in US2014 / 0112979. The relative amounts of DDA:MMG:poly(I:C) (w:w:w) are therefore 20:4:1, and a typical human dose contains 625 μg of DDA, 125 μg of MMG, and 31.25 μg of poly(I:C).

[0025] A "neoepitope" is an antigenic determinant (typically an epitope constrained to MHC class I or II) that does not exist as an expression product in a normal somatic cell in an individual, but does exist as an expression product in mutated cells of the same individual (e.g., cancer cells), due to the deletion of the gene encoding the neoepitope. As a result, from an immunological standpoint, a neoepitope is truly non-self despite its self-origin, and therefore, in the individual in which the neoepitope constitutes an expression product, the neoepitope can be characterized as a cancer-specific antigen. Because it is non-self, a neoepitope can potentially trigger a specific adaptive immune response in an individual, and in this case, the induced immune response is specific to the antigen and cells that possess the neoepitope. On the other hand, a neoepitope is individual-specific because the likelihood of the same neoepitope being expressed in other individuals is low. Thus, neoepitopes have several characteristics that distinguish them from, for example, cancer-specific antigen epitopes: the latter are typically found in multiple cancers of the same type (because they may be expression products from activated oncogenes), and / or may be present in non-malignant cells, albeit in small amounts, due to the overexpression of related genes in cancer cells.

[0026] A "neopeptide" is a peptide (i.e., a polyamino acid up to approximately 50 amino acid residues) that contains a neoepitope as defined in this text within its own sequence. Neopeptides are typically "natural," meaning their entire amino acid sequence constitutes a fragment of an expression product that can be isolated from an organism. However, neopeptides can also be "artificial," meaning they consist of a neoepitope sequence and at least one or two additional amino acid sequences that are not naturally related to the neoepitope sequence. In the latter case, the additional amino acid sequences may simply act as carriers for the neoepitope or may further enhance the neoepitope's immunogenicity (e.g., by improving the neopeptide's biological half-life or altering its solubility to facilitate processing of the neopeptide by antigen-presenting cells).

[0027] The term "amino acid sequence" refers to the order of amino acid residues linked by peptide bonds in peptides and proteins. Sequences are conventionally written from the N-terminus to the C-terminus.

[0028] An "immunogenic carrier" is a molecule or moiety that can bind to an immunogen or hapten in order to enhance or enable the induction of an immune response to the immunogen / hapten. Traditionally, immunogenic carriers have been relatively large molecules (e.g., tetanus toxoid, KLH, diphtheria toxoid, etc.) that can fuse or conjugate to immunogens / haptens that are not sufficiently immunogenic on their own. Typically, an immunogenic carrier can induce a potent T helper lymphocyte response to a substance bound to a T helper lymphocyte, which is composed of the immunogen and the immunogenic carrier, thus providing an enhanced response to the immunogen by B lymphocytes and cytotoxic lymphocytes. More recently, large carrier molecules have been replaced to some extent by shorter peptides called promiscuous T helper epitopes, i.e., those recognized by the majority of a population's HLA haplotypes and that induce a T helper lymphocyte response.

[0029] "T helper lymphocyte response" is an immune response induced by a peptide, the peptide being able to bind to an MHC class II molecule (e.g., an HLA class II molecule) on an antigen-presenting cell, and the T cell receptor recognizing the complex of the peptide and the MHC class II molecule presenting the peptide, thereby stimulating T helper lymphocytes in animal species.

[0030] An "immunogen" is a substance that can induce an adaptive immune response in a host when the host's immune system is confronted with it. Thus, an immunogen is a subset of the larger "antigen" family that can be specifically recognized by the immune system (for example, when bound to an antibody, or when an antigen fragment bound to an MHC molecule is recognized by a T cell receptor), but does not need to be able to induce an immune response. However, antigens can always trigger immunity; that is, a host with established immunological memory against an antigen will develop a specific immune response to that antigen.

[0031] A "hapten" is a small molecule that cannot induce or trigger an immune response on its own, but when conjugated with an immunogenic carrier, an antibody or TCR that recognizes the hapten can be induced by contact between the immune system and the hapten carrier conjugate.

[0032] An "adaptive immune response" is an immune response that corresponds to contact with an antigen or immunogen, and the immune response is specific to the antigenic determinant of the antigen / immunogen. Examples of adaptive immune responses include the induction of antigen-specific antibody production, or antigen-specific induction / activation of T helper lymphocytes or cytotoxic lymphocytes.

[0033] A "protective, adaptive immune response" is an antigen-specific immune response induced within a subject as a response to an antigen (artificial or natural) and which can protect the subject from subsequent challenges by the antigen or pathology-related agents containing the antigen. Typically, vaccination aims to establish a protective, adaptive immune response against one or more pathogens.

[0034] "Immune system stimulation" means that a substance or composition of substances exhibits a general, non-specific immunostimulatory effect. Many adjuvants and putative adjuvants (e.g., certain cytokines) share the ability to stimulate the immune system. As a result of using an immunostimulant, the "alertness" of the immune system increases; that is, simultaneous or subsequent immunization by the immunogen induces a significantly more effective immune response compared to the use of the immunogen alone.

[0035] In the context of this text, the term "polypeptide" is intended to mean any of the following: short peptides of 2 to 50 amino acid residues, oligopeptides of 50 to 100 amino acid residues, and polypeptides of 100 amino acid residues or more. Furthermore, the term is also intended to include proteins, i.e., functional biomolecules comprising at least one polypeptide, and if a functional biomolecule comprises at least two polypeptides, these may form a complex, be covalently bonded, or be noncovalently bonded. Additionally, polypeptides in proteins may be glycosylated and / or lipidized and / or have prosthetic groups.

[0036] A specific embodiment of the present invention. Treatment method of the present invention - First aspect The first aspect of the present invention relates generally to the induction of immunity, and as such, also to methods relating to the treatment, prevention, and improvement of diseases, and in particular to methods relating to the treatment and improvement of cancer. That is, the first aspect of the present invention generally relates to methods for treating neoplastic diseases, such as malignant neoplastic diseases, in mammalian patients, wherein the tumor cells in the patient exhibit T cell epitopes (neoepitopes) that do not show non-tumor cells, and the method is 1) At least one peptide (neopeptide) containing the amino acid sequence of a neoepitope in the patient's tumor cells, 2) Solvent, and 3) Cationic liposome adjuvants This involves administering an immunologically effective dose of a liposome composition containing the following:

[0037] Many cationic liposomal adjuvants are known. Of these, preferred in the present invention are those developed at the Statens Serum Institute in Denmark and part of a line of immunoadjuvants known as CAFs (Cationic Adjuvant Formulations); a recent review describes the general characteristics of adjuvants in the CAF line: Pedersen GK et al. (2018), Semin Immunol 39:4-13. doi:10.1016 / j.smim.2018.10.00. All CAFs are characterized by containing the surfactant dimethyldioctadecylammonium (DDA), and most CAFs exist as liposomal formulations (CAF01, CAF04, CAF05, CAF06, CAF09, CAF10, and CAF11), although a few recently developed CAFs are in the form of emulsions containing oil (squalene) (CAF19 and CAF24).

[0038] Particularly interesting cationic liposome adjuvants in the present invention are those comprising or consisting of DDA, poly(I:C), and MMG (including synthetic analogs of MMG such as 3-hydroxy-2-tetradecyl-octadecanoate-2,3-dihydroxypropyl ester). In other words, they are CAF09 adjuvants.

[0039] In preferred cationic liposome adjuvants, the DDA:MMG ratio is 5:1 (w / w), and the DDA:poly(I:C) ratio is between 5:1 and 20:1 (w / w). For example, in the present invention, in the case of "traditional" CAF09, the relative weight of DDA:MMG:poly(I:C) may be 5:1:1, and in the case of CAF09b adjuvant, the relative weight of DDA:MMG:poly(I:C) may be 20:4:1.

[0040] To facilitate the formulation of neopeptides into vaccines, it is preferable that neopeptides be water-soluble. For this purpose, neopeptides are carefully selected to meet specific solubility criteria before being incorporated into vaccine formulations. For example, it is preferable that at least one neopeptide is water-soluble in the sense that, when the NTU (nephelometric turbidity unit) is measured according to Section 2.2.1 of the European Pharmacopoeia, 1.0 mg / ml of neopeptide in 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO exhibits a maximum NTU of 50, and 0.1 mg / ml of neopeptide in 25 mM TRIS pH 7.4 and 0.5% (v / v) DMSO exhibits a maximum NTU of 25.

[0041] More specifically, neopeptides are tested in an assay involving spectrophotometric measurements at 620 nm to determine the degree of opalescence (i.e., turbidity) of peptide solutions in TRIS buffer at various concentrations, and from there, the corresponding NTU values ​​(using a calibration curve from reference suspension measurements with Primary Opalescent Suspension, Fisher Scientific; EPPOS01) are calculated. This can be used to evaluate whether a given neopeptide is "soluble" (NTU < 25 at a concentration of 0.1 mg / mL; NTU < 50 at a concentration of 1.0 mg / mL) or "insoluble" (NTU > 25 at a concentration of 0.1 mg / mL; NTU > 50 at a concentration of 1.0 mg / mL). This method is used to determine the solubility of HPLC-purified (purity > 95%) linear peptides consisting of natural L-amino acids (typically excluding cysteine ​​residues) by compounding each test peptide at 0.1 mg / mL in TRIS buffer (containing 0.5% DMSO). Furthermore, test samples can be measured in a concentration range of 0.1 to 4.0 mg / ml. For example, in this case, the neopeptide is dissolved in DMSO at 20 mg / ml, and then serially diluted 5-fold, 10-fold, 20-fold, and 200-fold to 4.0, 2.0, 1.0, and 0.1 mg / ml in TRIS buffer.

[0042] The turbidimetric assay then determines the clarity of the sample based on Ph Eur 2.2.1. If the neopeptide is insoluble, aggregates or particles will form, which are measured by examining the increase in scattering as light passes through the sample at 620 nm. The measurements are then compared to a standard curve to determine the final NTU score indicating the solubility of the single peptide. One procedure for determining the solubility of a single peptide is as follows:

[0043] The weight of a single peptide is measured and dissolved in DMSO at a concentration of 20 mg / ml. 1.0 mg / mL sample: Dilute 25 μl of 20 mg / mL sample in 475 μl of 25 mM TRIS pH=7.4 to 1.0 mg / mL (final 5% DMSO). 0.1 mg / mL sample: Dilute 2.5 μl of 20 mg / mL sample in 497,5 μl of 25 mM TRIS pH=7.4 to 0.1 mg / mL (final 0.5% DMSO). Place 100 μl of diluted sample into an ELISA plate and measure the optical density at 620 nm where agglutination occurs. The NTU value is calculated by linear regression of the standard curve.

[0044] To provide preferred neopeptides in liposome compositions, further solubility assays are performed on mixtures of multiple neopeptides: when at least one neopeptide is present in a mixture of multiple neopeptides and 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO, its concentration decreases by up to 50% upon sterile filtration, where each of the multiple peptides is preferably at a concentration of 0.1 mg / ml in the mixture before sterile filtration. This approach provides for the exclusion of neopeptides from liposome compositions that may be shown to be less suitable for vaccines due to their ability to aggregate with other neopeptides, although the turbidimetric method for a single neopeptide described above cannot be readily used to demonstrate this.

[0045] Generally, to make the vaccine as effective as possible, this method is based on the use of multiple neopeptides. Typically, the number of neopeptides in the liposome composition is selected 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, and 30 neopeptides, but in principle, the number may be higher as long as the physicochemical properties of the liposome composition allow.

[0046] Multiple neopeptides are identified by one of several methods known to those skilled in the art. The simplest approach is to sequence (typically by "deep sequencing") a genome derived from a sample of malignant cells from a patient and compare it in slico to a whole genome sequence (likewise, typically by "deep sequencing") obtained from the patient's normal cells or a standard "healthy" genome. The differences in the two sequence datasets are then analyzed to look for candidate mutations that 1) express (i.e., are parts of the open reading frame) and 2) provide expression products containing T cell epitopes not typically seen in the patient. For this purpose, data on the patient's MHC type is generally included to ensure that the prediction of T cell epitopes correctly provides only MHC-bound sequences. An overview of T cell epitope prediction methods, including MHC binding prediction, can be found, for example, in Desay DV and Kulkami-Kale U (2014), Methods Mol Biol 1184: 333-364 (doi: 10.1007 / 978-1-4939-1115-8_19) and Soria-Guerra RE et al. (2015), Journal of Biomedical Informatics 53: 405-414 (doi: 10.1016 / j.jbi.2014.11.003).

[0047] To optimize the identification and selection of neoepitopes, any predictive method available for this purpose is indeed effective. One example of a state-of-the-art predictive algorithm is NetMHCpan-4.0 (www.cbs.dtu.dk / services / NetMHCpan-4.0 / ; Jurtz V et al., J Immunol (2017), ji1700893; DOI:10.4049 / jimmunol.1700893). This method is trained on a combination of conventional MS-derived ligand and pMHC affinity data. Another example is NetMHCstabpan-1.0 (www.cbs.dtu.dk / services / NetMHCstabpan-1.0 / ; Rasmussen M et al., Accepted for J of Immunol, June 2016). This method is trained on a dataset of in vitro pMHC stability measurements using assays that synthesize each peptide and complex it with MHC molecules in vitro. In this assay, no cell treatment is performed, and the environment in which pMHC stability is measured is somewhat artificial. This method is generally less accurate than NetMHCpan-4.0. U.S. Patent 10,055,540 describes a method for identifying neoepitopes using ligands detected by conventional MS. Other patent applications using similar techniques are WO2019 / 104203, WO2019 / 075112, WO2018 / 195357 (MHC class II specific), and WO2017106638. Finally, MHCflurry:(DOI: doi.org / 10.1016 / j.cels.2018.05.014;https: / / github.com / openvax / mhcflurry) is similar to NetMHCpan, trained on ligand data detected by MS and pMHC affinity. Furthermore, a method for predicting peptide-MHC class II interactions is disclosed in the recent publication Garde C et al., Immunogenetics, DOI: doi.org / 10.1007 / s00251-019-01122-z.In this publication, naturally processed peptides eluted from MHC class II are used as part of the learning set and assigned a binding target value of 1 if confirmed as a ligand and 0 if negative.

[0048] Generally, these prediction systems employ artificial neural networks (ANNs): ANNs can identify nonlinear correlations: Quantifying nonlinear correlations is not an easy task because it is difficult to compute with simple calculations. This is mainly because nonlinear correlations are described by more parameters than linear correlations and may only become apparent when all features are considered together. Therefore, all features must be taken into account in order to capture the dependencies between features.

[0049] To further enhance the likelihood that selected neoepitopes will provide an effective immune response, techniques disclosed in European Patent Applications No. 19197295.9 and 19197306.4, both filed on September 13, 2019, may be preferably used. These applications disclose techniques that enable the determination of the binding stability between peptides and MHC molecules, and that enable the determination of the MHC binding stability of neoepitopes as part of the detection and selection of neoepitopes. Briefly, data obtained from stability determination can be used, for example, in a learning set for an ANN, and the ANN can then rank the identified peptides according to their predicted binding stability to the relevant MHC molecules.

[0050] Therefore, in the present invention, it is preferable that at least one neopeptide discussed herein includes neoepitopes exhibiting above-average, for example, top-quarter or higher MHC binding stability from among the neoepitopes identified in tumor cells. Specific selection of neoepitopes included in this neopeptide is facilitated by the techniques disclosed in European Patent Applications Nos. 19197295.9 and 19197306.4. In the present invention, the identified (long) neopeptide may be prepared as a cleaved version, where convenient, to optimize the characteristics and / or manufacture and / or stability of the final product ultimately administered to the patient. Therefore, in this preferred embodiment, the following cleavage rules are generally applied to any identified neopeptide.

[0051] If the neopeptide contains a carbon atom anywhere in its sequence, it is cleaved to remove the carbon atom, preferably leaving the longest fragment of the original neoepitope. For example: QIETQ C RKFKASRASILSEMKMLKEKR (Sequence ID 17) →RKFKASRASILSEMKMLKEKR (Sequence ID 18)

[0052] If there is a Q or N at the N-terminus, remove the Q / N. For example, as follows: Q IETQQRKFKASRASILSEMKMLKEKR (Sequence ID 19) →IETQQRKFKASRASILSEMKMLKEKR (Sequence ID 20) and N IETQQRKFKASRASILSEMKMLKEKR (Sequence ID 21) →IETQQRKFKASRASILSEMKMLKEKR (Sequence No. 20).

[0053] If a neopeptide contains a DG motif at its N-terminus, remove the D while retaining the G, for example, as follows: DGETQQRKFKASRASILSEMKMLKEKR (Sequence ID 22) →GETQQRKFKASRASILSEMKMLKEKR (Sequence ID 23)

[0054] As a result, the neopeptides discussed herein as useful immunogens in the compositions disclosed herein do not contain cysteine ​​residues and / or do not contain Q and N as N-terminal amino acid residues and / or do not contain the amino acid sequence DG at the N-terminus. In particularly preferred embodiments, none of the neopeptides in the composition contain cysteine ​​residues, none of the neopeptides in the composition contain Q or N as N-terminal amino acid residues, and none of the neopeptides in the composition contain the amino acid sequence DG at the N-terminus.

[0055] Following identification, the (multiple) neopeptides are synthesized and, preferably, subjected to methods for determining their solubility before incorporation into liposomal formulations, as discussed above.

[0056] In some embodiments, at least one neopeptide is preferentially associated with the discontinuous phase in the liposome composition; that is, at least one neopeptide is captured or bound to the liposome to a significantly higher degree than when they are present in the continuous phase (solvent). In other embodiments, instead of being associated with or bound to the liposomes in the liposome composition, at least one neopeptide is preferentially dissolved in the continuous phase of the liposome composition.

[0057] Typically, an immunologically effective dose is administered to a patient multiple times; typically, this involves administering the immunologically effective dose in a series of separate doses separated by at least one day. In a preferred embodiment of this aspect, the immunization scheme involves a mammal (e.g., human) receiving a single priming dose and one or more subsequent booster doses, but an equally attractive alternative is to use an immunization scheme of "cluster immunization," i.e., administering doses of the immunogen repeatedly at short intervals early in the immunization regimen before a memory immune response is established; and then performing a late immunization similar to the conventional booster immunization used in prime-boost immunization regimens.

[0058] When the immunologically effective dose constitutes a relatively large volume of the composition discussed in this text, it is advantageous to administer the immunologically effective dose in several "sub-doses," i.e., by dividing the immunologically effective dose into two or more parts, each typically administered to a different location in the patient. For example, when administering an immunogenic composition in a volume of 2000 μl by injection, it is convenient to do so as two substantially simultaneous injections (i.e., within the same 12-hour period) at different injection sites.

[0059] Liposome compositions may contain pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, such as a polypeptide. This term refers to any pharmaceutically acceptable carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without excessive toxicity. Suitable carriers may be large, slowly metabolized polymers, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, and inactivated viral particles. Such carriers are well known to those skilled in the art.

[0060] Medicinally acceptable salts can also be used as excipients, such as mineral salts like hydrochloride, hydrobromide, phosphate, and sulfate, and organic salts like acetate, propionate, malonate, and benzoate. A detailed discussion of medicinally acceptable excipients is available in Remington's Pharmaceutical Sciences (MackPub.Co., NJ. 1991).

[0061] A pharmaceutically acceptable carrier in a liposome composition may include liquids such as water, saline, glycerol, and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present in the vehicle. Typically, liposome compositions are prepared as either liquid solutions or suspensions for injection; they may also be prepared in solid form suitable for dissolving or suspending in a liquid vehicle before injection. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

[0062] In a first aspect of the present invention, the solvent in the liposome composition is typically aqueous and preferably a buffer solvent that maintains a pH within the physiological range for humans. Since the neopeptide is conveniently preserved and initially dissolved in DMSO (dimethyl sulfoxide), this solvent component is present in the solvent of the liposome composition, preferably at a relatively small concentration. Furthermore, the buffer used in the aqueous solvent is typically a TRIS (tris(hydroxymethyl)aminomethane) buffered aqueous solvent. Finally, this solvent may conveniently contain a small amount of glycerol. Thus, a preferred liposome composition contains 15% or less DMSO, for example 10% or less DMSO, preferably less than 5% DMSO (v / v) and about 2% (v / v) glycerol. Furthermore, the liposome composition conveniently contains TRIS at a concentration between 15 and 18 mM, preferably between 16.1 and 16.4 mM, but it should be emphasized that the TRIS concentration is not essential and can vary, for example, between 5 and 50 mM.

[0063] Typically, liposome compositions have a pH in the range of 7.0 to 7.6, preferably about 7.4.

[0064] A particularly preferred liposome composition used in the method of the first aspect of the present invention includes or comprises the following: 1) Multiple neopeptides with a concentration of 100 μg / ml per neopeptide. 2) DMSO with a concentration of less than 5% (v / v) 3) Approximately 1250μg / ml DDA, 4) Approximately 250 μg / ml MMG, 5) Approximately 62.5 μg / ml poly(I:C), 6) Approximately 2% (v / v) glycerol, and 7) TRIS with a concentration of approximately 16.25 mM and a pH of approximately 7.4.

[0065] When performing the method of the first embodiment, the immunological effective amount of each neopeptide administered is preferably at least 10 μg, for example, at least or more 15, at least or more 20, at least or more 25, at least or more 30, at least or more 35, at least or more 40, at least or more 45, at least or more 50, at least or more at least or more 55, at least or more 60, at least or more 65, at least or more 70, at least or more 75, at least or more 80, at least or more 85, at least or more 90, at least or more 95, at least or more 100, at least or more 105, at least or more 110, at least or more 115, at least or more 120, at least or more 125, at least or more 130, at least or more 135, at least or more 140, at least or more 145, at least or more 15 0, at least or more than 155, at least or more than 160, at least or more than 165, at least or more than 170, at least or more than 175, at least or more than 180, at least or more than 185, at least or more than 190, at least or more than 195, at least or more than 200, at least or more than 205, at least or more than 210, at least or more than 215, at least or more than 220, at least or more than 225, at least and at most 230, at least or at most 235, at least or at most 240, at least or at most 245, at least or at most 250, at least or at most 255, at least or at most 260, at least or at most 265, at least or at most 270, at least or at most 275, at least or at most 280, at least or at most 285, at least or at most 290, at least or at most 295, at least or at most 300, at least or at most 305,The amounts are at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, and at least 400 μg. Therefore, preferably, the immunological effective dose of each administered peptide is 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 5 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 ,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154 ,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 ,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297,298,299,300,30 1, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 3 Selected from the group consisting of 53, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400 μg.

[0066] In this regard, the volume of liposome composition administered per immunization to humans is typically between 400 and 2000 μl, with typical volumes being approximately 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, and 74 0, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 120 0, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 161 The available volumes are 0, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, and 2000 μl. As indicated above, each dose of an immunologically effective amount of liposomal composition can consist of at least two sub-doses, particularly two sub-doses per immunization; in this case, the immunologically effective amount is divided into a number of fractions, and each fraction is thus typically administered to a different site.For example, when administering 2000 μl, this is preferably done as two sub-administrations of 1000 μl of the composition, preferably at different locations in the patient.

[0067] As mentioned in the text, preferred vaccines of the present invention induce cellular immunity, particularly CD8+ T cell responses, especially CTL responses, but induction of CD4+ T cell responses is also valuable.

[0068] Regarding the route of administration, any route that is convenient and effective for the administration of peptide-based vaccines can be used. Preferred routes are intradermal, subcutaneous, intraperitoneal, and intramuscular routes, but intrapulmonary, intraocular, intrathecal, and intracerebral routes are also possible.

[0069] Unit dose of composition - Second aspect A second aspect of the present invention relates to a unit dose of a (liposome) composition, wherein the unit dose comprises an immunologically effective amount of at least one peptide (neopeptide) containing the amino acid sequence of a neoepitope of a patient's tumor cell, a cationic liposome adjuvant, and a buffering solvent. Generally, the properties and components of the unit dose composition correspond to the properties and components of the composition used in the first aspect of the present invention, and generally, all considerations and details relating to the liposome composition disclosed above apply mutatis mutandis to the composition forming the unit dose.

[0070] Therefore, in line with the fact that this method involves the administration of a specific preferred amount of neopeptide discussed above, the unit dose is preferably at least 10 μg, for example, at least or more 15, at least or more 20, at least or more 25, at least or more 30, at least or more 35, at least or more 40, at least or more 45, at least or more 50, at least or more at least or more 55, at least or more 60, at least or more 65, at least or more 70, at least or more 75, at least or more 80, at least or more 85, at least or more 90, at least or more 95, at least or more 100, at least or more 105, at least or more 110, at least or more 115, at least or more 120, at least or more 125, at least or more 130, at least or more 135, at least or more 140, at least or more 145, at least or more 150, at least or more 155, at least or more 160, at least or more 165, at least or more 170, at least or more 175, at least or more 180, at least or more 185, at least or more 190, at least or more 195, at least or more 200, at least or more 205, at least or more 210, at least or more 215, at least or more 220, at least or at most 225, at least or at most 230, at least or at most 235, at least or at most 240, at least or at most 245, at least or at most 250, at least or at most 255, at least or at most 260, at least or at most 265, at least or at most 270, at least or at most 275, at least or at most 280, at least or at most 285, at least or at most 290, at least or at most 295, at least or at most 300,At least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least or many It comprises at least 380, at least or more than 385, at least or more than 390, at least or more than 395, and at least or more than 400 μg of each neopeptide, and preferably 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 ,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,1 03, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 13 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 ,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 25 2, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 3 08, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, Contains an amount of each neopeptide selected from 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400 μg – however, it should be noted again that the upper limit of the total mass of neopeptides contained is determined by the physicochemical properties of the composition.

[0071] Similarly, along with the preferred volume of the liposome composition to be administered, the unit dose is preferably between 400 and 2000 μl, for example, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 7 60, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 121 0, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1 It consists of 620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1700, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1800, 1810, 1820, 1830, 1840, 1850, 1860, 1870, 1880, 1890, 1900, 1910, 1920, 1930, 1940, 1950, 1960, 1970, 1980, 1990, and 2000 μl.

[0072] Similarly, a unit dose contains many neopeptides, preferably a number of neopeptides selected 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, and 30, but the number may be greater if the physicochemical properties of the liposome composition allow it.

[0073] Composition of the present invention - Third aspect The liposome composition used in the first aspect of the present invention is considered to be an invention in itself. Accordingly, the third aspect of the present invention relates to the liposome composition disclosed in the context of the first aspect of the present invention, and all disclosures made in the first aspect apply mutatis mutandis to the third aspect of the present invention to the extent that they relate to the properties and components of the liposome composition.

[0074] Other aspects As is evident from the claims, the present invention also relates to a unit dose of a second embodiment or a composition of a third embodiment for use in therapy, particularly for use in the method of the first embodiment. Similarly, the present invention includes the use of a unit dose of a second embodiment or a liposome composition of a third embodiment for the manufacture of a pharmaceutical composition for use in any method of the first embodiment of the present invention. [Modes for carrying out the invention] [Examples]

[0075] Example 1 Induction of neoepitope-specific T cells by peptides in the CAF09b adjuvant. In two recent, confidential preclinical oncology trials (data not presented), immunization with a vaccine consisting of the adjuvant CAF09b mixed with 2.0 μg each of several predicted neopeptides did not inhibit the growth of tumors from which these neopeptides originated.

[0076] In this study, a single neopeptide (referred to as C22 in this text) was tested at higher doses. The minimal epitope of C22 has been defined, and therefore, neopeptide-specific CD8+ T cells can be detected by MHCI multimer staining. The amino acid sequence of C22 is QIETQQR KFKASRASI It possesses LSEMKMLKEKR (SEQ ID NO: 1), and its minimal MHC class I-constrained epitope is composed of the underlined 9 amino acid KFKASRASI (SEQ ID NO: 2).

[0077] Therefore, the objectives of this study are as follows: i) Test whether high doses of neopeptides mixed with CAF09b induce neopeptide-specific spleen T cells. ii) To test whether neopeptide-specific CD8+ cells can be detected in unconcentrated whole blood samples, which will allow for tracking of the response over time. iii) Test whether "cluster priming" is superior to standard prime-boost immunization.

[0078] From the perspective of promoting the CD8+ T cell immune response with peptide-based vaccines, the Statens Serum Institute (SSI) has developed CAF09b, a cationic liposome adjuvant that has shown induction in both CD4+ and CD8+ T cells. This liposome consists of positively charged dimethyldioctadecylammonium (DDA) to promote repulsion / stability and cell entry, monomicylglycerol (MMG) as a PAMP signal for CD4+ cell generation, and polyinosinate:polycytidylic acid ("poly(I:C)"), a Toll-like receptor 3 agonist that may induce reactive cytotoxic T cells.

[0079] Neopeptides were identified by whole-exome sequencing of the mouse colorectal cancer cell line CT26 and normal tissue samples from BALB / c mice, and by selecting peptides found only in cancer cells.

[0080] The purpose of this experiment is to test whether a predicted neopeptide mixed with the CAF09b adjuvant can induce reactive T cells in mice.

[0081] According to Wick DA et al. (2011), Vaccine 29(5):984-993, cluster priming with 3.7 μg of a 19-amino acid peptide derived from HPV E7 mixed with poly(I:C) was reported to be superior to conventional prime-boost immunization. In addition, a recent paper by Ott PA et al. (2017), Nature 547(7662):217-221, reports clinical responses in melanoma patients cluster-primed with Hiltonol adjuvant and neopeptide.

[0082] Therefore, it was hypothesized that cluster priming with neopeptides would elicit a stronger response compared to standard prime-boosted immunization.

[0083] material and method BALB / c mouse Six-to-eight-week-old BALB / c JrJ female mice were obtained from Janvier Labs. These mice were acclimatized for one week prior to the start of the experiment. The mice had free access to water and standard feed. The experiment was conducted under the Danish Animal Experimentation Act, which is more stringent than and therefore fully compliant with European Directive (2010 / 63 / EU), and under license 2017-15-0201-01338 from the Danish Animal Experimentation Inspection Service. The mice were tagged with ear tags for individual identification.

[0084] Experimental Design - Mouse Assignment and Treatment To avoid the "cage effect," all treatment groups were placed in separate cages. Mice from groups 1, 2, 3, and 4 (cluster immunization groups) were immunized intraperitoneally (ip) with C22 mixed with CAF09b at doses of 0.4, 2.0, 10.0, and 50.0 μg, respectively, on days 0, 1, 2, 3, 7, 14, and 21. The corresponding groups (5, 6, 7, and 8) were vaccinated on days 0, 14, and 21 using the same solution as groups 1, 2, 3, and 4, following the conventional prime-boost strategy. The dose administered to mice was 200 μl for all vaccines. See Figure 1 for details.

[0085] vaccine formulation The C22 peptide was synthesized by GenScript and dispensed as a lyophilized 0.8 mg dose. The peptide was dissolved in DMSO to a concentration of 10 mg / ml by adding 80 μl of DMSO to each dose. The stored peptide was then diluted 10-fold with sterile water (80 μl stored peptide + 720 μl sterile water). Peptides were prepared for each administration day and stored at -20°C until use. On the day of immunization, the peptide was thawed and added to TRIS buffer and CAF09b in the indicated volumes. The final vaccine for mice contained 200 μg of DDA, 40 μg of MMG, and 10 μg of Poly(I:C) per dose. See the table below for further details of the vaccine formulation.

[0086] [Table 1]

[0087] Preparation of a single-cell suspension derived from the spleen For the endpoint, the spleens of all mice participating in the study were collected. Single-cell suspensions were prepared according to the following protocol.

[0088] material RPMI (Gibco RPMI 1640) FCS (Gibco) 10xRBC lysis buffer (BioLegend, #420301) 70μm Cell Strainer (Corning, #43175) GentleMACS C Tube (Miltenyi, #130-093-237) 50ml tube cryovial

[0089] Equipment GentleMACS Dissociator (Miltenyi) LAF bench Mr. Frosty or other freezing boxes

[0090] procedure 1. Collect the tissue in a pre-labeled C tube containing R10 (RPMI containing 10% FCS). Recommendation: Use 3 ml of culture medium per spleen. Store on ice until processing. Maintain sterile conditions. 2: Securely close the C tube and invert it into the sleeve of the gentleMACS dissociator. Run program m_spleen_01 (for 1-2 spleens per tube). 3. (Optional) After dissociation, perform a short centrifuge of the C tube to collect the sample material that has accumulated at the bottom of the tube. Place a 4:70 μm cell strainer on top of a pre-labeled 50 ml tube and pre-wet it with R10. 5. Resuspend the sample in the C tube and apply the cell suspension to a 70 μm cell strainer. 6. (Optional) Wash the C tube with 2 ml of R10 and apply it to a cell strainer to obtain residual cells. Wash the cell strainer with 7.5 ml of R10. Remember to pipette any remaining liquid from the bottom of the filter. 8. Centrifuge the cells: 1500 rpm, 5 minutes, 4°C. Discard the supernatant. Crush the pellet. 9: (Optional) Lyse red blood cells for 2 minutes using 1 ml of 1x RBCL buffer (10x stock, diluted with PBS). Wash with 5 ml of R10 and remove cells from the RBCL buffer. Resuspend in 10:1 ml R10 and count the cells. 11. Wash again with 5 ml of R10. (Optional) Further filter through a 70μm cell strainer to remove any debris. 12: Place the cells in FCS + 10% DMSO at an appropriate concentration (e.g., 20x10). 6 Resuspend in (cells / ml). Dispense 13:1 ml of cell suspension into pre-labeled cryovials. 14. Transfer the cells to Mr. Frosty and leave them at -80°C for 24 hours, then transfer them to nitrogen storage.

[0091] To detect C22-specific CD8+ T cells, whole EDTA blood samples and splenocytes were stained with fluorescently labeled MHCI multimers loaded with KFKASRASI (SEQ ID NO: 2), the smallest peptide derived from C22, which was identified as the strongest MHCI conjugate embedded within the 27 amino acids of the C22 sequence. The MHCI multimer assay is shown in Figure 2. The detailed staining protocol is as follows.

[0092] material Eppendorf Tube Deep 96-well plate (2ml, Sigma, #575653) 10x Lysis Solution (BD#349202) MHC polymers labeled with C22 APC and PE, and unlabeled (C30) MHC polymers labeled with APC and PE. FACS buffer (PBS+2%FCS)

[0093] procedure 1. Collect blood from the mouse: Insert a vein (tail or saphenous vein) into an EDTA-coated tube - BD Vacutainer EDTA blood collection tube. 2. Stain 50 μl of blood in a deep 96-well plate according to the plate settings. 3. Spin the thawed polymer (quick spin). 4. Dilute 1 μl of each macromer with 29 μl of FACS buffer (per sample) in an Eppendorf tube. 5. Spin the polymer in an Eppendorf tube (3300g, 5 minutes). 6. Add 30 μl of diluted polymer according to the plate settings. Add 30 μl of FACS buffer to the "sample without polymer" according to the plate settings. 6.1: Avoid pellets in polymer tubes, as they may contain aggregates of MHC polymers. 7. Add 50 μl of FACS buffer to the sample according to the plate settings. 8. Incubate in a dark place at 37°C for 15 minutes. Add 9.1 μl of anti-CD16 / CD32 to block FC binding, and incubate in the dark at room temperature for 10 minutes. 10. Prepare the antibody master mix according to the calculations - see Appendix 2. 11. Add 20 μl of antibody master mix to the wells according to the plate settings and stain. Incubate at 12:4°C for 30 minutes. 13. To lyse the red blood cells, add 1 ml of lysis / fixative (diluted with H2O) per well. Incubate in the dark at room temperature for 5-10 minutes. 14. Wash twice with FACS buffer (spin at 1500 rpm for 5 minutes). Transfer to a FACS tube with 15:200 μl of FACS buffer. 16. Analyze using a flow cytometer.

[0094] result Detection of circulating neopeptide-specific CD8+ T cells On day 21, EDTA-treated blood was collected and stained with MHCI multimer according to the assay outlined above and in Figure 2. As this was the first attempt to detect neopeptide-specific CD8+ T cells in whole blood samples, only samples from two mice per group were analyzed; therefore, this analysis was considered a pilot experiment. For the endpoint (day 28), blood samples from all mice in the experiment were used.

[0095] On day 21, dose-dependent responses were observed with both cluster priming and the standard prime-boost schedule; neopeptide-specific CD8+ T cells were detected in mice administered 10.0 and 50.0 μg of C22 mixed with CAF09b, while lower doses did not induce any neopeptide-specific CD8+ T cells (Figure 3). At the endpoint, the response was similar to that on day 21, even after the mice received an additional boost (Figure 4). Notably, at the endpoint, while few peptide-specific CD8+ cells were observed with the standard prime-boost schedule, a low frequency of MHC:C22-positive CD8+ cells was detected in the blood of mice cluster-primed with 2.0 μg of C22.

[0096] To investigate the specificity of the assay, blood samples from mice administered CAF09b were stained with MHC:C22 polymers. No signal was detected in these samples. Similarly, blood samples stained with MHCI tetramers loaded with an unrelated peptide derived from neopeptides (referred to as "C25") were also negative, indicating high specificity of the assay.

[0097] Detection of neopeptide-specific CD8+ T cells in the spleen On the final day, 28 days after the initial immunization, mouse spleens were collected, single-cell suspensions were prepared, and stored at -80°C. In addition, the spleens included those from two naive mice and two mice that received ip-administered CAF09b.

[0098] On the final day, only splenocytes derived from cluster-primed mice were analyzed.

[0099] Neopeptide-specific CD8+ T cells were detected in the spleens of mice immunized with 10.0 and 50.0 μg of C22+CAF09b, while lower doses did not induce a detectable response. Similar to observations in whole blood samples, neopeptide-specific CD8+ T cells were not detected in CAF09b-treated mice. In addition, no signal was observed when spleen cells were stained with MHC tetramers loaded with an unrelated peptide (referred to as "C25"). See Figure 5.

[0100] Animal health To monitor the effects of the vaccine on the general health of the mice, the animals were carefully examined after vaccination, and their weight was measured at least three times a week throughout the experiment. Immediately after immunization, the mice showed signs of abdominal pain, but these subsided within 1-2 hours, and they subsequently returned to normal behavior. Immunization on consecutive days did not have a significant impact on the health of the mice compared to standard prime-boost. In both experimental settings, the mice lost approximately 5% of their initial body weight after the first immunization, but subsequent immunizations did not affect body weight (data not presented).

[0101] discussion Immunization with 10.0 and 50.0 μg of C22 mixed with CAF09b strongly induced neopeptide-specific CD8+ cells identified by MHCI tetramer staining, but surprisingly, did not induce them at lower doses. On the other hand, no significant advantage of cluster priming was observed compared to the conventional prime-boost schedule. However, a slight increase was observed in the response to 2.0 μg of C22 with cluster priming compared to the conventional prime-boost.

[0102] Mice immunized with a vaccine containing CAF09b lost approximately 5% of their initial body weight, suggesting the presence of a bioactive component in the vaccine. The general health impact of the vaccine on mice is considered acceptable, as the weight loss was temporary and all mice returned to normal within 3-4 days, using both cluster priming and prim-boost methods.

[0103] Example 2 Preliminary data from clinical trials A Phase 1 / 2a clinical trial testing the present invention's neoepitope-targeting approach commenced in March 2019 with the first dose administered to patients. This trial includes patients with malignant melanoma, non-small cell lung cancer (NSCLC), and bladder cancer.

[0104] In short, the procedure involves identifying a group of neoepitopes derived from malignant cells in each registered patient, then synthesizing, compounding, and administering the neoepitopes to the patient according to the present invention; first, three intraperitoneal immunizations at two-week intervals, followed by three intramuscular immunizations at two-week intervals, with a two-week rest period after the last intraperitoneal administration. Thus, all vaccinations are performed at preferred two-week intervals.

[0105] The following data was obtained from two patients after the completion of intraperitoneal immunization and before intramuscular immunization.

[0106] [Table 2]

[0107] Patients 1 and 2 each received three intraperitoneal immunizations using a vaccine prepared from a pool of peptides identified as neoepitopes containing amino acid sequences from the patient's malignant tissue.

[0108] For patient 1, the peptide pool contains the following nine peptides (SEQ ID NOs: 3-11) dissolved in DMSO.

[0109] [Table 3]

[0110] For patient 2, the peptide pool contains the following five peptides (SEQ ID NOs: 12-16) dissolved in DMSO.

[0111] [Table 4]

[0112] Peptides marked with an asterisk were present in each pool at approximately twice the concentration of the remaining peptides. The measured concentrations of low-concentration peptides ranged from 1.6 to 1.8 mg / ml, while high-concentration peptides ranged from 3.1 to 3.5 mg / ml.

[0113] The vaccine was prepared by diluting 0.12 ml of the patient's peptide pool composition with 1.08 ml of 25 mM TRIS buffer. From this solution, 1 ml of the TRIS buffer pool dilution was mixed with 1 ml of CAF09b to create the vaccine composition. For 1× dose IP administration, 0.50 ml of the vaccine composition per immunization was injected intraperitoneally.

[0114] Blood was collected from each patient immediately before the first intraperitoneal immunization, and similarly after three intraperitoneal immunizations, immediately before the first intramuscular immunization. PBMC cells derived from these blood samples were then subjected to pre-immunization and post-immunization ELISPOT assays and ICS / restimulation assays.

[0115] The ELISPOT assay is a standard technique for indirectly detecting reactive T cells. In a setting similar to ELISA, peripheral blood mononuclear cells (PBMCs) isolated from patients are stimulated with each peptide of the vaccine, and the secreted cytokines (in this case, IFN-γ) are measured.

[0116] In the ICS / restimulation assay, patient-derived PBMCs are stimulated with the entire pool of vaccine peptides and subsequently subjected to cell counting by flow cytometry, which detects the presence of a subpopulation of cytokine-releasing T cells by testing for the presence of cell surface markers (CD3, CD4, CD8) and cytokines (IFN-γ and TNF-α) in them.

[0117] The results obtained from these experiments are summarized below: Patient 1: In pre-immunization experiments, neither the ELISPOT nor the ICS / restimulation assay for IFN-γ showed a significant number of reactive T cells to any vaccine peptide compared to the positive control (data not shown). In contrast, in post-intraperitoneal immunization experiments, the ELISPOT assay showed the presence of reactive T cells to 4 out of 9 vaccine peptides (Figure 6A), and the ICS / restimulation assay showed the presence of INFγ and TNF-α CD4+ cells (Figure 6B), rather than CD8+ cells (data not shown). The strongest IFN-γ response was observed with ELISPOT for twice the dose of the peptide.

[0118] Patient 2: In pre-immunization experiments, neither the IFN-γ ELISPOT nor the ICS / restimulation assay showed a significant number of reactive T cells to any vaccine peptide compared to the positive control (Figures 7A and 7C, the latter showing data only for CD4+ cells). In contrast, in post-intraperitoneal immunization experiments, the ELISPOT assay showed the presence of reactive T cells to three of the five vaccine peptides (Figure 7B), and the ICS / restimulation assay showed the presence of INFγ and TNF-α CD4+ cells (Figure 7D), rather than CD8+ cells (data not shown). The strongest IFN-γ response was observed with ELISPOT for twice the dose of the peptide.

[0119] In all patients, it can be concluded that cocktail vaccines containing neoepitopes induce an adaptive immune response.

Claims

1. A pharmaceutical composition for use in a method of treating malignant tumors in human patients, Here, the tumor exhibits a T-cell epitope (neoepitope) that does not show non-tumor cells in the patient. Here, the pharmaceutical composition 1) At least one peptide (neopeptide) containing the amino acid sequence of a neoepitope in the patient's tumor cells, 2) Solvent, and 3) Cationic liposome adjuvants A liposome composition containing, Here, the cationic liposome adjuvant comprises or consists of dimethyldioctadecylammonium (DDA), polyinosinic acid:polycytidylic acid (poly(I:C)), and MMG. Here, the MMG is monomycoylglycerol or its synthetic analog 3-hydroxy-2-tetradecyl-octadecanoate-2,3-dihydroxypropyl ester, And here each neopeptide is administered in an immunologically effective dose, at least 100 μg. Pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the DDA:MMG ratio is 5:1 (w / w) and the DDA:poly(I:C) ratio is between 5:1 and 20:1 (w / w).

3. The pharmaceutical composition according to claim 2, wherein the relative weight of DDA:MMG:poly(I:C) is 5:1:

1.

4. The pharmaceutical composition according to claim 2, wherein the relative weight of DDA:MMG:poly(I:C) is 20:4:

1.

5. The pharmaceutical composition according to claim 4, wherein the MMG is 3-hydroxy-2-tetradecyl-octadecanoic acid-2,3-dihydroxypropyl ester.

6. When NTU (nephelometric turbidity unit) is measured according to Section 2.2.1 of the European Pharmacopoeia, 1.0 mg / ml neopeptide in 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO exhibits a maximum NTU of 50, and 0.1 mg / ml of neopeptide in 25 mM TRIS pH 7.4 and 0.5% (v / v) DMSO exhibits a maximum NTU of 25. In that sense, the pharmaceutical composition according to any one of claims 1 to 5, wherein the at least one neopeptide is water-soluble.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein when the at least one neopeptide is present in a mixture of a plurality of neopeptides and 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO, its concentration decreases by up to 50% when subjected to sterile filtration, each of the plurality of peptides is present at a concentration of 0.1 mg / ml in the mixture before sterile filtration.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the number of neopeptides is selected 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, and 30 neopeptides.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the at least one neopeptide comprises a neoepitope that exhibits MHC binding stability exceeding the average value among neoepitopes identified in tumor cells.

10. The pharmaceutical composition according to claim 9, wherein the at least one neopeptide comprises a neoepitope exhibiting MHC binding stability that ranks in the top quarter of neoepitopes identified in tumor cells.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the at least one neopeptide does not contain a cysteine ​​residue.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the at least one neopeptide does not contain Q and N as N-terminal amino acid residues.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the at least one neopeptide does not contain amino acid sequence DG at its N-terminus.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the at least one neopeptide preferentially associates with a discontinuous phase in the liposome composition.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein at least one neopeptide is preferentially dissolved in the continuous phase of the liposome composition.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the immunologically effective amount is administered to the patient multiple times.

17. The pharmaceutical composition according to claim 16, wherein separate administrations are spaced at least one day apart.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the liposome composition has a pH in the range of 7.0 to 7.

6.

19. The pharmaceutical composition according to claim 18, wherein the liposome composition has a pH of about 7.

4.

20. The immunological effective dose of each neopeptide administered is at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, At least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least or many at least 250, at least or more than 255, at least or more than 260, at least or more than 265, at least or more than 270, at least or more than 275, at least or more than 280, at least or more than 285, at least or more than 290, at least or more than 295, at least or more than 300, at least or more than 305, at least or more than 310, at least or more than 315, at least or more than 320, at least or more than 325, The amounts are at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, and at least 400 μg.A pharmaceutical composition according to any one of claims 1-19.

21. The immunological effective dose of each neopeptide administered is 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 ,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 2 19, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 2 50, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 28 1, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312 ,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,A pharmaceutical composition according to claim 20, selected from the group consisting of 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400 μg.

22. The pharmaceutical composition according to any one of claims 1 to 21, wherein each administration of an immunologically effective amount of the liposome composition is made as at least two sub-administrations.

23. The pharmaceutical composition according to claim 22, wherein each administration of an immunologically effective amount of the liposome composition is made as two sub-administrations per immunization.

24. The pharmaceutical composition according to claim 22, wherein 2000 μl is administered as two supplemental doses of 1000 μl each.

25. A unit dose immunogenic composition comprising an immunologically effective amount of at least one peptide (neopeptide) containing the amino acid sequence of a neoepitope of a patient's tumor cell, a cationic liposome adjuvant, and a solvent, Here, the cationic liposome adjuvant comprises or consists of dimethyldioctadecylammonium (DDA), polyinosinic acid:polycytidylic acid (poly(I:C)), and MMG. Here, the MMG is monomycoylglycerol or its synthetic analog 3-hydroxy-2-tetradecyl-octadecanoate-2,3-dihydroxypropyl ester, and a unit dose immunogenic composition wherein the unit dose immunogenic composition comprises at least 100 μg of each of the at least one neopeptide.

26. At least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least or many At least 180, at least or more than 185, at least or more than 190, at least or more than 195, at least or more than 200, at least or more than 205, at least or more than 210, at least or more than 215, at least or more than 220, at least or more than 225, at least or more than 230, at least or more than 235, at least or more than 240, at least or more than 245, at least or more than 250, at least or more than 255, less At most 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least An immunogenic composition in a unit dose according to claim 25, comprising 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, and at least 400 μg of each neopeptide.

27. １００、１０１、１０２、１０３、１０４、１０５、１０６、１０７、１０８、１０９、１１０、１１１、１１２、１１３、１１４、１１５、１１６、１１７、１１８、１１９、１２０、１２１、１２２、１２３、１２４、１２５、１２６、１２７、１２８、１２９、１３０、１３１、１３２、１３３、１３４、１３５、１３６、１３７、１３８、１３９、１４０、１４１、１４２、１４３、１４４、１４５、１４６、１４７、１４８、１４９、１５０、１５１、１５２、１５３、１５４、１５５、１５６、１５７、１５８、１５９、１６０、１６１、１６２、１６３、１６４、１６５、１６６、１６７、１６８、１６９、１７０、１７１、１７２、１７３、１７４、１７５、１７６、１７７、１７８、１７９、１８０、１８１、１８２、１８３、１８４、１８５、１８６、１８７、１８８、１８９、１９０、１９１、１９２、１９３、１９４、１９５、１９６、１９７、１９８、１９９、２００、２０１、２０２、２０３、２０４、２０５、２０６、２０７、２０８、２０９、２１０、２１１、２１２、２１３、２１４、２１５、２１６、２１７、２１８、２１９、２２０、２２１、２２２、２２３、２２４、２２５、２２６、２２７、２２８、２２９、２３０、２３１、２３２、２３３、２３４、２３５、２３６、２３７、２３８、２３９、２４０、２４１、２４２、２４３、２４４、２４５、２４６、２４７、２４８、２４９、２５０、２５１、２５２、２５３、２５４、２５５、２５６、２５７、２５８、２５９、２６０、２６１、２６２、２６３、２６４、２６５、２６６、２６７、２６８、２６９、２７０、２７１、２７２、２７３、２７４、２７５、２７６、２７７、２７８、２７９、２８０、２８１、２８２、２８３、２８４、２８５、２８６、２８７、２８８、２８９、２９０、２９１、２９２、２９３、２９４、２９５、２９６、２９７、２９８、２９９、３００、３０１、３０２、３０３、３０４、３０５、３０６、３０７、３０８、３０９、３１０、３１１、３１２、３１３、３１４、３１５、３１６、３１７、３１８、３１９、３２０、３２１、３２２、３２３、３２４、３２５、３２６、３２７、３２８、３２９、３３０、３３１、３３２、３３３、３３４、３３５、３３６、３３７、３３８、３３９、３４０、３４１、３４２、３４３、３４４、３４５、３４６、３４７、３４８、３４９、An immunogenic composition in a unit dose according to claim 25 or 26, comprising each neopeptide in an amount selected from 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400 μg.

28. An immunogenic composition in a unit dose according to any one of claims 25-27, wherein the number of neopeptides is selected 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, and 30.

29. An immunogenic composition in a unit dose according to any one of claims 25-28, wherein the DDA:MMG ratio is 5:1 (w / w) and the DDA:poly(I:C) ratio is between 5:1 and 20:1 (w / w).

30. The unit dose immunogenic composition according to claim 29, wherein the relative weight of DDA:MMG:poly(I:C) is 5:1:

1.

31. The unit dose immunogenic composition according to claim 29, wherein the relative weight of DDA:MMG:poly(I:C) is 20:4:

1.

32. The unit dose immunogenic composition according to claim 31, wherein the MMG is 3-hydroxy-2-tetradecyl-octadecanoic acid-2,3-dihydroxypropyl ester.

33. When NTU (nephelometric turbidity unit) is measured according to Section 2.2.1 of the European Pharmacopoeia, 1.0 mg / ml neopeptide in 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO exhibits a maximum NTU of 50, and 0.1 mg / ml of neopeptide in 25 mM TRIS pH 7.4 and 0.5% (v / v) DMSO exhibits a maximum NTU of 25. In that sense, the immunogenic composition in a unit dose according to any one of claims 25-32, wherein at least one neopeptide is water-soluble.

34. The unit dose immunogenic composition according to any one of claims 25-33, wherein when the at least one neopeptide is present in a mixture of a plurality of neopeptides and 25 mM TRIS pH 7.4 containing 5% (v / v) DMSO, its concentration decreases by up to 50% when subjected to sterile filtration, wherein each of the plurality of peptides is present at a concentration of 0.1 mg / ml in the mixture before sterile filtration.

35. The immunogenic composition in a unit dose according to any one of claims 25-34, wherein the at least one neopeptide comprises a neoepitope that exhibits MHC binding stability exceeding the average value among neoepitopes identified in tumor cells.

36. The unit dose immunogenic composition according to claim 35, wherein the at least one neopeptide comprises a neoepitope exhibiting MHC binding stability that ranks in the top quarter of neoepitopes identified in tumor cells.

37. The immunogenic composition in a unit dose according to any one of claims 25-36, wherein the at least one neopeptide does not contain a cysteine ​​residue.

38. The immunogenic composition in a unit dose according to any one of claims 25-37, wherein the at least one neopeptide does not contain Q and N as N-terminal amino acid residues.

39. The immunogenic composition in a unit dose according to any one of claims 25-38, wherein at least one neopeptide does not contain amino acid sequence DG at its N-terminus.

40. The unit dose immunogenic composition according to any one of claims 25-39, wherein the at least one neopeptide is preferentially associated with a discontinuous phase in the immunogenic composition.

41. The unit dose immunogenic composition according to any one of claims 25-39, wherein at least one neopeptide is preferentially dissolved in the continuous phase of the immunogenic composition.

42. The immunogenic composition in a unit dose according to any one of claims 25-41, wherein the immunogenic composition has a pH in the range of 7.0 to 7.

6.

43. The immunogenic composition in a unit dose according to claim 42, wherein the immunogenic composition has a pH of about 7.4.

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