Cancer vaccine based on mhc-ii epitope selection

Abstract

The present disclosure provides a vaccine against cancers that express MHC-II. This includes hormone-positive breast cancer, some prostate cancers, and melanoma. Based on an individual's genomic test, one can either (1) design a personalized vaccine consisting of a polytope based on epitopes from TAAs that, in most cases, are displayed on MHC-II but not on MHC-I, or (2) select previously manufactured sequences based on epitopes from TAAs as components of the vaccine, so that after vaccination the cancer will reveal itself to the immune system and be eradicated, while side effects are minimal because the vaccine will not cause the immune system to harm healthy tissue.

Description

Background Technology

[0001] Background of the Invention

[0002] This invention relates to utilizing the human immune system to recognize and kill or prevent diseases such as cancer—hereinafter referred to as "cancer." Key functions related to immune system function will be described below to explain the background of the invention, rather than to explain the immune system to those unfamiliar with it. This invention relates to a cancer vaccine and a method for producing a cancer vaccine. The invention also relates to specific epitopes and tandem epitopes and their use in treating cancer. The invention further relates to libraries of tandem epitopes.

[0003] Cancer is a leading cause of death for many, and most cancers cannot be completely eradicated once they develop because current treatments rarely utilize the immune system to eliminate them. In some cases, they may leverage the immune system to make cancer treatments more effective, such as by making T cells more efficient through checkpoint inhibitory therapy, which dominates current immunological treatments for cancer. However, previous utilization of the immune system has rarely enabled it to completely eliminate cancer after it is discovered, or prevent its development as it does with other diseases. One reason is that cancer develops within cells that express the same information about themselves to the immune system as healthy cells, and the immune system, through its functions—such as T-cell selection—is prevented from attacking healthy cells (to prevent the development of autoimmune diseases).

[0004] For an immune response to be triggered, cancer must be recognized on major histocompatibility complex I or II (MHC-I or MHC-II). This is achieved through epitope presentation, where an epitope is part of an antigen encapsulated within an MHC molecule. This epitope-encapsulated complex in MHC acts as a ligand for the T cell receptor (TCR), such that if the ligand matches the T cell's TCR, the T cell is attracted and its activation is initiated.

[0005] The function and appearance of MHC-I and MHC-II molecules are determined by the following genes: MHC-I: HLA-A, HLA-B, HLA-C; and those with no significant interaction with T cells: HLA-E, HLA-F, HLA-G, and HLA-H.

[0006] MHC-II: HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5.

[0007] Selection by T cells in the thymus ensures that if a protein is naturally present in the tissue at levels above a certain threshold, it is unlikely to match a TCR (unless in the case of an autoimmune disease). This process is often referred to as “negative selection,” in which T cells are exposed to substances similar to healthy tissue and then killed (negative selection) of T cells that would attack healthy tissue before being allowed to leave the thymus and enter the body (if they are “self-reactive” and therefore choose to attack healthy tissue, they are killed). However, this negative selection is not perfect; it is more stringent for MHC-I-associated T cells (CD8+, where an estimated 1% of self-reactive T cells are allowed to pass) and less stringent for MHC-II-associated T cells (CD4+, where an estimated 15% of self-reactive T cells are allowed to pass). These percentages (approximately 1% for CD8+ and 15% for CD4+) are not definitive and vary depending on the degree of expression of the substance represented by T cells in healthy tissues via their T cell receptors (TCRs). This suggests that if the substance is strongly expressed in healthy tissues, the percentage is lower, and conversely, if the substance is only vaguely expressed, the percentage is higher. In most cases, the number of T cells that, despite being autoreactive, escape this “negative selection” is high enough to fulfill the role of killing antigen-presenting cells (APCs) as described below, even if it is insufficient to trigger an effective immune response.

[0008] It is known that CD8+ autoreactive T cells that attack substances on healthy tissues, such as the "invariant chain" (Ii), are almost completely negatively selected or eliminated (the percentage is significantly less than 1%).

[0009] CD8+ T cells—cytotoxic T lymphocytes (CTLs)—potentially bind to MHC-I, specifically to a ligand composed of epitopes encapsulated within the MHC-I molecule. CD8+ CTLs can then kill the cells that present the ligand.

[0010] CD4+ T cells—helper T cells (Th), and possibly other CD4+ T cells and self-cytotoxic CD4+ cells (CD4+ CTLs)—potentially bind to MHC-II, that is, to ligands composed of epitopes encapsulated in MHC-II molecules. The fact that CD4+ cells themselves can be cytotoxic is a relatively new discovery and is not reflected in most overviews of the CD4+ phenotype, where CTLs are currently generally considered to be CD8+ T cells.

[0011] The immune system has several response mechanisms, such as the following: Professional antigen-presenting cells (APCs) patrol the body, digesting everything they encounter along the way and presenting it—partly—as epitopes on MHC-I and MHC-II. Each APC has millions of MHC-I molecules with epitopes on its surface and presents them before and after entering the lymph nodes (LNs).

[0012] CD8+ T cells kill APCs even before they reach the LN if their presentation has already been overridden by the immune response. In this case, there are already many CD8+ T cells in the periphery to prevent an overreaction of the immune response.

[0013] When an APC reaches the LN (where navigator T cells reside), it attracts both types of T cells (CD8+ and CD4+), provided they have escaped negative selection (in most cases, they have escaped to some extent)—this is based on the match between the TCR and epitopes encapsulated in MHC-I or MHC-II, respectively. MHC-I and MHC-II contents are present in all naturally occurring substances, so when digested by an APC, the APC always has both MHC-I and MHC-II on its surface and always attracts both types of T cells.

[0014] Then APCs send “signal 2” or “co-stimulation signal” to T cells, which then begin the activation process.

[0015] CD8+ T cells are faster than CD4+ T cells in all activities, but otherwise, the two types of T cells are quite similar—they specialize into CD8+ or CD4+ at a very late stage of thymic development. They both require and respond to the same set of co-stimulatory signals.

[0016] CD8+ T cells complete their activation process first (before CD4+ T cells), at which point or at this stage, they become cytotoxic—and thus they kill APCs. At this point, the CD4+ T cells attached to the APCs have not yet reached the stage where they become cytotoxic (in non-cancer cases, this is useless anyway, as there is no tissue expressing MHC-II that should be killed). Therefore, CD4+ T cells stop at this “intermediate” stage, where they have developed into “helper cells,” one of whose roles is to “call out” (by producing cytokines) B cells, as B cells are supposed to kill the invaders (the normal, non-cancer source of epitopes on the APC surface). Thus, in normal, non-cancer cases, CD8+ T cells are prepared to kill infected organism cells (which express the MHC-I portion of the invaders), a role they are specialized in doing so, while CD4+ T cells mobilize B cells to kill the invaders before they infect the body—a role B cells are specialized in doing so. In normal, non-cancer cases, these two cytotoxic roles work together to effectively eliminate the infection.

[0017] Issues related to immune response in cancer Many cancers have “learned” (through Darwinian selection) to downregulate MHC-I presentation (they “turn off channel 1”), thus becoming invisible to CD8+ T cells.

[0018] Most cancers, in turn, don't shut down "channel 2" (they don't inactivate the MHC-II presentation mechanism)—because nothing happens there anyway (they're not threatened with being killed based on MHC-II expression). Therefore, CD4+ T cells can easily see the cancer, they tune to channel 2, but they only call out (to B cells); they're not cytotoxic, so they don't harm the cancer cells.

[0019] However, B cells are not as good at killing cancer cells (which are inside) as they are at killing foreign invaders.

[0020] Therefore, we hypothesize that if we make CD4+ T cells cytotoxic, they will kill cancer (surprise the cancer). And this killing will happen much faster than it takes for cancer cells to "learn" to turn off "channel 2".

[0021] Because CD4+ T cells respond to antigens presented in all cancer cells (as is the case with tumor-associated antigens (TAAs), but not with mutated tumor-specific antigens (TSAs), which are not present in all cancer cells), they have the potential to kill all cancer cells.

[0022] Therefore, CD4+ cells can do one or more of the following: (a) they can attract CD8+ cytotoxic T cells, which in turn kill cells presenting the ligands, which consist of epitopes encapsulated in MHC-II; (b) (as described above) they can directly kill cells (as CD4+ cytotoxic T cells); (c) they can attract B cells by secreting cytokines, which kill cells carrying the antigen directly or through antibodies they produce; or (d) some other immune response. It is highly likely that a significant immune response, i.e., one or more of (a), (b), (c), and (d), will occur because, according to multiple studies, certain MHC-II gene alleles have a strong influence on the development of breast cancer and, in some cases, even a protective effect. Whether the immune response consists of one or more of the mechanisms described above—including, regardless of the presence of other mechanisms triggered by MHC-II and CD4+ T cells—does not affect the present invention.

[0023] There is growing interest in the use of MHC-II, whether as a supplement to MHC-I (“activating the arms of the immune system”) or used alone.

[0024] All cells with a nucleus (i.e., all cells in the human body except for a few exceptions such as red blood cells) have the ability to express MHC on their surface.

[0025] Only specialized antigen-presenting cells (sometimes simply called antigen-presenting cells, APCs), certain cancer cells, and a few rarer cell types and conditions (including post-inflammatory epithelial cells) are capable of expressing MHC-II on their surface. APCs include dendritic cells (DCs), macrophages, and B cells. The fact that some cancers can present MHC-II is a relatively new discovery and is not reflected in most overviews, and it is generally believed that MHC-II is presented only on the surface of APCs. Cancers that can express MHC-II include: Breast cancer (BC) Prostate cancer (PCa) melanoma Colorectal cancer, Ovarian cancer Classic Hodgkin's lymphoma, Gliomas, and Non-small cell lung cancer (NSCLC).

[0026] We are currently unsure whether all MHC-II is expressed in these cancers, or whether all the variants of the aforementioned cancers express MHC-II.

[0027] Therefore, the field needs to focus on novel vaccine technologies based on epitopes presented by MHC-II molecules.

[0028] Proteins are cleaved into epitopes before being attempted to be encapsulated into MHC molecules. These epitopes are typically 9 amino acids long in MHC I, ranging from 8 to 14, and 15 amino acids long in MHC-II, ranging from 13 to 25. These epitopes can then be presented on the cell surface encapsulated in MHC molecules, one of the steps in the antigen presentation pathway (APP), if they pass through several further steps.

[0029] The APP of MHC-II is called the MHC-II presentation pathway, such as Figure 10 As shown in the figure, proteins cleaved into epitopes (similar to peptides, as shown) must find a pathway to enter the MHC-II loading compartment (“MIIC”), where the peptide of the protein must replace the “CLIP” sequence derived from the invariant chain in order to enter the MHC-II molecule and subsequently be presented on the cell surface.

[0030] Proteins that can be cleaved into epitopes and that, after being encapsulated in MHC molecules, can be presented and match the TCRs of T cells to trigger an immune response are called antigens. Therefore, antigens are either (1) purely exogenous (e.g., representing pathogens of disease), (2) mutant proteins that were not present before the mutation, or (3) proteins whose expression levels are significantly higher than normal.

[0031] Tumor-specific antigens (TSAs) are (primarily) mutated proteins (i.e., produced by mutated genes). Therefore, they are highly immunogenic candidate antigens because they are not associated with normal, “healthy” expression levels, making TSAs a focus of much cancer vaccine work. However, TSAs present two major problems: (1) to capture them, tumor samples are required and must be extracted, for example, via biopsy; and (2) because they are mutated, cancer is likely to mutate again, moving away from the protein, thus allowing cancer to continue to survive in cells that do not express the TSA. In any case, a vaccine requires the presence of cancer, therefore it can only be therapeutic, not preventative.

[0032] Tumor-associated antigens (TAAs) are instances of proteins expressed at low levels in normal tissues but at high levels in cancerous tissues. The fact that cells with normal levels of these antigens still express them implies either the existence of a threshold mechanism, an acceptable low level of killing of healthy cells, or that they are ineffective as antigens at any level because the selection of T cells in the thymus would make it difficult to find T cells whose TCR matches that antigen. This mechanism is not causally determined, but rather empirical. In any case, the effectiveness of TAAs as vaccine candidates depends on whether there is a significant gap between their expression levels in cancer and in normal tissues—so that an appropriate vaccine dosage can be determined to "teach" the immune system to kill cancer cells without killing healthy tissue. In this case, "teaching" means adjusting (enhancing) the number of T cells so that they either ignore or substantially do not kill healthy tissue.

[0033] All (nuclear) cells present epitopes of their proteins on MHC-I. This includes APCs, all cancer cells, and all other (nuclear) cells. See also Figure 1 .

[0034] APCs can engulf and digest external antigens (a process called phagocytosis), causing them to be presented on the APC's MHC-II (and MHC-I), along with the APC's own internal proteins (which are cleaved into epitopes and encapsulated within MHC-II). The presentation of antigens cleaved into epitopes (also called peptides) and encapsulated within the MHC is called a "peptide-MHC complex" (pMHC).

[0035] APCs can also engulf and digest internal cells, including diseased or dying cancer cells, thereby presenting epitopes of antigens presented from cancer cells as pMHC in MHC-I and MHC-II on the surface of the APC.

[0036] Some cancer cells can present their internal proteins and, consequently, antigens on MHC-II, which are then cleaved into epitopes (as described in the cancer cell types mentioned above).

[0037] APCs migrate to organs such as lymph nodes (LNs) and are exposed to and attracted by T cells. If a match exists between the T cell receptor (TCR) and the pMHC on the surface of the APC, this match is referred to as "Signal 1" in the "2-signal (or sometimes 3-signal) theory." "Signal 1" must be followed by one or more signals in "Signal 2" to activate the T cell and allow it to develop from its "initial" state to the stage where it should be, expanding and proliferating to perform its functions. Examples of these signals are provided in Table 1 below, and a more comprehensive list of signals is given in the references at the URL "https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3786574 / ". If "Signal 1" is not followed by "Signal 2", the T cell may become unresponsive.

[0038] Table 1

[0039] The focus of existing technologies has traditionally been on the MHC-I mechanisms used in cancer vaccines and cancer immunotherapy. This is likely because there is a simpler mechanism behind this anti-cancer immunity, namely that CD8+ CTLs are killed directly, without the knowledge that MHC-II does the same. Most of the focus has been on (1) turning “cold” tumors into “hot” tumors (helping MHC-I function to make cancer visible to CD8+ CTLs) and checkpoint inhibition of these CD8+ CTLs (avoiding two mechanisms that can slow down or inactivate CD8+ CTLs: PD-1 and CTLA-4 receptors).

[0040] Recent technological focus has shifted further towards the TSA (Tracking Authority) rather than the TAA (Tracking Authority). This focus on the TSA has the following drawbacks: Cancer may mutate away from specific antigens This antigen may not represent all cancers. Errors may occur during the extraction and detection process. Cyclin D1 (genetic code and abbreviation CCND1) is an instance of TAA, and its expression is particularly elevated in the following cancers, including: Breast cancer (especially hormone-positive BC, i.e., ER+ BC, which accounts for more than half of all BCs, see https: / / v17.proteinatlas.org / ENSG00000110092-CCND1 / pathology and https: / / www.genome.jp / pathway / hsa05224 (top right)). Prostate cancer (see https: / / v17.proteinatlas.org / ENSG00000110092-CCND1 / pathology and https: / / www.genome.jp / pathway / hsa05215 (bottom right)). Melanoma (see https: / / v17.proteinatlas.org / ENSG00000110092-CCND1 / pathology and https: / / www.kegg.jp / pathway / hsa05218 (top right)). Liver cancer Kidney cancer, and Urothelial carcinoma Therefore, cyclin D1 and MHC-II are expressed in the following cancers: Breast cancer (ER+ BC) Prostate cancer, and Melanoma.

[0041] In the current technology, it is not possible to create a situation on the surface of APC with pure MHC-II and no MHC-I, so as to observe the effect on CD4+ cells and their potential developmental cytotoxicity when there is no "interference" from CD8+ T cells (i.e. they do not kill APCs).

[0042] For example, a paper at URL "https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2017.00194 / full" describes a condition associated with influenza infection in the lungs, particularly if the infection involves reduced MHC-I presentation as a viral immune escape mechanism. CD4+ cells, which normally "stop developing into virulence" in the LN (we call this "point A"), can continue their "activation journey" from the "helper cell phase" to virulence (e.g., Figure 7As shown, we call this "point B"), if they are continuously exposed to the antigens of APCs (which can continue to provide "co-stimulatory signals" or signal 2), especially if CD8+ T cells are blocked due to MHC-I reduction. Therefore, if "point A" is reached in the LN and "point B" is reached in the lungs (because MHC-I is blocked in the lungs), then if MHC-I is blocked in the LN, such as... Figure 8 As shown, reaching "point B" can also occur in LN.

[0043] Other TAAs besides cyclin D1 (CCND1) include: mucin-1 (MUC1), mucin 16, carcinoembryonic antigen (CEA—usually specifically in the form of CEACAM5, but also in the form of CEACAMx, where x is 1, 3, 4, 6, 7, 8, 16, 18, 19, 20, or 21), human epidermal receptor 2 (HER2 or ERBB2), telomerase reverse transcriptase (TERT), Wilms' tumor gene (WT-1), sialic acid-Tn, Myc proto-oncogene protein (MYC), and prostate-specific antigen (PSA). Prostate membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate-specific antigen (PSA), cancer / testis antigen 1 (NY-ESO-1), melanoma antigen preferentially expressed in tumors (PRAME), melanoma-associated antigen 1 (MAGEA1), melanoma-associated antigen 3 (MAGEA3), prostate stem cell antigen (PSCA), B lymphocyte antigen CD19 (CD-19), cell tumor antigen p53 (TP-53), GTPase KRas (KRAS), keratin type I cytoskeleton 19 (Cyfrax). 21-1), Cellular retinoic acid binding protein 1 (CRABP1), Cellular retinoic acid binding protein 2 (CRABP2), Folate receptor (FOLR1), Kallikrein-10 (KLK10), Baculovirus IAP repeat protein 5 (BIRC5 or survivin), Insulin, Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), Epidermal growth factor receptor (EGFR), Protein LYRIC (Metadherin), Tumor protein D52, Liver ligand A receptor 2 (EphA2), Tyrosinase, and Alpha-fetoprotein (AFP).

[0044] These TAAs all have the ability to be cleaved into epitopes presented by MHC II molecules, which are not presented by MHC I molecules. This ability depends on the genotype of the human subject. Different genotypes may promote the production and presentation of different epitopes from a particular TAA.

[0045] The TAAs listed above are shown in Table 2 below, which indicates which cancers expressing MHCII may be associated with (by which cancers express them). Any TAA may also be expressed by other cancers other than those shown with an "X" in the figure.

[0046] Table 2

[0047] Compared with other TAAs, cyclin D1 is presented weakly on MHC-I compared with its presentation on MHC-II. See [link to relevant documentation]. Figure 4 For all the TAAs mentioned, MHC-I presentation per unit protein length (measured by the number of amino acids) appears to be similar, but MHC-II presentation per unit length varies considerably, such as... Figure 5 As shown in the figure, the right half depicts those TAAs that are "loud" on MHC-II, i.e., TAAs that are likely to be presented more extensively on MHC-II than on MHC-I. TAAs that are "loud" on MHC-II are most likely suitable for this invention.

[0048] Figure 5 The implications of the assumed immunogenicity of TAAs in the mid-vertical diagram are unknown: (1) high immunogenicity implies the presence of more of both types of T cells available, depending on the ability to avoid CD8+ T cell interactions; (2) low immunogenicity implies fewer of both types of T cells available, and the immune response depends on whether it is possible to completely prevent CD8+ T cells from being used while simultaneously having enough CD4+ T cells to proliferate. It is difficult to determine which of (1) or (2) is "better," or whether other factors need to be considered.

[0049] Perhaps due to its "loudness" on MHC-II and the fact that there is a general focus on MHC-I, the antigenic cyclin D1 is rarely listed in the overview of potential TAAs. Other TAAs such as MUC1, CEA, HER2, and TERT are listed in the overview.

[0050] Compared to other TAAs, cyclin D1 is well distinguished, meaning it is highly expressed in cancerous tissues compared to normal tissues. See https: / / v17.proteinatlas.org / ENSG00000110092-CCND1 / pathology and https: / / www.antibodypedia.com / gene / 3660 / CCND1 (click “More gene data” and scroll down to “Tissue RNA expression”).

[0051] Cancers typically have the ability to silence MHC-I, but for cancers that express MHC-II, MHC-II remains unaffected.

[0052] This fact and Figure 5 The fact that the TAA on the right side is presented worse on MHC-I than on MHC-II means that whenever the expression of the TAA epitope occurs—especially when it occurs on MHC-II—it is most likely to originate from cancer cells (except for weak expression on APC).

[0053] This could explain why some MHC-II alleles are protective against breast cancer; as reported in multiple publications, the alleles that produce MHC-II molecules appear to be adept at binding certain TAAs. This fact suggests that if CD4+ T cells have an increased ability to “see” TAA epitopes in the remaining cases, then cancers expressing MHC-II in these cases may trigger an immune response that kills them.

[0054] Therefore, there is a need in this field to focus on novel vaccine technologies that utilize epitopes derived from tumor-associated antigens, presented by MHC II molecules, and not by MHCI molecules, to maintain CD8+ T cells from killing APCs and prevent CD4+ T cells from becoming cytotoxic.

[0055] A key step in the field of cancer vaccines is predicting whether a protein, as an antigen or a subset of antigens (called epitopes), can pass through the APP (Active Cell Apparatus). This involves determining whether the antigen can be cleaved into epitopes, and whether one or more of these epitopes will bind to MHC and potentially be presented on the cell surface. This analysis depends on the alleles of MHC genes—in MHC-I, genes named HLA-A, HLA-B, and HLA-C, and to some extent other MHC-I genes—and in MHC-II, genes named HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5, and to some extent other MHC-II genes. This prediction can be performed as a computer-based program, one of which is available as a service for MHC-I (hereinafter referred to as "URL MHC-I") at the URL https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / (or a subsequently approved version) and for MHC-II (hereinafter referred to as "URL MHC-II") at the URL https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.3 / (or a subsequently approved version). It is known that if the prediction shows that the epitope cannot bind to the MHC in question, it will not be present on the cell surface. Therefore, epitope binding is a necessary condition for them to be displayed.

[0056] This predictive analysis is based on knowledge of the alleles of the vaccine-bearing humans under consideration, or on a significant set of alleles that occur at a significant frequency in the population.

[0057] This predictive analysis is considered to have converged to accuracy or optimality, but may be updated in the future to become even better. For MHC-I or MHC-II alleles, each possible epitope of the protein is associated with a score that places the epitope into one of the following categories: "Non-binding agent" "Weak binder" (WB), or "Strong binder" (SB) Table 3 below shows some examples of TAA CEACAM5 ("Strong Bonding Agents Only").

[0058] Table 3

[0059] “Strong binders” are statistically considered to almost certainly bind to the corresponding MHC molecules, thus potentially moving to the cell surface and being displayed to the immune system; “weak binders” may bind to MHC molecules; and “non-binding agents” are certain not to bind to MHC molecules and therefore will not be displayed to the immune system on the cell surface. Summary of the Invention

[0060] This invention belongs to the field of vaccination, namely, injecting chemical reagents containing active substances that can trigger a response in the human immune system.

[0061] The subject of this disclosure generally relates to killing cells (i.e., apoptosis induced by immune system cells attaching to cancer cells and other immune system responses).

[0062] This invention utilizes a computational environment to calculate the correct vaccine components. This computational environment uses a computational model that simulates the function of the immune system, including alleles of the genes behind the immune system and the proteins that the system reacts to.

[0063] This invention also utilizes techniques for producing vaccines based on the aforementioned computational model and vaccine component data. This invention does not include any of these further vaccine production techniques.

[0064] Therefore, in a first aspect, the present invention relates to a method for producing a cancer vaccine composition for treating or preventing cancer in a human subject, the method comprising the steps of: a) selecting one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) such that the selected epitopes bind to the subject's MHC-II, thereby providing a library of epitopes and tandem epitopes that bind to the subject's MHC-II; b) removing epitopes and tandem epitopes containing epitopes that bind to the subject's MHC-I from the library of epitopes and tandem epitopes selected in step a), thereby providing a library of epitopes and tandem epitopes that bind to the subject's MHC-II but not MHC-I; c) optionally, combining one or more epitopes and tandem epitopes provided in step b) into a multi-epitope comprising one or more selected epitopes and tandem epitopes (but observed that no MHC-I binding epitope is generated in the combination); d) optionally, providing an information-carrying molecule capable of promoting in vivo generation of one or more epitopes and tandem epitopes provided in step b) or the multi-epitope provided in step c); e) One or more epitopes and tandem epitopes selected from the library of epitopes and tandem epitopes that bind to the subject’s MHC-II but not MHC-I, provided in step b), or multiple epitopes provided in step c), or information-carrying molecules provided in step d), are combined with suitable pharmaceutically acceptable excipients.

[0065] In this regard, it should be noted that the term "tandem epitope" refers to a peptide sequence containing more than one predicted epitope within the same sequence. Epitopes are tandemly linked within the same sequence and may overlap relative to each individual peptide sequence. For example, an epitope consisting of sequence ABC and another epitope consisting of sequence BCD can produce a tandem epitope ABCD. As another example, an epitope consisting of sequence ABC and another epitope consisting of sequence DEF can produce a tandem epitope ABCDEF. As yet another example, an epitope consisting of sequence ABC and another epitope consisting of sequence EFG can produce a tandem epitope ABCDEFG. Typically, when identifying predicted epitopes according to the present invention, the most suitable prediction is identified as a hotspot sequence containing a tandem epitope. Analyzing the binding of the identified hotspot (tandem epitope) to MHC-II, followed by analyzing the binding to MHC-II with the relevant subject genotype, forms the basis of the present invention.

[0066] Preferably, steps a) and b) are implemented by using an algorithm based on allele data of the subject's genes to select a suitable TAA, preferably the TAA provided in this specification, and analyzing the possible MHC-II and MHC-I binding epitopes of the corresponding TAA, the algorithm predicting immune system behavior against the corresponding TAA and any epitope sequences believed to constitute the corresponding TAA.

[0067] More preferably, the step of analyzing the possible MHC-II and MHC-I combined or tandem epitopes of the corresponding TAA is implemented by using appropriate algorithms that can be used to calculate the binding probability of each epitope of the TAA with MHC-II and MHC-I. Appropriate algorithms are provided for MHC-I at URL MHC-I or a later improved version thereof, and for MHC-II at URLMHC-II or a later improved version thereof.

[0068] In a second aspect, the present invention relates to the use of one or more epitopes or tandem epitopes derived from tumor-associated antigens in the treatment or prophylactic treatment of a subject with cancer, wherein the epitopes or tandem epitopes are specifically selected to activate an immune response via the subject's major histocompatibility complex II (MHC-II), and wherein the epitopes or tandem epitopes are specifically selected not to activate an immune response via the subject's major histocompatibility complex I (MHC-I). This aspect can also be defined as a method for the treatment or prophylactic treatment of a subject with cancer.

[0069] This aspect can also be defined as the use of one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) in the treatment or prophylaxis of a subject's cancer, wherein the epitopes or tandem epitopes are specifically selected to activate an immune response via major histocompatibility complex II (MHC II) in the subject, wherein the subject is characterized by having a genotype that promotes non-binding of the specifically selected epitopes or tandem epitopes to the subject's major histocompatibility complex I (MHC I). This aspect can also be defined as a method for the treatment or prophylaxis of a subject's cancer.

[0070] In a third aspect, the present invention relates to a cancer vaccine comprising one or more specifically selected epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), the selected epitopes or tandem epitopes activating an immune response by the subject’s major histocompatibility complex II (MHC II), said epitopes being characterized in that a) they consist of more than 13 amino acids but less than 25 amino acids, preferably 15 amino acids.

[0071] Preferably, the epitope is one or more tandem epitopes of a tumor-associated antigen (TAA). Each epitope or tandem epitope must consist of no more than 25% of the amino acids in a TAA. Tandem epitopes consisting of more than 25% of the amino acids in a TAA have a high probability of containing epitope sequences that bind to and activate the MHC-I portion of the immune system, and are therefore unsuitable for the purposes of this invention. Similarly, tandem epitopes consisting of more than 50 amino acids (e.g., more than 45 of the amino acids in a TAA) have a high probability of containing epitope sequences that bind to and activate the MHC-I portion of the immune system, and are therefore undesirable for the purposes of this invention. Preferably, the tandem epitopes according to the invention contain fewer than 50 amino acids, for example, fewer than 45.

[0072] However, the multi-epitopes according to the present invention may be longer than 50 amino acids. Care should be taken not to introduce epitope sequences that bind to and activate the MHC-I portion of the immune system by any sequence connecting individual epitopes and tandem epitopes.

[0073] It should be noted that the epitopes and tandem epitopes provided in the sequence listing may include additional amino acids in the N-terminal and / or C-terminal portions of the sequence without departing from the invention. However, it must be evaluated whether any such additional amino acids would result in the formation of an MHC-I epitope.

[0074] It should also be noted that epitopes and tandem epitopes identified and appearing in the sequence listing according to the present invention can generally be shortened at the N-terminus and / or C-terminus of the sequence without departing from the present invention. However, it must be evaluated whether any such shortening will lead to the elimination of MHC-II epitope binding. Generally, shortening by 1, 2, 3, 4, or 5 amino acids at the N-terminus and / or C-terminus of the sequence will not lead to the elimination of MHC-II epitope binding and therefore will not deviate from the present invention.

[0075] Preferably, the vaccine is used for the treatment or preventive treatment of cancer in a subject who has a genotype that promotes non-binding of the specifically selected epitope or tandem epitope to the subject's major histocompatibility complex I (MHC I).

[0076] In a fourth aspect, the present invention relates to a library or sequence catalog of cancer vaccine epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), wherein the epitopes or tandem epitopes are specifically selected to activate an immune response via major histocompatibility complex II (MHC II) in human subjects.

[0077] In a fifth aspect, the present invention relates to a tandem epitope comprising a peptide sequence or a derivative thereof as shown in a sequence listing, wherein the sequence is shortened or lengthened by 1, 2, 3, 4, or 5 amino acids at the N-terminal portion of the tandem epitope, and / or shortened or lengthened by 1, 2, 3, 4, or 5 amino acids at the C-terminal portion of the tandem epitope. The tandem epitope may further comprise an adjuvant sequence. The adjuvant sequence may enhance MHC-II binding of the epitope and the tandem epitope according to the present invention.

[0078] Preferably, the library or sequence catalog consists of epitopes or tandem epitopes specifically selected to not activate an immune response via major histocompatibility complex I (MHC I) in human subjects. The library preferably contains at least four, for example, at least five, for example, at least six, for example, at least seven epitopes or tandem epitopes for each relevant TAA. Preferably, these epitopes and tandem epitopes are selected from tandem epitopes provided as sequences in the sequence listing. The library preferably contains a total of at least 20, for example, at least 30, for example, at least 40, for example, at least 50 epitopes or tandem epitopes. Preferably, these epitopes and tandem epitopes are selected from tandem epitopes provided in the sequence listing.

[0079] Preferably, in all aspects of the invention, the epitope is a tandem epitope. Even more preferably, the one or more tandem epitopes are selected from the tandem epitopes provided in the sequence listing or derivatives thereof, wherein the sequence is shortened by 1, 2, 3, 4 or 5 amino acids at the N-terminal portion of the tandem epitope and / or shortened by 1, 2, 3, 4 or 5 amino acids at the C-terminal portion of the tandem epitope. Tandem epitopes comprising the peptide sequences shown in the sequence listing are preferred. Even more preferably, the one or more tandem epitopes are selected from the sequence listing as sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, 281, 28 2, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412, 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803, 8 The serial table positions provided are 05, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

[0080] As a personalized vaccine, the specifically selected epitope should activate an immune response via major histocompatibility complex II (MHC II) in the subject and should not activate an immune response via major histocompatibility complex I (MHC I) in the same subject.

[0081] These epitopes derived from TAAs can be selected based on a subject's specific genotype regarding the alleles of major histocompatibility complex I (MHC I) and MHC II. As described below, a specific genotype in a particular subject leads to different processing of TAAs and the generation of different associated epitopes that bind to MHC II and MHC I.

[0082] Selected epitopes related to the activation of an immune response via major histocompatibility complex II (MHC II) in the subject are listed in the sequence listing. Each individual may benefit from one or more of these epitopes as the active ingredient in a cancer vaccine, depending on the individual's genotype. Typically, up to four or more epitopes or tandem epitopes from each associated TAA constitute a vaccine. Therefore, a preferred vaccine according to the invention contains at least four epitopes. Typically, up to 16 or more epitopes or tandem epitopes constitute a vaccine. Therefore, a preferred vaccine according to the invention contains epitopes from more than one TAA, such as two or three TAAs. Preferably, the vaccine contains epitopes from at least two, such as at least three, or such as at least four TAAs.

[0083] The exact number depends on the specific circumstances, where the number 16 is derived from the following: (1) considering the individual genotype, selecting about 4 epitopes or tandem epitopes within the TAA, and (2) selecting about 4 TAAs for the type of cancer in question—4 multiplied by 4 equals 16.

[0084] Multiple epitopes

[0085] Multiple selected epitopes and tandem epitopes can be combined into so-called multiepitopes by adding them as chains to a string, potentially introducing connecting amino acids.

[0086] The cancer vaccine field also includes a vaccine technology in which selected multi-epitopes are used as vaccine reagents, for example, by converting their digital equivalents into mRNA as input to generate part of the vaccine reagent. The FDA has approved mRNA vaccines against COVID-19, and this method is currently frequently used in clinical trials; see, for example, a paper at URL "https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC10132791". Other vaccine approaches besides mRNA include: SAM: Self-amplified mRNA vaccine LNP mRNA: Lipid nanoparticle mRNA vaccine Viral vector vaccine DC (dendritic cell) vaccine ISV: In-situ vaccine Therefore, as an alternative, the active agent according to all aspects of the invention is a reagent that promotes the production of the one or more specifically selected epitopes in the body of a subject, for example, an mRNA molecule encoding the specifically selected epitope produced in vivo.

[0087] The epitopes of antigens that bind to MHC molecules and may therefore be presented to the immune system vary considerably among different HLA alleles. This means that the location of "strong-binding" epitopes in proteins representing antigens varies greatly depending on the allele. Therefore, each TAA may produce different epitopes presented by MHC molecules, depending on the subject's allele.

[0088] For the antigen cyclin D1, which has a protein length of 295 amino acids, the number of MHC-II “strong binders” varies depending on the selected MHC-II, ranging from none (0) to 11, with most falling in the 1–8 range, according to the prediction service on URL MHC-II. There are fewer “strong binders” in MHC-I. Any human expresses at most six different MHC-I molecules and at most 12 different MHC-II molecules, and while one molecule may not have a “strong binder,” another person is likely to have one. Based on the URL “https: / / allelefrequencies.net / hla.asp” (which appears to be based on the prediction service on URL MHC-II), we calculated an estimate of the proportion of the world population that, considering allele combinations, has no MHC-II strong binders in the epitopes of cyclin D1, which is less than 1% (0.3%).

[0089] Therefore, in most cases (for all TAAs), one or more epitopes from that TAA can be identified that are strongly bound to MHC-II and completely unbound to MHC-I. It is possible that a “strong binder” for MHC-I is completely embedded within a “strong binder” for MHC-II, and thus it is discarded (omitted in the final multiepitope section).

[0090] Allele frequencies for the world population can be derived from the URL "https: / / allelefrequencies.net / hla.asp". At the top (for reference), it includes: MHC-I: HLA-A — Sorted in descending order of probability, showing the first 5: HLA-A02:01 25% HLA-A01:01 12% HLA-A03:01 12% HLA-A24:02 10% HLA-B — Sorted in descending order of probability, showing the first 5: HLA-B07:02 10% HLA-B08:01 8% HLA-B15:01 7% HLA-B44:02 5% HLA-B35:01 5% HLA-C — Sorted in descending order of probability, showing the first 5: HLA-C07:01 3% HLA-C07:02 3% HLA-C04:01 3% HLA-C06:02 3% HLA-C12:03 2% The probability of having an MHC-I combination (assuming no cross-coupling or covariance, usually attributed to haplotype) — sorted in descending order of probability, showing the top 5: HLA-A02:01 HLA-B07:02 HLA-C07:01 0.079% HLA-A02:01 HLA-B07:02 HLA-C07:02 0.078% HLA-A02:01 HLA-B07:02 HLA-C04:01 0.070% HLA-A02:01 HLA-B08:01 HLA-C07:01 0.062% HLA-A02:01 HLA-B08:01 HLA-C07:02 0.062% Based on a very limited sample of allowed alleles (HLA-B represents 89% of the world’s population, while HLA-C represents only 23%), there are at least 9,000 such allele combinations on MHC-I.

[0091] MHC-II: HLA-DPx (where "x" is "A1" or "B1") — Haplotypes are sorted in descending order of probability, showing the first 5: HLA-DPA 102:02-DPB 105:01 21% HLA-DPA101:03-DPB104:01 15% HLA-DPA101:03-DPB102:01 14% HLA-DPA101:03-DPB104:02 10% HLA-DPA 101:03-DPB 103:01 3% HLA-DQx (where "x" is "A1" or "B1") — Haplotypes are sorted in descending order of probability, showing the first 5: HLA-DQA105:01-DQB103:01 9% HLA-DQA103:01-DQB103:02 9% HLA-DQA101:02-DQB106:02 8% HLA-DQA101:01-DQB105:01 8% HLA-DQA105:01-DQB102:01 7% HLA-DRx (where "x" is "B1", "B3", "B4" or "B5") — sorted in descending order of probability, showing the first 5: DRB1_07:01 10% DRB1_15:01 8% DRB1_03:01 7% DRB1_11:01 6% DRB1_01:01 5% The probability of having an MHC-II combination (assuming no further cross-coupling) — top 5, sorted in descending order of probability: HLA-DPA10202-DPB10501 HLA-DQA10501-DQB10301 DRB1_0701 0.18% HLA-DPA10202-DPB10501 HLA-DQA10301-DQB10302 DRB1_0701 0.17% HLA-DPA10202-DPB10501 HLA-DQA10102-DQB10602 DRB1_0701 0.17% HLA-DPA10202-DPB10501 HLA-DQA10101-DQB10501 DRB1_0701 0.16% HLA-DPA10202-DPB10501 HLA-DQA10501-DQB10301 DRB1_1501 0.14% There are at least 1,195,000 such allele combinations on MHC-II.

[0092] Then the probabilities of MHC-I and MHC-II can be multiplied together.

[0093] Brief Overview of the Invention

[0094] This invention is a cancer vaccine that, upon injection into the human body, triggers an immune response to fight or prevent cancer. It thus complements existing cancer treatments or renders some of them obsolete and / or makes treatments more effective.

[0095] This invention utilizes the fact that some cancers express MHC-II, and that the antigens overexpressed in these cancers were previously only visible on MHC-I (before cancer), while T cell responses have been weakened.

[0096] This invention utilizes the MHC-II of an antigen while simultaneously silencing and thus circumventing MHC-I based on individual allelic composition. For the antigen cyclin D1, binding sites (i.e., epitopes) are widely distributed across different alleles, allowing for the selection of a very small number of epitopes to form a polyepitope profile—which varies considerably with individual allelic composition. However, when targeting cyclin D1, it is feasible to identify and select epitopes that bind to MHC-II without any component binding to MHC-I, as it is poorly expressed on MHC-I (where "strong binders" are scarce). However, this concept is universal for all antigens in all cancers expressing MHC-II.

[0097] This invention educates the MHC-II portion of the immune system while avoiding damage to healthy tissue and minimizing the risk of autoimmune diseases, by making the MHC-II arm cytotoxic through non-matching with the MHC-I portion of the immune system. The fact that the MHC-II arm becomes cytotoxic when it does not match with the MHC-I portion of the immune system is... Figure 8 and Figure 9 The mechanism described in [the document].

[0098] Cancer also often silences MHC-I, so that the side effects on healthy tissues are unaffected because healthy tissues still express MHC-I.

[0099] T cells of the MHC-II portion of the immune system typically do not see these epitopes before cancer develops because healthy tissues (except for APCs) express only MHC-I.

[0100] Cancers that express MHC-II include: Breast cancer (BC) Prostate cancer (PCa) melanoma Colorectal cancer, Ovarian cancer Classic Hodgkin's lymphoma, Gliomas, and Non-small cell lung cancer It is possible that not all MHC-IIs are expressed, and this must be taken into account in such cases.

[0101] This invention utilizes the fact that MHC-II-responsive CD4+ cells can exhibit cytotoxicity (CD4+ CTLs), for example through... Figure 8 The mechanism described in the literature suggests that this cytotoxic behavior is reserved only for MHC-I, CD8+ CTLs. Alternatively, it may utilize a similar strong immune response, as suggested by references indicating the presence of a protective MHC-II allele.

[0102] Some TAAs are expressed at low levels in healthy tissues, so they may not be associated with inhibitory CD4+ Tregs (because at least outside the thymus they must come from APCs).

[0103] The overview of cancers that simultaneously express MHC-II and the TAA includes those listed in Table 2.

[0104] Predictions of MHC-I and MHC-II binding are based on the following calculations: (1) a list of epitopes and their respective MHC alleles, or (2) a computer algorithm that derives epitopes or tandem epitopes according to the invention (e.g., one or more epitopes or tandem epitopes or multiple epitopes according to the invention that bind MHC-II but not MHC-I) from an allelic composition determined from genomic testing. It is possible that, regardless of the amino acid placed between epitopes, “cutting” or transitioning at two substrings in a multiple epitope cannot be avoided, resulting in an epitope with MHC-I or MHC-II binding properties. In such cases, one of the substrings can be discarded from the multiple epitope, or the two separate multiple epitopes can be designed as separate proteins in a vaccine.

[0105] The vaccine is then produced to contain epitopes or tandem epitopes according to the invention (e.g., one or more epitopes or tandem epitopes or multiple epitopes according to the invention) as the active ingredient (or several multiple epitopes, see above). This is called a personalized vaccine.

[0106] Alternatively, a vaccine may be selected from a set of pre-tested and approved epitopes or tandem epitopes that exhibit the desired behavior. This is known as a sequence catalog. A selected set of pre-tested and approved epitopes or tandem epitopes is provided in the sequence listing attached to this application.

[0107] The personalized vaccine is manufactured for only one patient, based on the patient's MHC-I and MHC-II alleles. Based on the subject's alleles, suitable TAA epitopes or tandem epitopes that bind MHC-II and do not contain MHC-I binding sequences can be selected using, for example, a database provided herein as URL MHC-I or a subsequent improved version thereof (for MHC-I) and a database provided herein as URL MHC-II or a subsequent improved version thereof (for MHC-II). Preferably, the selection of suitable TAA epitopes or tandem epitopes that bind MHC-II and do not contain MHC-I binding sequences can be achieved by selecting one or more sequences from the sequence catalog provided herein. The personalized vaccine manufactured according to the present invention has the following characteristics: No need for sequencing and deducing (a set of) TSA uncertainties: therefore faster, cheaper, and more certain; Both methods require inexpensive and rapid (1) genomic testing of 11 genes (each with two alleles) (“HLA typing”). And optionally (2) express analysis.

[0108] Creating "personalized vaccines" will always be more expensive and time-consuming than selecting a sequence.

[0109] Sequences are selected from the sequence catalog based on the following factors: Any sequence must contain an epitope triggered by one or more MHC-II alleles of a human subject.

[0110] No sequence may contain any epitope triggered by any MHC-I allele of a human subject.

[0111] Each TAA's sequence catalog includes both manually identified and automatically generated sequences.

[0112] The sequence catalog contains validated sequences, i.e., tandem epitope sequences that have been validated in specific allele subjects to bind to and be presented by MHC-II without containing MHC-I binding epitope sequences, as well as sequences predicted to have these characteristics but not yet validated. Sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 279, 280, 281, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364. Sequences 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412, 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, and 440 are derived from CEACAM5; sequences 51, 469, 495, and 498... Sequences 499 and 500 are derived from cyclin D1; sequences 101, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, and 627 are derived from MAGEA3; sequences 105, 106, 108, 629, and 631 are derived from NY-ESO-1; sequences 173, 174, 176, 177, 178, 179, 180, 803, 805, and 806 are derived from cyclin D ...01, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, and 627 are derived from MAGEA3; sequences 105, 106, 108, 629, and 631 are derived from NY-ESO-1; sequences 173, 174, 176, 177, 178, 179, 180, 803, 805, and 806 are derived from cyclin D1; sequences 105, 106, 108, 629, and 606 are derived from cyclin D1; sequences 105, 106, 108, 629, and 606 are derived from cyclin D1; sequences 105, 106, 108, 629, and 606 are Sequences 807, 895, 896, 897, 904, 905, and 906 are derived from TERT; sequences 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607 are derived from HER2 and have all been verified.

[0113] The vaccine can be a preventative vaccine (before cancer is detected) or a therapeutic vaccine (after cancer is detected). The vaccine may contain an active agent (i.e., one or more epitopes, tandem epitopes, or multiple epitopes according to the invention) alone, or may also contain other active ingredients, or may be designed in combination with other vaccines. If the vaccine is administered in multiple injections, it can be adjusted, i.e., the dosage of subsequent injections can be determined based on efficacy measurements, etc.

[0114] The vaccine may be one of the following vaccine technologies, or others: mRNA vaccine, SAM: Self-amplified mRNA vaccine LNP mRNA: Lipid nanoparticle mRNA vaccine Viral vector vaccines, DC vaccine, or ISV: In situ vaccine.

[0115] This invention takes into account physiological limitations and sequence ordering; it may be possible to replicate “good” sequences (those that have been verified).

[0116] This invention performs dose calculations, taking into account both unpresented and validated epitopes.

[0117] This invention supports the recalculation of master data and its operation on multiple versions—so that it can be used in multiple countries or regions based on the approval of different master data and at different speeds based on the approval of new master datasets.

[0118] One or more epitopes, tandem epitopes, or multiple epitopes according to the invention can be enhanced to promote function by adjuvants. These adjuvants may be specific peptide sequences added as “intra-tandem adjuvants,” wherein the adjuvant is linked to the active agent via an optional spacer sequence, or they may be added as separate active ingredients administered together with the vaccine. Attached Figure Description

[0119] Figure 1 Cell types and MHC-I and II. This figure shows that some cancer cells (such as breast cancer, prostate cancer, melanoma, and non-small cell lung cancer (NSCLC)) express MHC-II (communication on MHC-II)—which is normally reserved by APCs in the absence of cancer. All nucleated cells communicate on MHC-I.

[0120] Figure 2 CD4+ cells have the functions of (a) attracting CD8+ cytotoxic T cells, (b) being cytotoxic themselves, and (c) helping B cells.

[0121] Figure 3The first step in the antigen presentation pathway: protein binding to MHC (I or II). Part A: Protein, such as "cyclin D1," and its amino acid composition, one amino acid per square. Part B: The modified protein contains only strong binders. Part C: Same as Part B, except that a new set of amino acids is now present and will become epitopes. These epitopes should not be "strong binders" or "weak binders" and produce unpredictable presentation consequences—see Part E. Part D: The modified protein from Part C is cleaved into epitopes by phagocytosis (shown as an example of 3 amino acids in length; in reality, epitopes are longer, typically 15 for MHC-II). Part E: Epitopes with new amino acid transitions in the modified protein, thus requiring examination of their binding to MHC. Part F: The binding of the epitope to the MHC molecule—allowing the epitope, along with the MHC molecule, to migrate to the cell surface and be displayed to the immune system (as a ligand for the T cell receptor TCR).

[0122] Figure 4 The alleles in MHC-I and MHC-II—proteins that are candidates for breast cancer antigens—were ranked according to the predicted levels of proteins indicated in the presentation titles (cyclin D1, mucin 1, CEA, and TERT). It can be seen that, particularly for cyclin D1, the alleles behind MHC-II are ranked higher than those behind MHC-I, while for other proteins, some alleles behind MHC-I (A, B, and C) are ranked higher. The ranking of ) is higher than some of the alleles behind MHC-II.

[0123] Figure 5 TAAs are classified according to (1) the extent to which they express MHC-II (as the number of strong binders divided by the length of the TSS) – also known as their “loudness” on MHC-II, and (2) how immunogenic they are assumed to be (based on the inverse of the expression intensity in healthy tissue).

[0124] Figure 6 When T cells (CD8 corresponds to MHC-I, and CD4 corresponds to MHC-II) encounter APCs (such as dendritic cells) in lymph nodes, they are activated via activation signals. Since CD8 kills APCs at the end of its activation but before CD4 is activated, CD4 T cells cease their activation in the "CD4 helper T cell" phase.

[0125] Figure 7 The continuation of the CD4 helper T cell activation process, for example in the lungs, and in the absence of CD8 T cells, until CD4 T cells are fully activated as "cytotoxic T cells".

[0126] Figure 8When APCs do not contain MHC-I on their surface, in the absence of CD8 T cells, they undergo a complete activation process in lymph nodes, from "naïve" T cells all the way to cytotoxicity.

[0127] Figure 9 Overview of vaccination and the process of action, including main steps 1 through 4.

[0128] Figure 10 Overview of the MHC-II delivery route, with a focus on the MHC-II loading compartment (“MIIC”).

[0129] Figure 11 An example of the total probability or proportion of alleles of a sequence and its triggering epitopes that are part of that sequence.

[0130] Figure 12 : Sequences and examples of alleles that trigger epitopes—detailed Figure 11 The bottom.

[0131] Figure 13 Sequence 1, or SEQ1, is an example of a joined-up sequence consisting of (1) “CEACAM1A val”, (2) “CEACAM 5A”, (3) “CEACAM 11 val”, and (4) “CEACAM 20 val”, with a set of spacer sequences between them—making it necessary for humans with certain MHC-I alleles to avoid “SEQ1” to prevent an MHC-I response. SEQ2 is generated by extending each element of “SEQ1” to include more MHC-I alleles.

[0132] Figure 14 Comparison of personalized vaccines with manually and automatically generated sequences. Detailed Implementation

[0133] This invention relates to triggering an immune response by injecting, once or several times, a reagent containing an antigen (protein) or a portion of an antigen (so-called a polyepitope) or several polyepitopes. The purpose of the immune response is to partially or completely eradicate cells representing a disease, which may be cancer, referred to below as "the cancer".

[0134] If tested on humans, and if cancer is shown to express MHC-II or a portion thereof, along with the antigen, the multiepitope can be engineered to bind to it and thus potentially be displayed on MHC-II but not on MHC-I. This elicits an immune response that may lead to complete or partial eradication of the cancer without involving cells representing healthy tissue that express MHC-I. This excludes humans with cancers expressing MHC-II where the antigen does not bind to MHC-II and therefore is not displayed. For cyclin D1, it is estimated that 0.3% of humans lack a strong MHC-II binder.

[0135] We believe that one suitable antigen protein for this purpose is cyclin D1 (genetic code CCND1). It is expressed at low levels on MHC-I and in healthy tissue cells. It is likely that the immune system has not yet generated any MHC-II-targeting T cells with multi-epitope-matched T cell receptors (TCRs) based on cyclin D1 prior to vaccination, since only low levels of cyclin D1 in APCs are exhibited on MHC-II prior to cancer. Other TAAs are disclosed herein. Preferred TAAs are CEACAM5, cyclin D, MAGEA3, NY-ESO-1, TERT, and HER2. CEACAM5 is a particularly preferred TAA.

[0136] This invention is based on selecting a subset of TAA epitopes or tandem epitopes for vaccination, which are completely or almost completely silenced on MHC-I but presented by MHC-II. There will then be no new CD8+ CTLs; the new CD4+ cytotoxic T cells and other immune responses on MHC-II will simply kill cancer cells (and perhaps a few additional APCs).

[0137] If the invention is used in a therapeutic setting, it may further involve testing samples from cancer (e.g., biopsy) or other expertise to determine (1) whether the cancer expresses MHC-II or a portion thereof, and (2) whether the cancer overexpresses the antigen (e.g., cyclin D1), i.e., significantly higher than in healthy tissue. A subset of breast cancer, prostate cancer, and melanoma are expected to express MHC-II and overexpress cyclin D1.

[0138] Genome sequencing is preferred to deduce the allelic composition of human HLA genes (e.g., oral swabs). This test includes a subset or the complete set of HLA gene alleles: MHC-I: HLA-A, HLA-B, HLA-C.

[0139] MHC-II: HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5.

[0140] Personalized vaccines: Computer programs currently defined in URL MHC-I or their subsequent improved versions (for MHC-I), and computer programs defined in URL MHC-II or their subsequent improved versions (for MHC-II), can be used to predict the binding data of antigens (e.g., cyclin D1 and subsequently derived multiple epitopes, see description below) for a given specific allele or haplotype (allele combination). The computer program provides one attribute as part of its output for each epitope and each allele, which is one of the following: “non-binding,” “weak binding” (WB), and “strong binding” (SB), representing the likelihood that the epitope binds to the MHC molecule (given allele): “strong binding” (SB) is highly likely to bind to the MHC in question (see description below). Figure 3 The epitopes (F in the original text) can be presented to the immune system along with MHC molecules on the cell surface. "Weak binders" (WB) may bind to MHC and be presented to the immune system along with MHC molecules on the cell surface, with a probability greater than 1% but less than 100%. "Non-binding agents" do not bind to MHC and therefore are almost certain not to be presented to the immune system along with MHC molecules on the cell surface. Subsequently, epitopes or tandem epitopes that are "strong binders" (or "weak binders" in specific cases) for a particular allele of the subject and are non-binding agents for MHC-I are selected.

[0141] Multi-epitope antigens are used as vaccine agents to trigger an immune response on MHC-II of the adaptive immune system for the prevention or treatment of cancers expressing MHC-II. Multi-epitope antigen vaccines can be used in conjunction with other agents in the vaccine that trigger other immune responses on MHC-I or MHC-II, or both.

[0142] The epitopes, tandem epitopes, or multiple epitopes according to the present invention are determined by the following steps 1 to 6: Step 1: We use the output of the URL MHC-II service, a standalone service, or a similar or optimized MHC-II service to construct a database or a list of combined properties with the following combinations: All epitopes of the antigen on MHC-II (eptopes of 13-25 amino acids in length, or subsets of these epitopes, such as only one of 15 length, derived from the full length of the antigen, see [link to MHC-II epitopes]). Figure 3 A) and All alleles (or haplotypes) that make up different MHC-II molecules.

[0143] This means identifying an epitope as one of the following: (1) a non-binding epitope, (2) a “weakly binding epitope” (WB), or (3) a “strongly binding epitope” (SB) – having any specific allele or combination of alleles.

[0144] Step 2: Select epitopes that are “strong binders” (SBs) and have a specific combination of alleles of the subject, for example, by their position in the full-length antigen sequence. See also Figure 3 In section B, the "strong binding agent" can form a sequence as tandem epitopes, such as consecutive epitopes longer than the individual epitope—in Figure 3 In the example, a table length of 3 is used as a simple example; see [link to example]. Figure 3 D in the formula. If there is no "strong binding agent" for a particular allele combination, then WB is included.

[0145] Step 3: A "priori polytype" is constructed by appending the aforementioned epitopes—marking the "cuts" of the epitopes within the polytype, i.e., the connection points of the epitopes. All cuts are located within the polytype. See also Figure 3 C in the middle. Then modify the a priori multi-tablet as follows: Step 4: Using the services or standalone services or similar or optimized services of URL MHC-I and URL MHC-II, examine both MHC-I and MHC-II to determine whether the prior polyepitopes (focusing on epitopes now generated around the cut) trigger a "strong binder" (SB) or a "weak binder" (WB) on MHC-I or MHC-II. See also Figure 3 E in this context. This would constitute unprecedented or anomalous and untested biological behavior. Although the figure shows the typical epitope length (3 in the figure), this check needs to be performed on all possible epitope lengths, namely 8 to 14 on MHC-I and 13 to 25 on MHC-II.

[0146] Step 5: Padding introduces strong or weak binding agents into cleavages on MHC-I or MHC-II or both: (1) If the "cleavages" are wide (positions far apart), any sequence either (1A) silences the cleavage (making it non-binding) or (1B) extends the epitope to include more amino acids from the original protein (padding with amino acids from the original string) until the cleavage is silenced (non-binding). (2) If the "cleavages" are close (positions near each other), the original string of the full-length protein must be used to connect the epitopes into a continuous substring of the protein, thus resembling the result produced from the full-length protein. See also Figure 3 The progression between A, B, and C involves adding more parts of A to create a longer substring in B, and "filling" gaps when concatenating in C.

[0147] If the prior polyepitope removes all WBs or SBs from both MHC-I and MHC-II after the filling, then the A prior polyepitope is updated to include the filling.

[0148] If it is impossible to prevent a substring in the prior polyetope from generating a WB or SB on MHC-I, MHC-II, or both after padding, then the substring is removed from the prior polyetope, i.e., the prior polyetope is updated to exclude the substring. In this case, we return to and repeat step 4, i.e., we need to check the prior polyetope again on MHC-I and MHC-II.

[0149] Consider using the substring extracted therefrom as a separate multiepitope, thus as a separate protein in the vaccine, provided that it passes through subsequent step 6.

[0150] Step 6: If there are “strong binders” on MHC-I that have been selected for the prior polyepitopes (these SBs on MHC-I are likely related to epitopes embedded in SB epitopes on MHC-II, since in most cases the length of epitopes on MHC-I is shorter than the length of epitopes on MHC-II, and we have changed or removed those SB epitopes from “cutting” in the steps above), then these epitopes on MHC-II must be discarded, i.e., the prior polyepitopes must be updated to not contain epitopes that constitute SBs on MHC-II, if the epitopes contain epitopes that are SBs on MHC-I (or are related to epitopes that are SBs on MHC-I).

[0151] If this introduces a new cut, repeat step 4, which means we need to check the prior polyepitopes again on MHC-I and MHC-II.

[0152] If this results in no epitopes remaining in the multi-epitope group, then either (1) they are retained (and the side effect of triggering an immune response on MHC-I) or (2) a “weak binder” on MHC-II is included. We prefer (1) because it is highly likely that no TCR match will occur anyway.

[0153] Vaccines are made using selected epitopes, tandem epitopes, or multiple epitope sequences as active ingredients, for example, by converting them into mRNA. If converted into mRNA, it is administered (usually by injection) so that the body will produce proteins similar to epitopes, tandem epitopes, or multiple epitopes in, for example, APCs expressing MHC-II, thereby prompting the immune system to respond to that protein, for example by producing CD4+ T cells that can also attach to and kill cancer.

[0154] Repeated vaccinations may be necessary to achieve the desired effect (the desired effect of prevention or treatment).

[0155] The effectiveness of the vaccine can be monitored and evaluated to see if CD4+ CTLs or antibodies are produced (in preventative cases) or if cancer cells are further killed by the immune system (in therapeutic cases). The vaccine dosage can be adjusted based on this monitoring or evaluation.

[0156] Sequence identification: The sequence catalog contains sequences representing the selected tandem epitopes for MHC-II binding in multiple MHC-II alleles for each TAA, consisting of the original sequence and shortened sequences, as follows: Original: A substring derived from a TAA, wherein the epitope is triggered by multiple MHC-II alleles (which are strong binders if a human has one of these alleles), and wherein the probability or proportion of the MHC-II allele is high, the epitopes are tandemly linked into a substring, wherein the tandem is then considered a “hotspot” of the TAA.

[0157] Shortening: If they are still longer than 18 amino acids after shortening, and if shortening them can avoid epitopes triggered by one or more MHC-I alleles that have a high probability or proportion in the world population, then the original sequence is shortened.

[0158] Each sequence in the sequence catalog is either validated or unvalidated: a sequence is validated if it contains one or more epitopes that have been empirically proven to be present on human cells and to be immunogenic (attracting T cells). Validation is typically associated with a specific allele, but this invention chooses to rely on predictions of URL MHC-I and URL MHC-II to generate the correct association between epitopes and the alleles required for their presentation.

[0159] Automatic generation of the original sequence: Original: A substring derived from a TAA, wherein the epitope is triggered by at least a threshold number of alleles (or a strong binder if a human has one of these alleles), the threshold number being specific for each of DP, DQ, and DR, and the probability or proportion of the allele within DP, DQ, or DR being at least a threshold probability, the threshold probability being specific for each of DP, DQ, and DR, the epitopes being concatenated into a substring, which is then considered a “hotspot” of that TAA.

[0160] Shortening: If the shortened sequences are still longer than the threshold number of amino acid lengths, and if shortening can avoid epitopes triggered by one or more MHC-I alleles that are likely or occur in a certain percentage of the world population, then the original sequences are shortened.

[0161] The purpose of shortening the original sequence is to reduce the likelihood that the shortened sequence will be eliminated due to the presence of MHC-I alleles in a given human subject, while retaining as much or as little of the MHC-II epitopes of the shortened sequence as possible, or retaining them completely, relative to the original sequence, so that the shortened sequence may be just as effective as the original sequence in evoking an MHC-II immune response, but will not be eliminated or removed as frequently as the original sequence.

[0162] Automatically generated shortened sequences can be further shortened—becoming another shortened sequence—if they meet the shortening requirements.

[0163] The sequences in the sequence catalog can be combined or connected as follows: (1) they are equal (if they start and end at the same position in the TAA) (in which case the same sequence is represented only once in the sequence catalog) or (2) if they are nearly equal, an algorithm is performed to prove their validity (if it does not create or recreate them, resulting in their shortening, etc.) (in which case the sequences are concatenated). Sequences in the sequence catalog can be linked together with spacer sequences (where each sequence retains its full number of amino acids) and would, in most cases, be selected together if they were triggered by nearly identical alleles. Linking them with spacer sequences avoids the need for separate administration as individual injections of a vaccine, instead allowing them to be administered as a single injection.

[0164] Select a sequence from the sequence catalog: Sequences are selected from the sequence catalog based on the following factors: Any sequence must contain an epitope triggered by one or more human MHC-II alleles. This is called an MHC-II sequence list.

[0165] No sequence may contain any epitope triggered by any human MHC-I allele (this is referred to as "removing" the sequence from the "MHC-II sequence list," resulting in a "net sequence list").

[0166] Physiological limitations, sequencing, and repetition: This invention may employ a limitation on the number of individual vaccines, wherein the number is calculated based on individual and clinical measurements and parameters. This limitation is referred to as a "physiological limitation".

[0167] The present invention can sort sequences and / or repeat some sequences (“good,” for example, verified) while adhering to the said physiological constraints to concentrate the immune response.

[0168] Adjuvant: This invention uses adjuvants to enhance function. These adjuvants are either "intra-string adjuvants," where the adjuvant is linked to the sequence via an optional spacer sequence, or they are separate active ingredients administered together with the vaccine.

[0169] Examples of “intra-string” adjuvants are shown in Table 4 below, where the “invariant chain” is attached to the beginning of the sequence, and in other cases the sequence constitutes the string (from TAA CEACAM5 in this figure).

[0170] Table 4

[0171] Administration: This invention calculates vaccine dosage based on the following factors: Has the sequence been validated (including validated epitopes)? How many tabletops are not presented? Personal and clinical conditions (measurements and parameters)

[0172] Best mode for carrying out the invention

[0173] We describe below examples of the use of the antigen cyclin D1 in personalized vaccines. Cyclin D1 multiepitopes are used as vaccine agents to trigger an immune response on MHC-II of the adaptive immune system as a means of prevention or treatment of (1) breast cancer, (2) prostate cancer, or (3) melanoma types according to Table 2. Other TAAs according to the invention may also be used.

[0174] Personalized vaccines: Epitopes, tandem epitopes, or multiple epitopes are determined by the following steps, which are special cases of steps 1-6 described above—the implementation of which is referred to and detailed below for cyclin D1: Step 1: A database table was constructed containing the binding properties of all epitopes of cyclin D1 on MHC-II (episodes of 15 amino acids in length, ranging from 13 to 25, from the full length of cyclin D1 295) to all MHC-II alleles (or haplotypes). This included identifying any epitope as (1) a non-binding agent, (2) a “weak binding agent” (WB), or (3) a “strong binding agent” (SB).

[0175] Of all epitopes of length 15, using the service on URL MHC-II, selecting the alleles that constitute the majority of the world's population based on their frequency at URL "https: / / allelefrequencies.net / hla.asp", we obtained the following "strong binding" counts: Position table (length 15) SB count 10 VETIRRAYPDANLLN 11 11 ETIRRAYPDANLLND 28 12 TIRRAYPDANLLNDR 41 13 IRRAYPDANLLNDRV 11 18 PDANLLNDRVLRAML 1 19 DANLLNDRVLRAMLK 2 22 LLNDRVLRAMLKAEE 2 23 LNDRVLRAMLKAEET 5 24 NDRVLRAMLKAEETC 2 25 DRVLRAMLKAEETCA 2 47 CVQKEVLPSMRKIVA 3 48 VQKEVLPSMRKIVAT 3 49 QKEVLPSMRKIVATW 3 55 SMRKIVATWMLEVCE 3 56 MRKIVATWMLEVCEE 11 57 RKIVATWMLEVCEEQ 2 79 PLAMNYLDRFLSLEP 2 84 YLDRFLSLEPVKKSR 1 85 LDRFLSLEPVKKSRL 2 86 DRFLSLEPVKKSRLQ 5 87 RFLSLEPVKKSRLQL 3 88 FLSLEPVKKSRLQLL 2 104 ATCMFVASKMKETIP 12 105 TCMFVASKMKETIPL 11 108 FVASKMKETIPLTAE 2 109 WASKMKETIPLTAEK 16 110 ASKMKETIPLTAEKL 19 111 SKMKETIPLTAEKLC 9 112 KMKETIPLTAEKLCI 6 116 TIPLTAEKLCIYTDN 2 119 LTAEKLCIYTDNSIR 6 120 TAEKLCIYTDNSYRP 8 121 AECLCIYTDNSIRPE 20 122 EKLCIYTDNSIRPEE 6 124 LCIYTDNSIRPEELL 7 125 CIYTDNSIRPEELLQ 10 126 IYTDNSIRPEELLQM 7 127 YTDNSIRPEELLQME 17 128 TDNSIRPELLQMEL 20 129 DNSIRPELLQMELL 14 133 RPEELLQMELLLVNK 4 134 PEELLQMELLLVNKL 3 135 EELLQMELLLVNKLK 17 144 LVNKLKWNLAAMTPH 6 145 VNKLKWNLAAMTPHD 16 146 NKLKWNLAAMTPHDF 1 148 LKWNLAAMTPHDFIE 2 149 KWNLAAMTPHDFIEH 2 152 LAAMTPHDFIEHFLS 4 153 AAMTPHDFIEHFLSK 13 154 AMTPHDFIEHFLSKM 13 155 MTPHDFIEHFLSKMP 4 159 DFIEHFLSKMPEAEE 2 160 FIEHFLSKMPEAEEN 3 161 IEHFLSKMPEAEENK 7 162 EHFLSKMPEAEENKQ 2 173 ENKQIIRKHAQTFVA 1 188 LCATDVKFISNPPSM 5 189 CATDVKFISNPPSMV 16 190 ATDVKFISNPPSMVA 40 191 TDVKFISNPPSMVAA 17 192 DVKFISNPPSMVAAG 6 193 VKFISNPPSMVAAGS 1 198 NPPSMVAAGSVVAAV 29 199 PPSMVAAGSVVAAVQ 63 200 PSMVAAGSVVAAVQG 79 201 SMVAAGSVVAAVQGL 42 202 MVAAGSVVAAVQGLN 18 205 AGSVVAAVQGLNLRS 1 206 GSVVAAVQGLNLRSP 2 214 GLNLRSPNNFLSYYR 2 218 RSPNNFLSYYRLTRF 1 219 SPNNFLSYYRLTRFL 1 220 PNNFLSYYRLTRFLS 1 247 CQEQIEALLESSLRQ 2 248 QEQIEALLESSLRQA 6 249 EQIEALLESSLRQAQ 1 256 ESSLRQAQQNMDPKA 1 262 AQQNMDPKAAEEEEE 1 263 QQNMDPKAAEEEEEE 9 264 QNMDPKAAEEEEEEE 15 265 NMDPKAAEEEEEEEE 38 266 MDPKAAEEEEEEEEE 19 267 DPKAAEEEEEEEEEV 8 Following step 1, we narrow down the MHC-II alleles based on specific combinations, such as the combination DPA1_0202+ DPB1_0501 + DQA1_0501 + DQB1_0301 + DRB1_0701, which will give us 8 “strong binders,” see step 3 below. As can be seen from the list above, the results of narrowing down the alleles will vary significantly for different combinations.

[0176] We estimate that approximately 0.3% of the world's population will have MHC-II allele combinations that do not provide a "strong binding agent" for cyclin D1 on MHC-II.

[0177] Step 2: Epitopes are selected that (e.g., positions within the full-length 295-word cyclin D1 protein sequence) constitute a “strong binding agent” (SB) of a specific combination of alleles in that person. The “strong binding agent” can form substrings of consecutive epitopes, whose combined length is greater than that of the individual epitopes.

[0178] If there is no “strong binding agent” for a particular combination of alleles, which occurs in about 0.3% of cases, then we include WB in that selection.

[0179] Step 3: A "prior polyphenotype" is constructed by attaching the above epitopes—the "cut" in the polyphenotype is marked, that is, the connection point of the epitope.

[0180] For the allele / haplotype combination DPA1_0202 + DPB1_0501 + DQA1_0501 + DQB1_0301 + DRB1_0701, we obtain: Location table distance additional 85 LDRFLSLEPVKKSRL LDRFLSLEPVKKSRL 86 DRFLSLEPVKKSRLQL 1 QL 197 SNPPSMVAAGSVVAAV 111 SNPPSMVAAGSVVAAV 199 PPSMVAAGSVVAAVQ 2 Q 200 PSMVAAGSVVAAVQG 1 G 201 SMVAAGSVVAAVQGL 1 L 202 MVAAGSVVAAVQGLN 1 N 206 GSVVAAVQGLNLRSP 4 LRSP The nucleic acids in the "Additional" column constitute the sequence of prior polyepitopes. These polyepitopes have a cut, located between the epitope starting at position 86 and the epitope starting at position 197.

[0181] Step 4: Examine both MHC-I and MHC-II to determine whether the prior polyepitopes (with a focus on epitopes now generated around the cleavage) trigger a “strong binder” (SB) or a “weak binder” (WB) on either MHC-I or MHC-II. This examination requires testing for all possible epitope lengths, i.e., 8 to 14 on MHC-I and 13 to 25 on MHC-II.

[0182] Step 5: Cuts introduced with strong or weak binding agents by filling MHC-I or MHC-II or both: (1) If the "cuts" are wide (positions far apart), any sequence either (1A) silences the cut (making it non-binding) or (1B) extends the epitope to include more amino acids from the original protein (filling with amino acids from the original string) until the cut is silenced (non-binding). (2) If the "cuts" are close (positions near each other), the original string of the full-length protein must be used to connect the epitopes into a continuous substring of the protein, thus resembling the result produced from the full-length protein. See also Figure 3 The progression between A, B, and C involves adding more parts of A to create a longer substring in B, and "filling" gaps when concatenating in C.

[0183] If the prior polyepitope removes all WBs or SBs from both MHC-I and MHC-II after the filling, then the prior polyepitope is updated to include the filling.

[0184] If it is impossible to prevent a substring in the prior polyetope from generating a WB or SB on MHC-I, MHC-II, or both after padding, then the substring is removed from the prior polyetope, i.e., the prior polyetope is updated to exclude the substring. In this case, we return to and repeat step 4, i.e., we need to check the prior polyetope again on MHC-I and MHC-II.

[0185] Step 6: If a “strong binder” exists on MHC-I that has been selected for the prior polyepitope, the prior polyepitope is updated to exclude epitopes constituting SB on MHC-II, if the epitope includes (or is associated with) an epitope that is an SB on MHC-I.

[0186] Continuing with the above example, for the MHC-II allele / haplotype combination DPA1_0202 + DPB1_0501 + DQA1_0501 + DQB1_0301 + DRB1_0701 and for the MHC-I allele combination A_0201 + B_0702 + C_0701, we obtain: Location table distance additional 197 SNPPSMVAAGSVVAAV 111 SNPPSMVAAGSVVAAV 199 PPSMVAAGSVVAAVQ 2 Q 200 PSMVAAGSVVAAVQG 1 G 201 SMVAAGSVVAAVQGL 1 L 202 MVAAGSVVAAVQGLN 1 N The nucleic acids in the "Additional" column constitute sequences with prior multi-epitopes.

[0187] If this introduces a new cut, repeat step 4, which means we need to check the prior polyepitopes again on both MHC-I and MHC-II.

[0188] If this results in no epitopes remaining in the multi-epitope group, they are retained (and the side effect of triggering an immune response on MHC-I is accepted).

[0189] Using the aforementioned multi-epitope sequences as active ingredients, they are converted into mRNA to manufacture vaccines. This can be carried out as an mRNA vaccine or a SAM vaccine, causing dendritic cells (“DCs” – a subset of APCs) to produce proteins similar to the aforementioned multi-epitopes, such that MHC-II expressed on said DCs will activate the immune system, for example, by causing it to produce CD4+ T cells that can also respond to cancer cells.

[0190] This can be implemented such that a reduced vaccine set will meet the needs of the majority of the world's population—without achieving the optimal combination of SBs on MHC-II for a specific allele combination, but not exceeding the length of SBs on MHC-II, and not inducing any WBs or SBs on MHC-I or MHC-II due to cleavage, nor inducing any SBs on MHC-I. This would mean that we could pre-approve vaccines with specified sequences and their allele combinations without relying on approval of the methods described in steps 1-6 above.

[0191] Sequence identification: Automatic generation of the original sequence: Original: A substring derived from a TAA, wherein the epitope is triggered by at least (6 alleles for DP and DQ, 4 alleles for DR) (a strong binder if humans have one of them), and wherein the probability or proportion of said allele in DP, DQ or DR is at least 1%, the epitopes are concatenated into a substring and then considered as a “hotspot” of said TAA.

[0192] Shortening: If the shortened sequence is still longer than 18 amino acids, and if shortening can avoid epitopes triggered by one or more MHC-I alleles with a probability or proportion of 10% in the world population, then the original sequence is shortened.

[0193] Automatically generated shortened sequences can be further shortened—becoming another shortened sequence—if they meet the shortening requirements.

[0194] Table 5 below provides a brief selection of several sequences based on TAA CEACAM5, outlining the probability of all sequences in the sequence catalog being selected by human subjects—the total probability given a proportion of the world population—by MHC-II and MHC-I, and further subdivided into genes (MHC-II: DP, DQ, and DR; for MHC-I: A, B, and C):

[0195] The sequences in the sequence catalog can be combined or connected as follows: (1) they are equal (if they start and end at the same position in the TAA) (in which case the same sequence is represented only once in the sequence catalog) or (2) if they are nearly equal, an algorithm is performed to prove their validity (if it does not create or recreate them, resulting in their shortening, etc.) (in which case the sequences are concatenated). Sequences in the sequence catalog can be linked together with spacer sequences (where each sequence retains its full number of amino acids) and would, in most cases, be selected together if they were triggered by nearly identical alleles. Linking them with spacer sequences avoids the need for separate administration as individual injections of a vaccine, instead allowing them to be administered as a single injection.

[0196] Sequence selection: This selection is based on an SQL query, which takes the following input: MHC-I and MHC-II alleles (22 in total for 11 genes: ("HLA-" omitted): (MHC-I:) A, B, C, (MHC-II:) DPA1, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5) Tumor-expressed TAAs (listed as a group of identities) An example might look like this: SELECT from mol_SB_AfterKnockOutMultipleAntigens_4 ('NP_444284_1_G1_','NP_001018016_1_','NP_060884_1_alp','tr_A0A024R0K5_A','NP_937983_2_tel','', -- Cyclin D1, Mucin 1 iso 2, Sialyl Tn iso 1, CEACAM5, TERT '0202', '0103', -- DP A1 '0101', '1101', -- DP B1 -- 2% 0.1% ------------------------ '0501', '0102', -- DQ A1 '0201', '0502', -- DQ B1 -- 7% 2% ------------------------ '1503', '0301', -- DR B1 -- 4% 7% '0101', '0108', -- DR B3 '0101', '0107', -- DR B4 '0101', '0102', -- DR B5 ------------------------ '2402', '0201', -- A --10% 25% '4001', '1501', -- B -- 5% 7% '0602', '1203' -- C -- 3% 2% )” The use of "-" indicates that a line is a comment if it begins with or starts from that point, which has no effect in SQL.

[0197] A comparison of manually and automatically generated sequence selection with personalized vaccines is shown in Figure 14 The findings are quite close; only one-fifth of the sequences in the optimal personalized vaccine (211-226) are not covered by the selection sequence for the TAA "mucin 1" and the selection of alleles. Since any cancer with a majority of alleles has many epitope sequences, and since any cancer expressing MHC-II has multiple TAAs, this is good enough that we will select a range of sequences for this vaccine. However, we will eventually reach physiological limitations because we will have to prioritize among the selected sequences.

[0198] Sequence List XML

[0199] The sequence directory contains the sequences appearing in the sequence table. The sequence table XML file was submitted as an XML file and is incorporated here by reference. The submitted sequence table XML file is named "Cancer Vaccine Based on MHC-II Epitope Selection.xml", created on January 13, 2025, and is 2,738 kilobytes in size.

[0200] project

[0201] This invention can be defined as follows: 1. A method for producing a cancer vaccine composition for treating or preventing cancer in a human subject, the method comprising the steps of: a) selecting one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) such that the selected epitopes bind to the subject's MHC-II, thereby providing a library of epitopes and tandem epitopes that bind to the subject's MHC-II; b) removing epitopes and tandem epitopes containing epitopes that bind to the subject's MHC-I from the library of epitopes and tandem epitopes selected in step a), thereby providing a library of epitopes and tandem epitopes that bind to the subject's MHC-II but not MHC-I; c) optionally combining one or more epitopes and tandem epitopes provided in step b) into a multi-epitope comprising the one or more selected epitopes and tandem epitopes; d) optionally providing an information-carrying molecule capable of promoting in vivo production of one or more epitopes and tandem epitopes provided in step b) or the multi-epitope provided in step c); e) One or more epitopes and tandem epitopes selected from the library of epitopes and tandem epitopes that bind to the subject’s MHC-II but not MHC-I, provided in step b), or multiple epitopes provided in step c), or information-carrying molecules provided in step d), are combined with suitable pharmaceutically acceptable excipients.

[0202] 2. The method according to Project 1, wherein the tabletop is a serial tabletop.

[0203] 3. The method according to Item 2, wherein the one or more tandem tabletops are selected from tandem tabletops provided in the sequence list.

[0204] 4. The method according to Item 3, wherein the one or more tandem epitopes are selected from the sequence list as sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, 28 1, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412, 4 13, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803, The serial table positions provided are 805, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

[0205] 5. The method according to any of the preceding items, wherein steps a) and b) are implemented by: using an algorithm based on allele data of the subject's genes to select a suitable TAA, preferably a TAA provided in this specification, and analyzing the possible MHC-II and MHC-I binding epitopes of the corresponding TAA, the algorithm predicting immune system behavior against the corresponding TAA and any epitope sequences believed to constitute the corresponding TAA.

[0206] 6. According to the method of Project 5, the step of analyzing the possible MHC-II and MHC-I binding epitopes or tandem epitopes of the corresponding TAA is implemented by using appropriate algorithms that can be used to calculate the binding probability of each epitope of the TAA to MHC-II and MHC-I.

[0207] 7. The method according to Item 6, wherein the appropriate algorithm is provided for MHC-I at the URL “https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ” or a later improved version thereof, and for MHC-II at the URL “https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.3 / ” or a later improved version thereof.

[0208] 8. Use of one or more epitopes or tandem epitopes derived from tumor-associated antigens in the treatment or prophylactic treatment of a subject with cancer, wherein the epitope or tandem epitope is specifically selected to activate an immune response via the subject's major histocompatibility complex II (MHC-II), and wherein the epitope or tandem epitope is specifically selected not to activate an immune response via the subject's major histocompatibility complex I (MHC-I).

[0209] 9. Use of one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) in the treatment or prophylactic treatment of a subject with cancer, wherein the epitope or tandem epitope is specifically selected to activate an immune response via the subject's major histocompatibility complex II (MHC II), wherein the subject is characterized by having a genotype that promotes non-binding of the specifically selected epitope or tandem epitope to the subject's major histocompatibility complex I (MHC I).

[0210] 10. Depending on the purpose of item 8 or 9, the tabletops therein are serial tabletops.

[0211] 11. As per the purpose of item 10, the tandem tabletop is a tandem tabletop provided in the sequence list.

[0212] 12. According to the purpose of item 11, the tandem epitopes are the sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, 281, 282 in the sequence list. 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412, 413 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803, 8 The serial positions provided by 05, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

[0213] 13. A cancer vaccine comprising one or more specifically selected epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), wherein the selected epitopes or tandem epitopes activate an immune response via the subject's major histocompatibility complex II (MHCII), said epitopes being characterized in that a) they consist of more than 13 amino acids but less than 25 amino acids, preferably 15 amino acids.

[0214] 14. The vaccine according to Item 13, wherein the epitope is one or more tandem epitopes of a tumor-associated antigen (TAA), each epitope or tandem epitope comprising the tumor-associated antigen (TAA) being composed of no more than XX% of the amino acids of the TAA.

[0215] 15. The vaccine according to item 14, wherein the one or more tandem epitopes are selected from tandem epitopes provided in the sequence listing.

[0216] 16. The vaccine according to item 15, wherein the one or more tandem epitopes are selected from the sequence list as sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, 2 81, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803 The serial table positions provided by 805, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

[0217] 17. The vaccine according to any one of items 13-16, used for the treatment or prophylactic treatment of cancer in a subject with a genotype that promotes non-binding of the specifically selected epitope or tandem epitope to the subject's major histocompatibility complex I (MHC I).

[0218] 18. A library or sequence catalog of cancer vaccine epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), wherein the epitopes or tandem epitopes are specifically selected to activate an immune response via major histocompatibility complex II (MHC II) in human subjects.

[0219] 19. The library or sequence catalog according to item 18, wherein the tabletop is a serial tabletop.

[0220] 20. A library or sequence catalog according to item 19, wherein the library or sequence catalog contains one or more tandem tabletops provided in the sequence list.

[0221] 21. A library or sequence catalog according to Item 20, wherein the library or sequence catalog contains sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, in a sequence list. 281, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412, 4 13, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803, 80 One or more serial table positions provided by 5, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

[0222] 22. The library or sequence catalog according to items 18-21, wherein the epitopes or tandem epitopes are specifically selected to prevent activation of an immune response by major histocompatibility complex I (MHC I) in human subjects.

[0223] 23. A tandem epitope comprising a peptide sequence or a derivative thereof as shown in a sequence listing, wherein the sequence is shortened by 1, 2, 3, 4 or 5 amino acids at the N-terminal portion of the tandem epitope and / or shortened by 1, 2, 3, 4 or 5 amino acids at the C-terminal portion of the tandem epitope.

[0224] 24. A tandem epitope, further comprising an adjuvant sequence.

[0225] Preferably, the cancer to be treated is selected from breast cancer (BC), prostate cancer (PCa), melanoma, colorectal cancer, ovarian cancer, classical Hodgkin lymphoma, glioma, and non-small cell lung cancer.

[0226] Preferably, the tumor-associated antigen is selected from cyclin D1 (CCND1), mucin-1 (MUC1), carcinoembryonic antigen (CEA), human epidermal receptor 2 (HER2 or ERBB2), telomerase reverse transcriptase (TERT), Wilms' tumor gene (WT-1), sialic acid-Tn, Myc proto-oncogene protein (MYC), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate-specific antigen (PSA), and cancer / testis antigen 1 (NY-ESO-1). Carcinoembryonic antigen (CEACAM5) is particularly preferred.

[0227] The present invention may alternatively be defined as follows: 1. A method for generating an active ingredient comprising one or more proteins in a vaccine, said vaccine preventing or treating human cancers that fully or partially express MHC-II (hereinafter referred to as “MHC-II”) on the cell surface, which prevents or treats cancers by: (1) activating the MHC-II portion of the adaptive immune system to detect cancer by maximizing the likelihood that certain epitopes in a first group of epitopes are displayed after injection of said vaccine, and said epitopes, when tandem (without repeating the amino acids constituting said epitopes), are similar to said active ingredient; (2) such that the displaying epitopes of the first group of epitopes are also substrings of antigens generated by said cancer, said epitopes being likely to be displayed on MHC-II; and (3) simultaneously not activating the adaptive immune system by maximizing the likelihood that any portion of said active ingredient will not be displayed on MHC-I when deriving any second group of epitopes (when tandem (without repeating the amino acids constituting said epitopes) constituting said active ingredient). The system's MHC-I portion, thereby minimizing any undesirable side effects in humans and minimizing the possibility of autoimmune diseases resulting from the injection of the vaccine, (4) while maximizing the likelihood that elements in the third group of epitopes (which are neither elements in the first group of epitopes nor the second group of epitopes, and which are not part of the antigen (because the epitopes of the third group of epitopes span connections or cleavages in the antigen, and therefore the epitopes are not present in the antigen)) will not be displayed on MHC-I or MHC-II, (5) based on an algorithm to specify the active ingredient, which takes into account data on the genetic alleles of the human, predicts the immune system behavior of the antigen and any epitope sequences considered to constitute the active ingredient in terms of calculating the binding probability of each epitope of the first group of epitopes to MHC-II and calculating the binding probability of each epitope of the second group of epitopes to MHC-I.

[0228] 2. According to the method described in Project 1, the cancer that fully or partially expresses MHC-II on the cell surface is one of breast cancer (BC), prostate cancer (PCa), melanoma, colorectal cancer, ovarian cancer, classical Hodgkin lymphoma, glioma, and non-small cell lung cancer.

[0229] Project 3. According to the method of Project 1, wherein the antigen, in addition to being produced by the cancer, is also present in some non-cancerous cells of the human body, and the non-cancerous cells, except for antigen-presenting cells (APCs), do not express MHC-II, and therefore the antigen is a tumor-associated antigen.

[0230] Project 4. The method according to Project 1, wherein the active ingredient is a protein, the protein being referred to as a multiepitope.

[0231] Project 5. The method according to Project 1, wherein the length (number of amino acids) of the epitopes in the first group of epitopes is one of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 amino acids, and the length (number of amino acids) of the epitopes in the second group of epitopes is one of 8, 9, 10, 11, 12, 13 and 14 amino acids.

[0232] Project 6. According to the method of Project 1, wherein the algorithm classifies the binding probability of an epitope to MHC-I and to MHC-II into one of the following: (1) non-binding agent, (2) weak binding agent (WB), and strong binding agent (SB), such that “maximizing the probability that an epitope in the first group of epitopes binds to MHC-II” is achieved by selecting the epitope as an SB on MHC-II, and such that “maximizing the probability that an epitope in the second group of epitopes does not bind to MHC-I” is achieved by selecting the epitope as one of (1) non-binding agent and (2) WB on MHC-I, and such that “maximizing the probability that an epitope in the third group of epitopes is not exhibited on either MHC-I or MHC-II” is achieved by selecting the epitope as a non-binding agent on both MHC-I and MHC-II.

[0233] Project 7. The method according to Project 1, wherein the algorithm is implemented as a service in URL MHC-I or a subsequent improved version thereof for use with MHC-I and a service in URL MHC-II or a subsequent improved version thereof for use with MHC-II, modified to take into account the possibility that not all MHC-II may be expressed by the cancer.

[0234] Project 8. According to the method of Project 6, wherein if the result after selecting MHC-II as SB in the first group of epitopes is to select empty (if there is no SB on MHC-II), then “maximizing the probability of epitopes in the first group of epitopes binding with MHC-II” is achieved by selecting said epitopes as WB on MHC-II.

[0235] Project 9. The method according to Project 6, wherein if the result after selecting MHC-I as a SB to bind to the second group of epitopes is that the active ingredient is empty (if no binding agent is left for MHC-II after removing the SB of MHC-I), then it is implemented as not requiring the identification of any second group of epitopes, and then marking this situation (allowing the method to continue without removing epitopes that may bind to MHC-I, because due to T cell selection, there may be a limited number of CD8+ T cells that bind to these epitopes in MHC-I, but with the warning of possible undesirable side effects or autoimmune diseases).

[0236] Item 10. The method according to Item 4, wherein the multi-tablet consists only of consecutive tabletops and the third group of tabletops is empty (therefore there is no "cut" in the multi-tablet).

[0237] Project 11. The method according to Project 3, wherein the tumor-associated antigen is cyclin D1 (genetic code CCND1) and as defined in Serial No. 1 entitled “Cycin D1”, or one or more of the following: cyclin D1 (CCND1), mucin-1 (MUC1), mucin 16, carcinoembryonic antigen (CEA—in the form of CEACAM5, or in the form of CEACAMx, where x is 1, 3, 4, 6, 7, 8, 16, 18, 19, 20 or 21), human epidermal receptor 2 (HER2 or ERBB2), telomerase reverse transcriptase (TERT), Wilmer The following genes are listed: WT-1 (Sialotinic Acid-Tn), Myc proto-oncogene protein (MYC), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate-specific antigen (PSA), cancer / testis antigen 1 (NY-ESO-1), melanoma antigen preferentially expressed in tumors (PRAME), melanoma-associated antigen 1 (MAGEA1), melanoma-associated antigen 3 (MAGEA3), prostate stem cell antigen (PSCA), B lymphocyte antigen CD19 (CD-19), cell tumor antigen p53 (TP-53), and GTPase. KRas (KRAS), keratin type I cytoskeleton 19 (Cyfra 21-1), cellular retinoic acid-binding protein 1 (CRABP1), cellular retinoic acid-binding protein 2 (CRABP2), folate receptor (FOLR1), kallikrein-10 (KLK10), baculovirus IAP repeat protein 5 (BIRC5 or survivin), insulin, insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), epidermal growth factor receptor (EGFR), protein LYRIC (metadherin), tumor protein D52, liver glycoprotein A receptor 2 (EphA2), tyrosinase, and alpha-fetoprotein (AFP).

[0238] Project 12. The method according to Project 1, wherein the vaccine is produced and administered to a human as one of an mRNA vaccine, SAM: self-amplifying mRNA vaccine, LNP mRNA: lipid nanoparticle mRNA vaccine, viral vector vaccine, DC vaccine, and ISV: in situ vaccine.

[0239] Project 13. The method according to Project 1, wherein the vaccine is combined with a treatment that enhances MHC-II, such as “CIITA treatment” (CIITA is a major transcription factor coactivator of MHC-II) or “IFN-γ” treatment.

[0240] Item 14. The method according to Item 1, wherein the active ingredient is combined with other active ingredients in the vaccine.

[0241] Item 15. The method of Item 1, wherein the vaccine is combined with a treatment targeting other parts of T cells, such as checkpoint inhibitory therapy targeting PD-1 or CTLA-4 receptors on T cells.

[0242] Item 16. The method according to Item 1, wherein the vaccine is administered more than once, and the amount of the active ingredient in non-first injections is adaptively calculated based on the following measurements: (a) immune response, such as one or a combination of: (1) expression of epitopes on MHC-II, (2) the number of CD4+ cells produced, and if used for treatment rather than prevention of cancer, (3) the number of tumor cancer cells killed; and (b) side effects caused by the vaccine.

[0243] Project 17. The method according to Project 1, wherein the vaccines are manufactured and approved with different active ingredients, such that there is a mapping method for allele combinations to a finite set of vaccines, each with a different active ingredient, such that the term “maximizing probability” is achieved as a trade-off relative to limiting the number of elements in the finite set of vaccines, such that all vaccines in the finite set can be approved without significantly reducing their effectiveness.

[0244] Item 18. According to the method described in Item 17, the limited vaccine set is a catalog consisting of [a subset of sequences in an XML file] or a combination of them with spacer sequences [as described in the third group of epitopes], wherein the spacer sequences are placed between them, taking into account the patient's MHC-I and MHC-II alleles, and the spacer sequences do not present any epitopes. [We do not mention here that this catalog is derived from (1) the “original” sequence and (2) “shortened” sequences based on the “original” sequence; the next item explains how they are generated automatically.]

[0245] Project 19. According to the method of Project 18, wherein the limited vaccine set portion is constructed by the following computerized generation method: based on (1) the “original sequence” of MHC-II (hotspots) as follows: (A) triggered by at least a threshold number of DP, DQ and DR alleles respectively; (B) the sum of the probabilities of the alleles in the world population is at least a threshold number; (C) [concatenated as long as there are no gaps in the string, using these criteria] and (2) the “shortened sequence” based on the original sequence to increase the probability of avoiding epitopes triggered by one or more MHC-I alleles, as follows: (D) the length of the shortened sequence is at least a threshold number; (E) the probability of triggering MHC-I alleles that have been excised in whole or in part is at least a threshold number, wherein the step in (2) may be repeated if the provisions in (E) and (D) are still valid for the resulting sequence.

[0246] Item 20. The method according to Item 19, wherein the threshold number of (1) is (A) at least 6 for DP, at least 6 for DQ, and at least 4 for DR, (B) the sum of the probabilities of the alleles in the world population is at least 1%, and wherein the threshold number of (2) is (D) the shortened sequence length is at least 19 amino acids, and (E) the probability of triggering the MHC-I allele of the epitope that has been completely or partially excised is at least 10%.

[0247] Item 21. According to the method of Item 20, the sequences in the catalog are merged (meaning linked without repeating amino acids) if they meet the following two criteria: (1) [they almost completely overlap] and (2) they are triggered by almost identical MHC-II and MHC-I alleles.

[0248] Project 22. According to the method of Project 1, wherein the vaccine is administered such that measuring which epitopes in the vaccine are immunogenic (meaning they present and attract T cells) refers to the immunogenic epitopes as validated.

[0249] Item 23. According to the method described in Item 17, the dosage of said vaccine takes into account (1) whether the sequence contains one or more validated epitopes, (2) the number of epitopes that may be presented in the injected vaccine sequence but are not validated, and (3) the number of epitopes that are determined [or highly likely] not to be presented, and (4) personal and clinical parameters and measurements.

[0250] Item 24. The method according to Item 17, wherein the vaccine has a limit on the number of sequences that can be administered as a single vaccine, taking into account physiological limitations calculated based on individual and clinical parameters or measurements.

[0251] Item 25. The method according to Item 17 [and Item 24], wherein the vaccine employs an algorithm to determine the ordering of the finite number of vaccine [sequences], and wherein some elements of the finite number of vaccines are repeated multiple times in the vaccine (possibly repeating "good" ones, such as those containing verified epitopes).

[0252] Item 26. According to the method of Item 1, wherein the active ingredient is extended with an adjuvant at the beginning, end or [point in the connection between epitopes - unlikely case] to promote one or more of the following: (1) the process of delivering the protein into the MHC-II loading compartment (MIIC), (2) avoiding MHC-I contamination in APCs (because it takes up other epitopes besides vaccines), (3) the presentation of epitopes to T cells, (4) the activation process of T cells and other cells of the immune system, thereby making CD4+ T cells cytotoxic, or (5) the effector process of killing cancer.

[0253] Item 27. The method according to Item 14, wherein the other active ingredient is an adjuvant to promote one or more of the following: (1) the process of delivering proteins into the MHC-II loading compartment (MIIC), (2) the avoidance of MHC-I contamination in APCs (as it takes up other epitopes besides vaccines), (3) the presentation of epitopes to T cells, (4) the activation process of T cells and other cells of the immune system, thereby making CD4+ T cells cytotoxic, or (5) the effector process of killing cancer.

[0254] Item 28. The method according to Item 23 (point 1 below), Item 26 (point 2), and Item 27 (point 3), wherein the data relating to (1) a dosage algorithm including the relative weights of validated epitopes, (2) a formula for determining the dosage limit of the vaccine administered, and (3) the sorting and repeating logic (1), (2), and (3) are referred to as “master data”, the master data being re-evaluated and assigned a version reference, and wherein the method is operated on the master data having different versions of the reference, the reference being [explicit]. [Different master data may be approved in different countries or regions.]

Claims

1. A method for producing a cancer vaccine composition for treating or preventing cancer in human subjects, the method comprising the following steps: a. Select one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) such that the selected epitopes bind to the subject's MHC-II, thereby providing a library of epitopes and tandem epitopes that bind to the subject's MHC-II. b. From the library of epitopes and tandem epitopes selected in step a), remove epitopes and tandem epitopes that contain epitopes binding to MHC-I of the subject, thereby providing a library of epitopes and tandem epitopes that bind to MHC-II of the subject but not to MHC-I. c. Optionally, combine one or more tabletops and cascaded tabletops provided in step b) into a multi-tabletop containing one or more selected tabletops and cascaded tabletops. d. Optionally, an mRNA molecule is provided that is capable of promoting the production in vivo of one or more epitopes and tandem epitopes provided in step b) or multiple epitopes provided in step c). e. One or more epitopes and tandem epitopes selected from the library of epitopes and tandem epitopes that bind to the subject’s MHC-II but not MHC-I, provided in step b), or multiple epitopes provided in step c), or information-carrying molecules provided in step d), are combined with suitable pharmaceutically acceptable excipients.

2. The method according to claim 1, wherein the tabletop is a serial tabletop.

3. The method of claim 2, wherein the one or more tandem tabletops are selected from tandem tabletops provided in the sequence list.

4. The method of claim 3, wherein the one or more tandem epitopes are selected from the sequence list as sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280. 281, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803 The serial table positions provided are 805, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

5. The method according to any one of claims 1-4, wherein steps a) and b) are performed by: selecting a suitable TAA, preferably the TAA provided herein, based on allele data of the subject's genes, using published data on peptides and alleles, and analyzing possible MHC-II and MHC-I binding epitopes of the corresponding TAA, the published data predicting immune system behavior against the corresponding TAA and any epitope sequences believed to constitute the corresponding TAA.

6. The method according to claim 5, wherein, The steps of analyzing the possible MHC-II and MHC-I binding epitopes or tandem epitopes of the corresponding TAA are implemented by using appropriate algorithms that can be used to calculate the binding probability of each epitope of the TAA to MHC-II and MHC-I.

7. The method according to claim 6, wherein, The appropriate algorithm is provided for MHC-I at the URL "https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / " or a later improved version thereof, and for MHC-II at the URL "https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-4.3 / " or a later improved version thereof.

8. Use of one or more epitopes or tandem epitopes derived from tumor-associated antigens in the treatment or prophylactic treatment of cancer in a subject, wherein, The epitope or tandem epitope is specifically selected to activate an immune response via the subject's major histocompatibility complex II (MHC-II), and wherein the epitope or tandem epitope is specifically selected to not activate an immune response via the subject's major histocompatibility complex I (MHC-I).

9. Use of one or more epitopes or tandem epitopes derived from tumor-associated antigens (TAAs) in the treatment or prophylactic treatment of cancer in a subject, wherein, The epitope or tandem epitope is specifically selected to activate an immune response via the subject's major histocompatibility complex II (MHC II), wherein the subject is characterized by having a genotype that promotes non-binding of the specifically selected epitope or tandem epitope to the subject's major histocompatibility complex I (MHC I).

10. The use according to claim 8 or 9, wherein the tabletop is a tandem tabletop.

11. The use according to claim 10, wherein the tandem epitope is selected from tandem epitopes provided in the sequence list.

12. The use according to claim 11, wherein the tandem epitopes are selected from the sequence list as sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 280, 2 81, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803 The serial table positions provided by 805, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

13. A cancer vaccine comprising one or more specifically selected epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), wherein the selected epitopes or tandem epitopes activate an immune response via the subject's major histocompatibility complex II (MHCII), wherein the epitopes are characterized by consisting of more than 13 amino acids but less than 25 amino acids, preferably consisting of 15 amino acids.

14. The vaccine of claim 13, wherein the epitope is one or more tandem epitopes of a tumor-associated antigen (TAA), each epitope or tandem epitope consisting of no more than 25% of the amino acids of the TAA.

15. The vaccine of claim 14, wherein the one or more tandem epitopes are selected from tandem epitopes provided in the sequence listing.

16. The vaccine of claim 15, wherein the one or more tandem epitopes are selected from sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, 2 80, 281, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 41 2, 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 80 The serial positions provided by 3, 805, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, and 1607.

17. The vaccine according to any one of claims 13-16, used for the treatment or prophylactic treatment of cancer in a subject with a genotype that promotes non-binding of the specifically selected epitope or tandem epitope to the subject's major histocompatibility complex I (MHC I).

18. A library or sequence catalog of cancer vaccine epitopes or tandem epitopes derived from tumor-associated antigens (TAAs), wherein the epitopes or tandem epitopes are specifically selected to activate an immune response via major histocompatibility complex II (MHCII) in human subjects.

19. The library or sequence catalog according to claim 18, wherein the tabletop is a serial tabletop.

20. The library or sequence catalog of claim 19, wherein the library or sequence catalog comprises one or more tandem tabletops provided in the sequence list.

21. The library or sequence catalog of claim 20, wherein the library or sequence catalog comprises sequences 2, 3, 4, 5, 6, 7, 8, 9, 18, 23, 29, 30, 33, 34, 43, 46, 47, 48, 49, 50, 51, 101, 105, 106, 108, 173, 174, 176, 177, 178, 179, 180, 279, in the sequence list. 280, 281, 282, 283, 284, 301, 316, 318, 319, 320, 321, 342, 350, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 379, 380, 394, 405, 406, 407, 408, 412 413, 421, 422, 424, 425, 426, 427, 428, 429, 430, 431, 432, 436, 437, 438, 439, 440, 469, 495, 498, 499, 500, 580, 581, 582, 620, 621, 622, 623, 624, 625, 626, 627, 629, 631, 803, 8 One or more serial table positions provided by 05, 806, 807, 895, 896, 897, 904, 905, 906, 1468, 1475, 1478, 1495, 1502, 1503, 1504, 1505, 1536, 1551, 1552, 1553, 1554, 1558, 1559, 1560, 1561, 1562, 1575, 1607.

22. The library or sequence catalog according to any one of claims 18-21, wherein the epitope or tandem epitope is specifically selected to prevent activation of an immune response by major histocompatibility complex I (MHC I) in human subjects.

23. A tandem epitope comprising a peptide sequence or a derivative thereof as shown in a sequence listing, wherein the sequence is shortened by 1, 2, 3, 4 or 5 amino acids at the N-terminal portion of the tandem epitope and / or shortened by 1, 2, 3, 4 or 5 amino acids at the C-terminal portion of the tandem epitope.

24. The tandem epitope of claim 23, further comprising an adjuvant sequence.

Images