Spacer peptide and use thereof

Abstract

The present application relates to a spacer peptide, which plays the role of spacing different antigens in a vaccine, and can be efficiently cleaved by a specific enzyme in vivo to release antigens, thereby laying a solid foundation for the effective presentation of antigens by antigen-presenting cells. The present application further relates to use of the spacer peptide.

Description

A spacer peptide and its uses

[0001] Cross-reference to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2024116690617, filed on November 21, 2024, entitled "A Spacer Peptide and Its Use Thereof", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more particularly to a spacer peptide and its uses. Background Technology

[0004] Spacer peptides are short peptides in vaccines that act as spacers between different antigens, and they have the following characteristics:

[0005] 1. The range of selectable spacer peptide lengths is relatively wide; based on the length of the spacer peptide, they are divided into large spacer peptides, medium spacer peptides, and small spacer peptides.

[0006] 2. Threonine (T), serine (S), proline (P), glycine (G), glutamic acid (E), aspartic acid (N), lysine (K), glutamine (Q), alanine (A), and arginine (R) are common optional amino acid residues in spacer peptides.

[0007] 3. Generally speaking, proline (P) appears more frequently. Due to the structural characteristics of proline, it can improve the rigidity and structural independence of antigen proteins. Threonine (T), serine (S), and glycine (G) have small chemical groups in their side chains, which helps to improve the flexibility of antigen proteins.

[0008] Multiepitope vaccines, also known as cocktail vaccines, are vaccines that simultaneously carry multiple epitopes associated with the target antigen and helper epitopes. Prepared based on the amino acid sequence of antigenic epitopes, multiepitope vaccines represent a unique vaccine design approach developed in recent years, signifying a new direction in vaccine design and making their research a hot topic in the field of genetic engineering vaccine research. Compared to traditional vaccines, multiepitope vaccines have many advantages: they can be recognized and bound by MHC molecules with diverse genetic backgrounds, resulting in highly efficient presentation; they also have unique advantages in cellular immunity, effectively addressing pathogenic microbial variations and many adverse factors in the immune response.

[0009] Generally, multiepitope vaccines are prepared by tandemly combining multiple DNA sequence fragments encoding antigenic epitopes using recombinant DNA technology and then recombining them into a vector. These include recombinant protein vaccines, nucleic acid vaccines, and live vector vaccines. The simplest method for designing multiepitope vaccines is to tandemly link the different epitopes using spacer sequences. This design allows different epitopes to function relatively independently. Therapeutic tumor vaccines are an important type of multiepitope vaccine, and most currently under development employ a design approach that uses spacer peptides to separate different antigens.

[0010] Therapeutic tumor vaccines, due to their ability to induce highly specific T-cell attacks against tumors, have become a significant breakthrough in the treatment of solid tumors and are increasingly attracting the attention of researchers. Scientists utilize specific gene mutations in patients' tumor cells to screen for a large number of tumor neoantigens not found in normal cells, and then design "personalized neoantigen vaccines" targeting the patient's tumor to stimulate a specific immune response against the neoantigens, enabling the body to specifically recognize and attack cancer cells. Personalized neoantigen vaccines are also considered a new frontier in global cancer immunotherapy, and MIT Technology Review selected them as one of the "Top 10 Breakthrough Technologies" of 2019.

[0011] A clinical report presented at the American Association for Cancer Research (AACR) Annual Meeting held from April 5-10, 2024, showed promising efficacy for BNT122, a personalized neoantigen pancreatic cancer mRNA vaccine jointly developed by BioNTech, a leading global mRNA vaccine developer, and Genentech, a subsidiary of Roche, a global biopharmaceutical giant. Patients receiving this therapy have seen their recurrence-free survival extended to over three years, doubling the results previously published in *Nature*. Another neoantigen tumor mRNA vaccine, mRNA-4157, jointly developed by Moderna and Merck, officially began a large-scale Phase III clinical trial on July 26, 2023, combining mRNA-4157 with Keytruda (pembrolizumab) as adjuvant therapy for resectable high-risk (stage IIB-IV) melanoma patients. This combination therapy has received Breakthrough Therapy Designation (BTD) from the US FDA and PRIME (Prime Medicines Initiative) in Europe, and is expected to be conditionally available as early as 2025.

[0012] As of April 14, 2024, data from the U.S. Clinical Trials Information website (Clinicaltrials.gov) shows that there are more than 90 clinical trials of personalized neoantigen cancer vaccines at different stages worldwide, most of which are targeting refractory and recurrent malignant tumors such as pancreatic cancer, non-small cell lung cancer, and brain cancer.

[0013] Personalized tumor mRNA vaccines typically require efficient expression of a dozen or even dozens of neoantigen fragments in their sequence design. Spacer peptides are generally needed between these neoantigens. These spacer peptides play a role in reducing conjugation immunogenicity and also crucially in maintaining the identity of each individual epitope during intracellular vaccine processing, thus ensuring moderate immunogenicity for each epitope. Multi-epitope vaccines without spacer peptides may lead to the generation of neoantigen epitopes with unknown properties. These issues will decisively affect the vaccine's immunizing efficacy. Therefore, selecting appropriate spacer peptides is critical for the design of personalized tumor mRNA vaccines, namely: 1. avoiding the generation of binding junction peptides with MHC molecules; 2. avoiding proteasome processing to generate binding junction peptides; and 3. being efficiently translated and processed by the proteasome.

[0014] Currently, commonly used spacer peptides include AAY, GGPPG, EAAAK, and REKR. For example, researchers have used REKR to prepare tumor vaccines, linking antigenic peptides to different epitopes with excellent results (Luigi Aurisicchio et al., 2014). However, these spacer peptides have not achieved satisfactory results in the development of personalized tumor mRNA vaccines. For instance, the content of enzymes capable of cleaving these spacer peptides and the optimal enzyme activity conditions vary greatly among different types of host cells; therefore, the effectiveness of cleavage and antigen release is highly unpredictable.

[0015] Therefore, researchers still need to develop suitable spacer peptides that can achieve better antigen presentation effects, specifically designed to address the characteristics of mRNA vaccine transfection into cells. Summary of the Invention

[0016] To address the shortcomings of existing technologies, this disclosure provides a spacer peptide that can effectively enhance the immunogenicity of neoantigen vaccines in vivo. For example, antigenic peptides whose corresponding sequences are translated in dendritic cells (DCs) tend to be preferentially cleaved at the spacer peptide, facilitating better processing and presentation of each epitope peptide.

[0017] In the first aspect, this application relates to a spacer peptide having the amino acid sequence shown in SEQ ID NO.1: ARKV.

[0018] Furthermore, this application relates to a synthetic nucleic acid molecule that encodes the spacer peptide of this application.

[0019] In some implementations, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2: GCTCGGAAGGTG.

[0020] In some implementations, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3: GCUCGGAAGGUG.

[0021] Secondly, the use of the spacer peptide or nucleic acid molecule of this application in the preparation of vaccines is provided.

[0022] In some implementation schemes, vaccines include: microbial vector-based vaccines, viral vector vaccines, cell-based vaccines, nucleic acid vaccines, and peptide-based vaccines.

[0023] In some implementations, the nucleic acid vaccine includes nucleic acid infectious disease vaccines, nucleic acid autoimmune disease vaccines, and nucleic acid cancer vaccines.

[0024] In some implementation schemes, the nucleic acid infectious disease vaccines include: pneumonia vaccines, rotavirus vaccines, varicella vaccines, hand-foot-and-mouth disease vaccines, influenza vaccines, COVID-19 vaccines, rabies virus vaccines, respiratory syncytial virus vaccines, and varicella-zoster virus vaccines.

[0025] In some implementations, the nucleic acid cancer vaccine targets cancers or tumors including, but not limited to, cysts, malignant tumors, metastatic cancers, or any disease or condition characterized by uncontrolled cell growth that would be considered cancerous. The cancer can be primary or metastatic.

[0026] In some implementations, cancer may include, but is not limited to, bile duct cancer, bladder cancer, brain cancer including glioblastoma and neuroblastoma; breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematologic malignancies (including acute lymphoblastic and myeloid leukemia), multiple myeloma, liver cancer, lung cancer, lymphoma (including Hodgkin's disease and lymphocytic lymphoma), neuroblastoma, oral cancer, including squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (including melanoma, basal cell carcinoma, and squamous cell carcinoma), testicular cancer, choriocarcinoma, stromal tumors and germ cell tumors, thyroid cancer (including thyroid adenocarcinoma and medullary carcinoma), and kidney cancer.

[0027] In some implementations, the nucleic acid autoimmune disease vaccine targets autoimmune diseases including: systemic lupus erythematosus (SLE), rheumatoid arthritis, psoriatic arthritis, myasthenia gravis, psoriasis, vitiligo, multiple sclerosis, neuromyelitis optica, Crohn's disease, and lupus nephritis.

[0028] In some specific embodiments, the nucleic acid cancer vaccine comprises: one or more nucleic acids, each having one or more open reading frames encoding 5-130, 20-40, 30-35, or 34 peptide epitopes. The peptide epitopes may be selected from portions of personalized cancer antigens and / or tumor-associated antigens (TAAs), and each of the peptide epitopes may have the same or different lengths.

[0029] In some specific embodiments, the minimum length of any peptide epitope is 8 amino acids; in some embodiments, the maximum length of any peptide epitope is 31 amino acids. In some embodiments, the minimum length of any or all peptide epitopes is 13 amino acids. In some embodiments, the maximum length of any or all peptide epitopes is 35 amino acids. In some embodiments, the length of any or all peptide epitopes is 25 amino acids.

[0030] In some specific implementations, each of the one or more nucleic acids encodes 3-130 peptide epitopes, such as 3-10 peptide epitopes, 5-10 peptide epitopes, 10-20 peptide epitopes, 20-30 peptide epitopes, 30-40 peptide epitopes, 40-50 peptide epitopes, 50-60 peptide epitopes, 60-70 peptide epitopes, 70-80 peptide epitopes, 80-90 peptide epitopes, 90-100 peptide epitopes, 100-110 peptide epitopes, 110-120 peptide epitopes, or 120-130 peptide epitopes.

[0031] In some more specific embodiments, the one or more nucleic acids each encode 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 peptide epitopes.

[0032] In some specific embodiments, each of the peptide epitopes is encoded by a separate open reading frame. In some embodiments, the peptide epitopes are in the form of a multivalent cancer antigen consisting of 3-130 peptide epitopes. In some embodiments, the cancer vaccine composition comprises an mRNA having an open reading frame encoding 15 peptide epitopes.

[0033] In some specific embodiments, one or more of the following conditions are met: a) each peptide epitope contains 8-31 amino acids and contains one or more SNP mutations; and / or b) each peptide epitope contains 8-31 amino acids and contains a mutation in the peptide sequence that causes unique expression; and / or c) at least 30% of the peptide epitopes have the highest affinity for class I MHC molecules from the subject; and / or d) at least 30% of the peptide epitopes have the highest affinity for class II MHC molecules from the subject. Affinity; and / or e) none of the peptide epitopes have the highest affinity for class II MHC molecules from the subject; and / or f) at least 50% of the peptide epitopes have a predicted binding affinity of IC50 < 500 nM for HLA-A, HLA-B, and / or DRB1; and / or g) the ratio of class I MHC molecule peptide epitopes to class II MHC molecule peptide epitopes is at least 1:1, 2:1, 3:1, 4:1, or 5:1; and / or h) no class II MHC molecule peptide epitopes are present.

[0034] In some implementations, the antigen sequence (e.g., peptide epitope) is preferably separated by spacer peptides.

[0035] In some implementations, all antigen sequences, except for the terminal antigen sequence, are arranged within antigen subunits, each subunit consisting of an antigen sequence and a spacer peptide. Because the antigen sequences are separated by the spacer peptide, each antigen is presented to the immune system in an optimal manner, achieving the best immune response.

[0036] In some specific implementations, the nucleic acid vaccine includes RNA vaccines and DNA vaccines.

[0037] In other specific embodiments, the RNA vaccine is preferably an mRNA vaccine, more preferably a multi-epitope mRNA vaccine, more preferably a neoantigen mRNA tumor (cancer) vaccine, and most preferably a DC-based neoantigen mRNA tumor (cancer) vaccine.

[0038] In some implementations, the mRNA vaccine further includes a 5'-UTR. The 5'-UTR refers to the region directly upstream of the start codon of the mRNA that does not encode a protein or peptide.

[0039] In some implementations, the mRNA vaccine further includes a 5' cap. Exemplary 5' caps include Cap0, Cap1, or Cap2.

[0040] In some implementations, the mRNA vaccine further includes a 3'-UTR. The 3'-UTR is the region located at the 3' end of the mRNA, downstream of the stop codon in the protein-coding region, and which is transcribed but not translated into a protein or peptide.

[0041] In some implementations, the mRNA vaccine further includes a polyA tail. A polyA tail refers to a sequence of adenosine residues typically located at the 3' end of an RNA molecule. Exemplary polyA tails include those having at least 20 and at most 500, such as 30, 40, 50, 60, 80, 100, 120, 150, 200, 300, or 400 A nucleotides.

[0042] In some other specific embodiments, the total length of the neoantigen mRNA antitumor vaccine encodes a total protein length of 50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400 amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 amino acids, 700-800 amino acids, 800-900 amino acids, 900-1000 amino acids, 1000-1100 amino acids, or 1100-1200 amino acids.

[0043] In the specific implementation plan, the vaccine is a multi-epitope vaccine.

[0044] In the specific implementation plan, the vaccine is a neoantigen tumor vaccine.

[0045] Thirdly, this application relates to the use of a variety of spacer peptides or nucleic acid molecules encoding them in combination for the preparation of vaccines, wherein the amino acid sequence of at least one of the spacer peptides is shown in SEQ ID NO.1.

[0046] In some embodiments, the spacer peptide may further be selected from one or more of the group consisting of GGPPG (SEQ ID NO.12), EAAAK (SEQ ID NO.13), AAY (SEQ ID NO.11), GGGS (SEQ ID NO.14), KK (SEQ ID NO.15), GGSGGGGSG (SEQ ID NO.16), GGS (SEQ ID NO.17), GS (SEQ ID NO.18), and REKR (SEQ ID NO.19).

[0047] Fourthly, this application relates to a nucleic acid cancer vaccine, wherein the vaccine encodes 3-40 peptide epitopes and spacer peptides between adjacent peptide epitopes, and the amino acid sequence of at least one spacer peptide is shown in SEQ ID NO.1.

[0048] In some embodiments, the spacer peptide may further be selected from one or more of the group consisting of GGPPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GS and REKR.

[0049] In some embodiments, the nucleic acid cancer vaccine comprises an mRNA vaccine encoding a plurality of peptide epitope antigens, wherein the plurality of peptide epitope antigens are arranged such that spacer peptides are disposed between adjacent peptide epitopes, or where spacer peptides are not disposed between some adjacent peptide epitopes, and they are directly linked to each other. The plurality of epitope antigens may include a mixture of class I MHC epitopes and class II MHC epitopes. As a non-limiting example, the plurality of peptide epitope antigens may be polypeptides having the following structure:

[0050] (XSX) 1-10 (YSY) 1-10 (SYSY) 1-10 (SXSX) 1-10 (YS) 1-10 (XS) 1-10 (SY) 1-10 (SX) 0-10 (XSXSX) 1-10 (YSYSY) 1-10 (XSXSXSX) 1-10 (SYSYSY) 1-10 (SXSXSX) 1-10 (YSYSYSY) 1-10 (X) 1-10 (Y) 1-10 (X) 0-10(Y) 0-10 (Y) 1-10 (X) 1-10 (Y) 0-10 (X) 0-10 (Y) 1-10 (X) 1-10 (XY) 1-10 (XS) 1-10 (X) 1-10 (XS) 1-10 (Y) 1-10 (YS) 1-10 (Y) 1-10 (YS) 1-10 (X) 1-10 (XSX) 0- 10 (SYSY) 0-10 (YSY) 0-10 (SXSX) 0-10 ;

[0051] Wherein X is a class I MHC epitope with a length of 5-100 amino acids (e.g., any length described herein, including 8-28 amino acids), Y is a class II MHC epitope with a length of 5-100 amino acids (e.g., any length described herein, including 8-31 amino acids), and S is the spacer peptide described in this application.

[0052] In some implementations, the epitope is a predictive epitope.

[0053] In some implementations, the epitope is a non-predictive epitope, which includes identified or experimentally confirmed T-cell epitopes or B-cell epitopes.

[0054] In some embodiments, the peptide epitopes comprise at least one class I MHC epitope and / or at least one class II MHC epitope. In some embodiments, at least 10% of the peptide epitopes are class I MHC epitopes. In some embodiments, at least 20% of the peptide epitopes are class I MHC epitopes. In some embodiments, at least 30% of the peptide epitopes are class I MHC epitopes. In some embodiments, at least 40% of the peptide epitopes are class I MHC epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 100% of the peptide epitopes are class I MHC epitopes. In some embodiments, none (0%) of the peptide epitopes are class II MHC epitopes. In some embodiments, at least 10% of the peptide epitopes are class II MHC epitopes. In some embodiments, at least 20% of the peptide epitopes are class II MHC epitopes. In some embodiments, at least 30% of the peptide epitopes are class II MHC epitopes. In some embodiments, at least 40% of the peptide epitopes are class II MHC epitopes. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 100% of the peptide epitopes are class II MHC epitopes.

[0055] In some embodiments, the ratio of Class I MHC epitopes to Class II MHC epitopes is selected from the following ratios: approximately 10% : approximately 90%; approximately 20% : approximately 80%; approximately 30% : approximately 70%; approximately 40% : approximately 60%; approximately 50% : approximately 50%; approximately 60% : approximately 40%; approximately 70% : approximately 30%; approximately 80% : approximately 20%; approximately 90% : approximately 10% (Class I MHC epitopes : Class II MHC epitopes). In one embodiment, the ratio of Class I MHC epitopes to Class II MHC epitopes is 1:1. In one embodiment, the ratio of Class I MHC epitopes to Class II MHC epitopes is 2:1. In one embodiment, the ratio of Class I MHC epitopes to Class II MHC epitopes is 3:1. In one embodiment, the ratio of Class I MHC epitopes to Class II MHC epitopes is 4:1.

[0056] In some implementations, the antigens include, but are not limited to: infectious disease virus antigens, tumor antigens, and autoimmune disease antigens.

[0057] In some embodiments, the tumor antigen includes tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). In some embodiments, the tumor-specific antigens (TSAs) include neotumor antigens, also known as tumor neoantigens.

[0058] In some implementations, tumor neoantigens include shared neoantigens and personalized neoantigens. Shared neoantigens can be expressed in different individuals or across different tumor types. Personalized neoantigens, on the other hand, are specifically expressed in a particular patient.

[0059] In some implementations, the universal neoantigen includes: KRAS G12 mutation, KRAS G13 mutation, NY-ESO-1 mutation, and MART1 mutation. Attached Figure Description

[0060] Figure 1: Experimental results of the percentage of NY-Jurkat positive cells.

[0061] Figure 2: Experimental results of GFP-MFI values ​​in NY-Jurkat cells.

[0062] Figure 3: Results of the experiment on the percentage of MART1-Jurkat positive cells.

[0063] Figure 4: Experimental results of GFP-MFI values ​​in MART1-Jurkat cells.

[0064] Figure 5: Experimental results of the percentage of G12V-Jurkat positive cells.

[0065] Figure 6: Experimental results of GFP-MFI values ​​in G12V-Jurkat cells.

[0066] Note: The cells mentioned above are Jurkat cells of different epitope types: NY-ESO-1 (SLLMWITQC), KRAS-G12V (VVVGAVGVGK), and MART1 (ELAGIGILTV). Detailed Implementation

[0067] definition

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, exemplary methods and materials are described.

[0069] The terms “patient,” “subject,” and “individual” are used interchangeably in this document. An individual may include, for example, mammals, primates; such as dogs, cats, pigs, cattle, sheep, goats, horses, rats, monkeys, mice, and humans.

[0070] The terms “spacer,” “linker,” “connector,” and “linking peptide” are used interchangeably herein. A spacer is a linking sequence added between two or more proteins or peptides (e.g., epitopes or vaccine sequences) by linking the proteins or peptides. Preferably, the spacer peptide sequence reduces steric hindrance between the two proteins or peptides, is well translated, and supports or allows epitope processing. Additionally, the spacer peptide should have little or no immunogenic sequence elements. Preferably, the spacer peptide should not generate non-endogenous new epitopes that could produce an undesirable immune response, such as those generated at the boundary between adjacent new epitopes. Therefore, multi-epitope vaccines should preferably contain a spacer peptide sequence that reduces the number of undesirable MHC binding confluences or boundary epitopes. When referred to herein as the spacer peptide or spacer peptide A155, it refers to the spacer peptide with the amino acid sequence shown in SEQ ID NO. 1. When a vaccine involves two or more spacer peptides, the spacer peptide of this application may be used in combination with other spacer peptides, also referred to as additional spacer peptides, further including spacer peptides.

[0071] Other spacer peptides may each contain 3 or more, 6 or more, 9 or more, 10 or more, 15 or more, 20 or more, and preferably up to 50, 45, 40, 35, or 30 amino acids. Preferably, the spacer peptides are rich in glycine and / or serine amino acids. Preferably, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the amino acids in the spacer peptides are glycine and / or serine. In a preferred embodiment, the spacer peptides are substantially composed of glycine and serine amino acids.

[0072] In some embodiments, the other spacer peptide may be selected from one or more of the group consisting of GGPPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GS, and REKR. In another preferred embodiment, the other spacer peptide sequence is REKR.

[0073] The term "neoantigen" refers to some tumor-specific abnormal proteins (generally polypeptides of 8-13 amino acids) produced due to gene mutations that can be recognized by the body's immune cells. These abnormal proteins are called tumor neoantigens.

[0074] The term "universal neoantigen" refers to a neoantigen that can be expressed in different individuals or tumor types.

[0075] The term "personalized neoantigen" refers to tumor-specific antigens, also known as neoantigens present in an individual's tumor that are not expressed or are expressed at low levels in the individual's normal, non-cancerous tissues. This antigen may or may not be present in the tumors of other individuals.

[0076] In some embodiments, this application provides a nucleic acid cancer vaccine comprising one or more nucleic acids, wherein each nucleic acid encodes at least one suitable cancer antigen, such as a personalized antigen specific to a cancer subject. For example, a nucleic acid cancer vaccine may comprise a nucleic acid encoding one or more cancer antigens (referred to as neoepitopes) specific to each subject. Antigens expressed in or by tumor cells are referred to as “tumor-associated antigens.” Specific tumor-associated antigens may or may not be expressed in non-cancerous cells. Many tumor mutations are well known in the art. Tumor-associated antigens that are not expressed or are rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is sufficiently reduced compared to their expression in cancerous cells, and which induce an immune response after vaccination, are referred to as neoepitopes. Neoepitopes generally do not elicit an immune response against healthy tissue and are not masked by protective components of the immune system.

[0077] In some implementations, personalized vaccines based on novel epitopes are desirable because such vaccine formulations maximize specificity against a patient's specific tumor. Mutant-originating novel epitopes can arise from: point mutations, resulting in nonsynonymous mutations of different amino acids in a protein; readthrough mutations, where a stop codon is modified or deleted, resulting in the translation of a longer protein with a novel tumor-specific sequence at the C-terminus; splice site mutations, resulting in the inclusion of introns in mature mRNA and thus unique tumor-specific protein sequences; chromosomal rearrangements, which produce chimeric proteins with tumor-specific sequences at the junction of two proteins (i.e., gene fusion); and frameshift mutations or deletions, resulting in new open reading frames and / or translocations with novel tumor-specific protein sequences.

[0078] Each peptide epitope can be of any reasonable length for an epitope. In some embodiments, the lengths of each peptide epitope are not necessarily equal. In some embodiments, each peptide epitope in the nucleic acid cancer vaccine has a different length. In some embodiments, at least two (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and up to and including all) of the peptide epitopes in the nucleic acid cancer vaccine have different lengths.

[0079] In some embodiments, at least one of the peptide epitopes has a length of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 amino acids. In other embodiments, at least one of the peptide epitopes has a length of 50 or fewer, 45 or fewer, 40 or fewer, 35 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids. In other embodiments, at least one of the peptide epitopes has a length of up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, or up to 10 amino acids.

[0080] In some more specific implementations, nucleic acids or mRNA may be specifically used to transfect antigen-presenting cells and as a tool for presenting antigens, the antigens to be presented corresponding to peptides or proteins expressed by the mRNA; antigen-presenting cells may be used to stimulate T cells, particularly CD4+ and / or CD8+ T cells, in vivo or in vitro.

[0081] In some embodiments, the antigen or its functional fragments include, but are not limited to, tumor neoantigens, tumor-associated antigens, tumor-specific antigens, and universal tumor mutation site antigens.

[0082] In some specific implementations, "peptide or protein" includes peptides and proteins that serve as antigens, i.e., peptides or proteins that elicit an immune response in a subject, which may be prophylactic or therapeutic or partially or completely protective.

[0083] In some specific implementations, RNA, especially mRNA, encoding an antigen such as a tumor-associated antigen, is administered to mammals, particularly if it is desired to treat mammals suffering from diseases involving that antigen. The RNA, especially mRNA, is taken into the mammal's antigen-presenting cells (monocytes, macrophages, dendritic cells, thymic cortical epithelial cells, or other cells). An antigenic translation product of the RNA, especially mRNA, is formed, and this product is displayed on the cell surface for recognition by T cells.

[0084] In some specific embodiments, the antigen is displayed on the cell surface for recognition by CAR-modified T cells targeting the antigen. In one embodiment, the antigen, or a product thereof, generated through optional processing, is displayed on the cell surface in the context of MHC molecules for recognition by T cells via T cell receptors.

[0085] In some specific embodiments, RNA expressing the antigen, particularly mRNA, is introduced into ex vivo antigen-presenting cells (e.g., antigen-presenting cells obtained from a patient), and the antigen-presenting cells, optionally cloned antigen-presenting cells, are transplanted back into the same patient. Transfected cells can be reintroduced into the patient in a sterile manner using any method known in the art, preferably via intravenous, intracavitary, intraperitoneal, or intratumoral administration.

[0086] In some specific embodiments, the methods of this disclosure may involve antigen-presenting cells for expressing RNA, particularly mRNA, encoding an antigen. To this end, the methods of this disclosure may involve introducing RNA, particularly mRNA, encoding an antigen into antigen-presenting cells such as dendritic cells (DC cells). For transfection of antigen-presenting cells (e.g., dendritic cells), pharmaceutical compositions comprising RNA, particularly mRNA, encoding an antigen can be used. Delivery carriers that target RNA, particularly mRNA, to dendritic cells or other antigen-presenting cells can be administered to a patient to complete transfection in vivo.

[0087] This document uses specific embodiments to illustrate the principles and implementation methods of this disclosure. The descriptions of the embodiments above are only for the purpose of helping to understand the methods and central ideas of this disclosure. It should be noted that those skilled in the art can make several improvements and modifications to this disclosure without departing from the principles of this disclosure, and these improvements and modifications also fall under the protection of the claims of this disclosure.

[0088] Example 1: Preparation of DC cells containing mRNA

[0089] 1.1 In vitro synthesis of mRNA

[0090] The in vitro synthesis of mRNA can be obtained using conventional techniques currently available, and the general steps are as follows:

[0091] (1) The antigen amino acid sequence was tandemly linked with a spacer peptide sequence and codon-optimized to construct an mRNA transcription template plasmid. The mRNA used a consistent cap, 5'UTR sequence, 3'UTR sequence, and polyA tail. The template plasmid was transformed into competent E. coli cells, and after overnight culture at 37°C, single clones were picked, and after 16 h of incubation at 37°C and 220 rpm, the plasmid was extracted using an endotoxin-free plasmid extraction kit.

[0092] Table 1: mRNA-related sequences involved in the examples

[0093] (2) The plasmid was added to the SpeI-HF restriction endonuclease reaction system and incubated at 37°C for 30-60 min. The linearized plasmid was purified using a DNA product purification kit to obtain a linearized plasmid precipitate, which was dissolved in sterile water for injection and collected by centrifugation. The concentration, purity, and percentage of the target band of the linearized plasmid were measured.

[0094] (3) Linearized plasmid, ATP, UTP, CTP, GTP, T7 RNA transcriptase, and cap analogue were added to the reaction buffer system in a certain proportion, mixed well, and incubated at 37°C for 2 h. After the reaction, the mixture was incubated with DNase I at 37°C for 15 min to obtain crude mRNA. Lithium chloride was added to a final concentration of 2.5 M, and the mixture was centrifuged at 4°C for 15 min in a stand-up pneumatic centrifuge to obtain mRNA precipitate. The precipitate was washed with 70% ethanol solution and then dissolved in sterile water for injection to obtain mRNA (A155 and control A142).

[0095] 1.2 DC cell culture and maturation promotion

[0096] (1) Immature dendritic cell (iDC) thawing: Remove iDC cells from the liquid nitrogen tank and place them in a 37°C water bath until the cryopreservation solution is completely thawed. Pipette a certain volume of AIM-V medium (Thermo Fisher Scientific) into a centrifuge tube, open the cryopreservation tube cap, aspirate the iDC cell suspension from the cryopreservation tube and add it to the medium, mix well, transfer the cell suspension to a centrifuge tube, cap the centrifuge tube, invert it several times to mix well, and centrifuge at 600×g at room temperature for 10 min.

[0097] (2) Resuspension and counting: Discard the supernatant, resuspend the cell pellet in DC cell culture medium, mix well, and count the cells. Based on the test results, add the appropriate culture medium to adjust the cell density to 1.00E+06 cells / mL.

[0098] (3) Preparation for electroporation: After mixing the cell suspension, dispense it into culture dishes or flasks, label them with the corresponding numbers, and incubate them in a 37.0℃, 5.0% CO2 incubator. After 24h of culture, collect mDC cells (mature dendritic cells), add fresh culture medium, count them, and use them for subsequent electroporation.

[0099] 1.3: Cell cryopreservation via electroporation

[0100] (1) The mRNAs used for electroporation were A155 and control A142. All mRNAs were identical except for the sequence encoding the linker peptide. The electroporation dose of each mRNA group was 100 μg, and the total volume of the electroporation system was 300 μL. The prepared electroporation system was transferred to an electroporation cuvette and electroporated using a cell electroporation instrument.

[0101] (2) After electroporation, dilute the cell suspension in the electroporation cup with culture medium and adjust the cell density to 1.00E+06 cells / mL. Dispense the diluted cell suspension into culture dishes and place them in a 37.0℃, 5.0% CO2 incubator for culture.

[0102] (3) Collect the cells from each group after electroporation and culture for 3 hours, centrifuge at 600×g for 10 min at room temperature. Discard the supernatant, resuspend the cells in CS10 cell cryopreservation solution, aliquot the cell suspension into cryovials and store at -80℃ for later use.

[0103] Example 2: Detection of the antigen presentation ability of DC cells to stimulate Jurkat cells

[0104] Experimental Principle: Jurkat reporter cells are stable Jurkat cell lines that express reporter genes. The reporter gene vector includes a specific T-cell antigen receptor (e.g., NY-ESO-1), a response element, and a GFP reporter gene. When antigen-presenting cells are co-incubated with Jurkat reporter cells, and the antigen-presenting surface presents a specific antigen (e.g., NY-ESO-1), the specific TCR on the Jurkat surface binds to the corresponding antigen, the transcription factor is activated and transported into the cell nucleus, promoting the expression of the downstream GFP reporter gene. The GFP positivity rate and GFP expression level of Jurkat cells are positively correlated with the amount of antigen presented on the dendritic cell surface, thus allowing for precise quantification of the amount of antigen presented on the dendritic cell surface.

[0105] (1) DC cell thawing: Cells frozen from electroporation at -80℃ were placed in a 37℃ water bath until the cryopreservation solution was completely thawed. A certain volume of AIM-V medium was aspirated into a centrifuge tube, the cap of the cryopreservation tube was opened, the iDC cell suspension in the cryopreservation tube was aspirated and added to the medium, mixed well, and the cells were counted;

[0106] (2) Based on the counting results, transfer the required cell suspension to centrifuge tubes and centrifuge at 600×g for 5 min at room temperature. Discard the supernatant, and resuspend the cell pellet in the corresponding volume of 1640 complete culture medium according to the number of experimental groups. Gently pipette to mix and set aside. Add appropriate culture medium to the remaining DCs and adjust the cell density to 1*102. 6 Cells / mL were collected and incubated continuously in a 37.0℃, 5.0% CO2 incubator.

[0107] (3) Jurkat cell preparation: Prepare Jurkat cells containing different epitope types (NY-ESO-1 (SLLMWITQC, SEQ ID NO.8), KRAS-G12V (VVVGAVGVGK, SEQ ID NO.9), and MART1 (ELAGIGILTV, SEQ ID NO.10). Take a certain amount of Jurkat cell suspension of different epitope types according to experimental needs, centrifuge at 1000×rpm for 5 min at room temperature. Discard the supernatant, add 1640 complete culture medium to resuspend the cell pellet, mix well, and count the cells.

[0108] (4) Co-culture plate making: After mixing the prepared DC cells and Jurkat cells separately, take 5*10 of each. 4 Cell suspension was added to a 96-well plate and mixed well; the positive control group was supplemented with 5 x 10⁸ DC cells and 5 x 10⁸ Jurkat cells. 4 In addition, 4 μg of the corresponding epitope antigen peptide should be added to each well; the negative control group should only have 1*10 μg added to each well. 5 The Jurkat cells were then placed in a 37.0°C, 5.0% CO2 incubator for culture.

[0109] (5) Cell collection for flow cytometry: DC cells that have been revived for 0 h (or cultured for a certain period after revival) are co-cultured with Jurkat cells for 20–26 h. Cell samples from each group are collected, centrifuged at 400 × rcf for 5 min at room temperature, and the supernatant is discarded. 1 mL of FACS buffer is added to each group to wash the cell pellet, and the pellet is centrifuged at 400 × rcf for 5 min at room temperature. The supernatant is discarded, and the cell pellet is resuspended in 100 μL of FACS buffer. APC anti-human CD8 and PE anti-human CD69 antibodies are added to each group, and the mixture is incubated at 4 °C for 20 min. After incubation, 1 mL of FACS buffer is added to each group to wash the cells, and the mixture is centrifuged at 400 × rcf for 5 min at room temperature. The supernatant is discarded. 100 μL of FACS buffer is added to each group to resuspend the cell pellet, and the mixture is incubated. The samples are then loaded into a flow cytometer for analysis.

[0110] The specific results are as follows:

[0111] Experimental results (see Figures 1-2 and Table 2) show that, compared to the control group spacer peptide (REKR), the A155 spacer peptide (ARKV) obtained in this application significantly enhances the antigen presentation ability of DC cells, maintaining a sustained high intensity and high level of presentation ability for up to 69 hours. The cleavage capacity of the ARKV spacer peptide is approximately 2-3 times that of the REKR control peptide. Moreover, this ability is not limited to a single target (NY-ESO-I). Experimental results based on other targets (e.g., MART1, KRAS-G12V, etc.) show (see Figures 3-6 and Tables 3-4) that, compared to the control, the A155 spacer peptide obtained in this application also significantly enhances the antigen presentation ability of DC cells and maintains a sustained high level of presentation ability for a relatively long period of time. Therefore, the A155 spacer peptide obtained in this application has achieved unexpected technical effects.

[0112] Table 2: Results of DC antigen presentation capability test (NY-ESO-I)

[0113] Table 3: Results of DC antigen presentation capability assay (MART1)

[0114] Table 4: Results of DC antigen presentation capability test (KRAS-G12V)

Claims

1. A spacer peptide having the amino acid sequence shown in SEQ ID NO.

1.

2. A synthetic nucleic acid molecule encoding the spacer peptide of claim 1, wherein the sequence of the nucleic acid molecule is preferably as shown in SEQ ID NO.

2.

3. Use of the spacer peptide of claim 1 or the nucleic acid molecule of claim 2 in the preparation of a vaccine.

4. The use as described in claim 3, wherein the vaccine includes, but is not limited to: microbial vector-based vaccines, viral vector vaccines, cell-based vaccines, nucleic acid vaccines, and peptide-based vaccines.

5. The use as described in claim 4, wherein the vaccine is a nucleic acid cancer vaccine, preferably an mRNA cancer vaccine, and more preferably a DC-based mRNA cancer vaccine.

6. The use according to any one of claims 4-5, wherein the nucleic acid vaccine encodes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 peptide epitopes.

7. Use of a plurality of spacer peptides or nucleic acid molecules encoding them in combination for the preparation of a vaccine, wherein the amino acid sequence of at least one of the plurality of spacer peptides is as shown in SEQ ID NO.

1.

8. The use of claim 7, wherein the plurality of spacer peptides further comprises one or more spacer peptides selected from the group consisting of GGPPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GS and REKR.

9. Use of the spacer peptide of claim 1 or the nucleic acid molecule of claim 2 in the preparation of antibody-drug conjugates (ADCs).

10. Use of the spacer peptide of claim 1 or the nucleic acid molecule of claim 2 in the preparation of fusion proteins.

11. A nucleic acid cancer vaccine, wherein, The nucleic acid cancer vaccine encodes 3-40 peptide epitopes and spacer peptides located between the peptide epitopes, and the amino acid sequence of at least one of the spacer peptides is as shown in SEQ ID NO.

1.

12. The nucleic acid cancer vaccine of claim 11, wherein the spacer peptide further comprises one or more spacer peptides selected from the group consisting of GGPPG, EAAAK, AAY, GGGS, KK, GGSGGGGSG, GGS, GS and REKR.

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