WO2025227519A1 - Tumor vaccine and use thereof - Google Patents

Abstract

A tumor vaccine for treating esophageal squamous cell carcinoma and other cancers, and a composition thereof. Specifically, provided is a polypeptide, which contains one or more antigen fragments selected from: ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10 and MAGEA11, wherein the one or more antigen fragments are linked via a linker peptide, and the antigen fragments contain one or more antigenic epitope peptides. Furthermore, provided are a linear epitope peptide, a nucleic acid encoding the polypeptide and the linear epitope peptide, and the use of the polypeptide or the linear epitope peptide in the preparation of a drug for preventing or treating cancers such as esophageal squamous cell carcinoma.

Description

A tumor vaccine and its application

priority

This application claims the benefit and priority of PCT International Application No. PCT/CN2024/090619, filed on April 29, 2024, the entire contents of which are incorporated herein by reference.

Technical Field

This disclosure pertains to the field of biotechnology, and in particular relates to tumor vaccines and their applications.

Background Technology

Cancer is a major disease affecting human health and is now the second leading cause of death worldwide. Cancer treatment mainly includes traditional surgical treatment, radiotherapy, chemotherapy, and rapidly developing new treatment methods such as targeted therapy and immunotherapy. Although existing immunotherapies have achieved significant results in the field of cancer treatment, cancer vaccines have unique advantages. For example, cancer vaccines can target intracellular antigens other than tumor-specific surface antigens and may even trigger new tumor-specific T-cell responses. However, the number of clinical trials currently underway for cancer vaccines is limited, and their therapeutic effects and detailed, well-defined mechanisms require further exploration and confirmation by researchers.

Due to the heterogeneity of tumors, screening for broad-spectrum, universal antigens is a major scientific challenge in mRNA vaccine design. The key to tumor vaccine design lies in selecting appropriate tumor antigens and epitopes to improve vaccine safety and immunogenicity, thereby effectively stimulating the body's immune response and generating anti-tumor effects. Solid tumors are highly heterogeneous; theoretically, tumor vaccines designed based on a multi-antigen target strategy can induce a wider range of specific T-cell responses, overcoming immune escape caused by antigen insufficiency or loss, and their clinical efficacy potential is superior to single-target vaccines. mRNA vaccines can encode multiple antigens, and their technological advantages are more fully demonstrated when using multi-epitope tandem design.

Summary of the Invention

This disclosure provides a polypeptide comprising one or more targeting antigen fragments, the antigen fragments comprising one or more antigenic epitope peptides, and the one or more antigen fragments being linked by linker peptides, wherein the antigen is selected from one or more of ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA11.

In some embodiments, the antigen further comprises one or more of TP53.175R/H, TP53.220Y/C, PIK3CA.545E/K, and KRAS.12G/D.

In some embodiments, the antigen fragment comprises an amino acid sequence selected from those shown in SEQ ID NOs: 108-162.

In some embodiments, the polypeptide comprises 20 antigen fragments, each of which is an amino acid sequence selected from SEQ ID NOs: 108-127.

In some embodiments, the polypeptide comprises 20 antigen fragments, each of which is an amino acid sequence selected from SEQ ID NOs: 128-147.

In some embodiments, the polypeptide comprises 15 antigen fragments, each of which is an amino acid sequence selected from SEQ ID NOs: 148-162.

In some embodiments, the antigenic epitope peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-107 or SEQ ID NOs: 163-225.

In some embodiments, the polypeptide comprises:

(1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NOs: 3 or 7;

(2) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 28;

(3) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 12;

(4) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 13;

(5) An antigenic epitope peptide targeting PLAC1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 163;

(ii) The amino acid sequence shown in SEQ ID NO: 164; and

(iii) The amino acid sequence shown in SEQ ID NO: 165;

(6) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 9;

(7) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 or SEQ ID NO: 169;

(8) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 70, SEQ ID NO: 170, SEQ ID NO: SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and

(ii) The amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 176;

(9) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 177, SEQ ID NO: 178, or SEQ ID NO: 179; and

(ii) The amino acid sequence shown in SEQ ID NO: 180;

(10) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 181, SEQ ID NO: 182 or SEQ ID NO: 183; and

(ii) The amino acid sequence shown in SEQ ID NO: 184;

(11) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 185, SEQ ID NO: 186 or SEQ ID NO: 187; and

(ii) The amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188 or SEQ ID NO: 189;

(12) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 14, SEQ ID NO: 190, SEQ ID NO: 191 or SEQ ID NO: 192; and

(ii) The amino acid sequence shown in SEQ ID NO: 193 or SEQ ID NO: 194;

(13) An antigenic epitope peptide targeting TP53.175R/H, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 195; and

(14) An antigenic epitope peptide targeting TP53.220Y/C, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 196.

In some embodiments, the polypeptide comprises:

(1) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NOs: 93, SEQ ID NOs: 101, or SEQ ID NOs: 106; and

(ii) The amino acid sequence shown in SEQ ID NOs: 104;

(2) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 46; and

(ii) The amino acid sequence shown in SEQ ID NO: 50;

(3) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 54 and SEQ ID NO: 55;

(4) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 53;

(5) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 40 and SEQ ID NO: 31;

(6) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 73 or SEQ ID NO: 78; and

(ii) The amino acid sequence shown in SEQ ID NO: 74;

(7) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 59 and SEQ ID NO: 60;

(8) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 197 and SEQ ID NO: 65;

(9) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72;

(10) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 57;

(11) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 198; and

(ii) The amino acid sequence shown in SEQ ID NO: 199, SEQ ID NO: 200 or SEQ ID NO: 201;

(12) An antigenic epitope peptide targeting PIK3CA.545E/K, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 202; and

(13) An antigenic epitope peptide targeting KRAS.12G/D, the antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 203.

In some embodiments, the polypeptide comprises:

(1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38; and

(ii) As shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 34 or SEQ ID NO: 40 amino acid sequence;

(2) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 54; and

(ii) The amino acid sequence shown in SEQ ID NO: 55;

(3) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218 or SEQ ID NO: 219; and

(ii) The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 222;

(4) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 208 or SEQ ID NO: 77;

(ii) The amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 223; and

(iii) The amino acid sequence shown in SEQ ID NO: 209;

(5) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 67;

(ii) The amino acid sequence shown in SEQ ID NO: 210, SEQ ID NO: 211 or SEQ ID NO: 212;

(iii) The amino acid sequence shown in SEQ ID NO: 213; and

(iv) The amino acid sequence shown in SEQ ID NO: 184 or SEQ ID NO: 65;

(6) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 185, SEQ ID NO: 94, SEQ ID NO: 105, SEQ ID NO: 95 or SEQ ID NO: 89;

(ii) The amino acid sequence shown in SEQ ID NO: 81 or SEQ ID NO: 99; and

(iii) The amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 80;

(7) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 70, SEQ ID NO: 19, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and

(ii) The amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176 or SEQ ID NO: 214;

(8) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 58 or SEQ ID NO: 60;

(9) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 43 or SEQ ID NO: 45.

In some embodiments, the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 226, SEQ ID NO: 227 or SEQ ID NO: 228.

In one specific embodiment, the polypeptide of this disclosure comprises 20 antigen fragments, wherein one or more antigen fragments are linked by linker peptides, wherein the antigen fragments are selected from ACTL8, SMC1B, FOXI3, GNGT1, PLAC1, BRDT, MAGEA1, and MAGEA4 as shown in SEQ ID NOs: 108-127. MAGEA6, MAGEA3, MAGEA11, MAGEA10, TP53.175R/H, and TP53.220Y/C, and the antigen fragment contains one or more antigenic epitope peptides, wherein the antigenic epitope peptides are:

(1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NOs: 3 or 7;

(2) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 28;

(3) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 12;

(4) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 13;

(5) An antigenic epitope peptide targeting PLAC1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 163;

(ii) The amino acid sequence shown in SEQ ID NO: 164; and

(iii) The amino acid sequence shown in SEQ ID NO: 165;

(6) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 9;

(7) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 or SEQ ID NO: 169;

(8) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 70, SEQ ID NO: 170, SEQ ID NO: SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and

(ii) The amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 176;

(9) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 177, SEQ ID NO: 178, or SEQ ID NO: 179; and

(ii) The amino acid sequence shown in SEQ ID NO: 180;

(10) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 181, SEQ ID NO: 182 or SEQ ID NO: 183; and

(ii) The amino acid sequence shown in SEQ ID NO: 184;

(11) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 185, SEQ ID NO: 186 or SEQ ID NO: 187; and

(ii) The amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188 or SEQ ID NO: 189;

(12) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 14, SEQ ID NO: 190, SEQ ID NO: 191 or SEQ ID NO: 192; and

(ii) The amino acid sequence shown in SEQ ID NO: 193 or SEQ ID NO: 194;

(13) An antigenic epitope peptide targeting TP53.175R/H, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 195; and

(14) An antigenic epitope peptide targeting TP53.220Y/C, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 196.

In one specific embodiment, the antigen comprises an amino acid sequence as shown in SEQ ID NO: 226.

In one specific embodiment, the polypeptide of this disclosure comprises 20 antigen fragments, wherein one or more antigen fragments are linked by linking peptides, wherein the antigen fragments are selected from SMC1B, BRDT, GNGT1, FOXI3, ACTL8, MAGEA6, MAGEA11, MAGEA3, MAGEA4, MAGEA10, MAGEA1, PIK3CA.545E/K, and KRAS.12G/D as shown in SEQ ID NOs: 128-147, and each antigen fragment comprises one or more antigenic epitope peptides, wherein the antigenic epitope peptides are:

(1) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NOs: 93, SEQ ID NOs: 101, or SEQ ID NOs: 106; and

(ii) The amino acid sequence shown in SEQ ID NOs: 104;

(2) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 46; and

(ii) The amino acid sequence shown in SEQ ID NO: 50;

(3) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 54 and SEQ ID NO: 55;

(4) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 53;

(5) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 40 and SEQ ID NO: 31;

(6) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 73 or SEQ ID NO: 78; and

(ii) The amino acid sequence shown in SEQ ID NO: 74;

(7) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 59 and SEQ ID NO: 60;

(8) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 197 and SEQ ID NO: 65;

(9) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72;

(10) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 57;

(11) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 198; and

(ii) The amino acid sequence shown in SEQ ID NO: 199, SEQ ID NO: 200 or SEQ ID NO: 201;

(12) An antigenic epitope peptide targeting PIK3CA.545E/K, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 202; and

(13) An antigenic epitope peptide targeting KRAS.12G/D, said antigenic epitope peptide comprising the antigenic epitope peptide shown in SEQ ID NO: 203 Amino acid sequence.

In one specific embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 227.

In one specific embodiment, the polypeptide of this disclosure comprises 15 antigen fragments, wherein the one or more antigen fragments are linked by linking peptides, wherein the antigen fragments are selected from ACTL8, GNGT1, MAGEA1, MAGEA6, MAGEA3, SMC1B, MAGEA4, MAGEA11 and BRDT as shown in SEQ ID NOs: 148-162, and each antigen fragment comprises one or more antigenic epitope peptides, wherein the antigenic epitope peptides are:

(1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38; and

(ii) The amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 34 or SEQ ID NO: 40;

(2) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 54; and

(ii) The amino acid sequence shown in SEQ ID NO: 55;

(3) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequences shown in SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218 or SEQ ID NO: 219; and

(ii) The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 222;

(4) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 208 or SEQ ID NO: 77;

(ii) The amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 223; and

(iii) The amino acid sequence shown in SEQ ID NO: 209;

(5) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 67;

(ii) The amino acid sequence shown in SEQ ID NO: 210, SEQ ID NO: 211 or SEQ ID NO: 212;

(iii) The amino acid sequence shown in SEQ ID NO: 213; and

(iv) The amino acid sequence shown in SEQ ID NO: 184 or SEQ ID NO: 65;

(6) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 185, SEQ ID NO: 94, SEQ ID NO: 105, SEQ ID NO: 95 or SEQ ID NO: 89;

(ii) The amino acid sequence shown in SEQ ID NO: 81 or SEQ ID NO: 99; and

(iii) The amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 80;

(7) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises:

(i) The amino acid sequence shown in SEQ ID NO: 70, SEQ ID NO: 19, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and

(ii) SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175, The amino acid sequence shown in SEQ ID NO: 176 or SEQ ID NO: 214;

(8) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 58 or SEQ ID NO: 60;

(9) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 43 or SEQ ID NO: 45.

In one specific embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 228.

In some embodiments, the C-terminus of the peptide further includes the Th cell epitope PADRE and the major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD).

In some embodiments, the N-terminus of the polypeptide contains a signal peptide.

In some embodiments, the linker peptide is a sequence as shown in SEQ ID NO: 235-236.

In some embodiments, the polypeptide comprises an amino acid sequence selected from those shown in SEQ ID NO: 229-231.

In some specific embodiments, the polypeptide is selected from the amino acid sequences shown in SEQ ID NO: 229-231.

On the other hand, this disclosure provides a linear epitope peptide comprising an amino acid sequence selected from the sequences shown in SEQ ID NOs: 1-107 and SEQ ID NOs: 163-225.

In some embodiments, the epitope peptide targets one or more antigens selected from the following: ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10 and/or MAGEA11.

In some embodiments, the linear epitope peptide is selected from one or more of the following:

(1) A linear epitope peptide targeting ACTL8, comprising an amino acid sequence selected from SEQ ID NOs: 1-7 and SEQ ID NOs: 29-41;

(2) A linear epitope peptide targeting BRDT, comprising an amino acid sequence selected from SEQ ID NOs: 8-11 and SEQ ID NOs: 42-52;

(3) A linear epitope peptide targeting FOXI3, comprising an amino acid sequence selected from SEQ ID NOs: 12 and SEQ ID NO: 53;

(4) A linear epitope peptide targeting GNGT1, comprising an amino acid sequence selected from SEQ ID NOs: 13 and SEQ ID NOs: 54-56;

(5) A linear epitope peptide targeting SMC1B, comprising an amino acid sequence selected from SEQ ID NOs: 24-28, SEQ ID NOs: 79-107 and SEQ ID NO: 185;

(6) A linear epitope peptide targeting PLAC1, comprising an amino acid sequence selected from SEQ ID NO: 23 and SEQ ID NOs: 163-165;

(7) A linear epitope peptide targeting MAGEA1, comprising an amino acid sequence selected from SEQ ID NOs: 16-17, SEQ ID NOs: 61-63, SEQ ID NOs: 166-169, SEQ ID NOs: 198-201, and SEQ ID NOs: 204-207;

(8) A linear epitope peptide targeting MAGEA3, comprising an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NOs: 64-68, SEQ ID NOs: 181-184, SEQ ID NO: 197 and SEQ ID NOs: 210-213;

(9) A linear epitope peptide targeting MAGEA4, comprising an amino acid sequence selected from SEQ ID NOs: 19-21, SEQ ID NOs: 69-72, SEQ ID NOs: 170-176, SEQ ID NO: 214 and SEQ ID NOs: 224-225;

(10) A linear epitope peptide targeting MAGEA6, comprising an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NOs: 73-78, SEQ ID NOs: 177-180, SEQ ID NOs: 208-209 and SEQ ID NO: 223;

(11) A linear epitope peptide targeting MAGEA10, comprising a subset of SEQ ID NO: 14, SEQ ID NO: 57 and The amino acid sequence of SEQ ID NOs: 190-194; and

(12) A linear epitope peptide targeting MAGEA11, comprising an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NOs: 58-60 and SEQ ID NOs: 185-189.

In another aspect, this disclosure provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide or a linear epitope peptide as described above.

In another aspect, this disclosure provides an RNA nucleic acid molecule comprising encoding a polypeptide or its open reading frame as described above.

In another aspect, this disclosure provides a tumor-associated antigen vaccine comprising RNA nucleic acid molecules as described above.

In some implementations, the RNA nucleic acid molecule is mRNA.

In another aspect, this disclosure provides a pharmaceutical composition comprising the polypeptide, the linear epitope peptide, the nucleic acid, the RNA nucleic acid molecule, or the tumor-associated antigen vaccine as described above.

Use of the aforementioned polypeptides, the aforementioned linear epitope peptides, the aforementioned nucleic acids, the aforementioned RNA nucleic acid molecules, the aforementioned tumor-associated antigen vaccines, or the aforementioned pharmaceutical compositions in the preparation of medicaments for the treatment and prevention of diseases.

In some implementations, the disease is selected from esophageal squamous cell carcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, colon cancer, and rectal cancer.

Beneficial effects

This disclosure is based on the de novo discovery and screening of tumor antigens using a database. It combines immunomass spectrometry and AI algorithms to screen high-frequency HLA-I antigen sequences. By tandemly combining multiple antigens and adding new functional elements, it can promote the presentation of multiple antigens and T cell responses, and overcome immune escape caused by insufficient or lost single antigens.

Attached Figure Description

This disclosure can be more fully understood with reference to the following figures.

Figure 1 illustrates the screening process for esophageal squamous cell carcinoma antigen targets and epitopes.

Figure 2 shows the results of bioinformatics analysis of esophageal squamous cell carcinoma targets. A shows the differential gene expression analysis; B shows the heatmap of screened genes; and C shows the TPM expression level of screened genes.

Figure 3 shows the analysis of antigen expression rates in various tumors.

Figure 4 shows the statistical graph of the high expression rate of ECVAC target RNA in tumor tissue of esophageal cancer patients, N=132.

Figure 5 shows the HLA-A*02:01 antigen peptide affinity detection results (cELISA).

Figure 6 shows the HLA-A*02:01 antigen peptide affinity test results (cell-loaded peptide).

Figure 7 shows the HLA-A*11:01 antigen peptide affinity detection results (cELISA).

Figure 8 shows the HLA-A*11:01 antigen peptide affinity test results (cell-loaded peptide).

Figure 9 shows a schematic diagram of the ECVAC-A2/11 mRNA design.

Figure 10 shows the results of mRNA migration detection by non-denaturing gel electrophoresis, where M1: marker, Lanes 1-7: ECVAC-A2/11-V1.0, ECVAC-A2/11-V1.1, ECVAC-A2/11-V1.2, ECVAC-A2/11-V1.3, ECVAC-A2/11-V1.4, ECVAC-A2/11-V1.5, ECVAC-A2/11-V1.6, and Lanes 8-14: ECVAC- A2-V1.0, ECVAC-A2-V1.1, ECVAC-A2-V1.2, ECVAC-A2-V1.3, ECVAC-A2-V1.4, ECVAC-A2-V1.5, ECVAC-A2-V1.6, Lane15～20 : ECVAC-A11-V1.0, ECVAC-A11-V1.1, ECVAC-A11-V1.2, ECVAC-A11-V1.3, ECVAC-A11-V1.4, ECVAC-A11-V1.5; M2: marker.

Figure 11 shows the intracellular expression level of mRNA detected by qPCR, where (A) is the intracellular expression level detected by qPCR of mRNA transfected with ECVAC-A2; (B) is the intracellular expression level detected by qPCR of mRNA transfected with ECVAC-A11; and (C) is the intracellular expression level detected by qPCR of mRNA transfected with ECVAC-A2/11.

Figure 12 shows the in vitro translation level of mRNA detected by the Cell-free system, where (A) is the in vitro translation level of ECVAC-A2; (B) is the in vitro translation level of ECVAC-A11; and (C) is the in vitro translation level of ECVAC-A2/11.

Figure 13 shows the frequency of antigen-specific T cells from single HLA epitope vaccines in HLA-A2.1 transgenic mice.

Figure 14 shows the frequency of antigen-specific T cells from single HLA epitope vaccines in HLA-A11.1 transgenic mice.

Figure 15 shows the frequency of vaccine antigen-specific T cells in HLA-A2.1 transgenic mice, where A represents the comparison results between peptide library groups; and B represents the overall comparison results between groups.

Figure 16 shows the frequency of vaccine antigen-specific T cells in HLA-A11.1 transgenic mice, where A represents the comparison results among peptide libraries with different optimized sequences; and B represents the comparison results among groups with different optimized sequences.

Figure 17 shows the bioactivity assay of human PBMCs after in vitro expansion via ECVAC. Figure A shows the flow cytometry results of intracellular cytokines. Appropriate amounts of scanning peptide library and brefeldin A were added to expanded T cells in both the control and experimental groups, and flow cytometry was performed after incubation at 37°C for approximately 16 hours. Figures B and C show the luciferase-based CTL cytotoxicity assay performed using KYSE-410 (B) or KYSE-410-A2.1 (C) transfected luciferin mRNA as target cells and expanded T cells in both groups as effector cells.

Figure 18 shows the killing effect of mouse spleen T cells on tumor target cells after ECVAC immunization. Figures A and B show the killing effect of T cells isolated from the spleen of HLA-A2.1 mice after immunization with PBS or ECVAC (labeled as Vaccine) and co-incubated with (A) MC38-A2.1-HHD or (B) MC38-A2.1-HHD-EC at effector-to-target ratios of 5:1 and 40:1 for 24 hours; Figures C and D show the killing effect of T cells isolated from the spleen of HLA-A11.1 mice after immunization with PBS or ECVAC (labeled as Vaccine) and co-incubated with (C) MC38-A11.1-HHD or (D) MC38-A11.1-HHD-EC at effector-to-target ratios of 10:1 and 40:1 for 24 hours.

Figure 19 shows the therapeutic effects of ECVAC in two transgenic mouse tumor-bearing models. Figure A shows a schematic diagram of the experimental design; Figure B shows the results of the HLA-A2.1 transgenic mouse tumor-bearing model; Figure C shows the results of the HLA-A11.1 transgenic mouse tumor-bearing model.

Figure 20 shows the tumor growth curves of experimental animals after the start of treatment. *P<0.05, **P<0.01.

Detailed Implementation

The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

As used herein, the term "antigen" refers to a molecule that elicits an immune response, which may involve antibody production or activation of specific immune-active cells. Those skilled in the art will understand that any macromolecule, including all proteins or peptides, can be used as an antigen. Antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, encoding what is referred to herein as an "antigen." Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that this disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in different combinations to elicit a desired immune response. The term "peptide" in this application comprises one or more antigen fragments selected from the following, consisting of 10-50, such as 20-50 or 25-30 amino acids: ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, MAGEA11, TP53.175R/H, TP53.220Y/C, PIK3CA.545E/K, and KRAS.12G/D, wherein the one or more antigen fragments are linked by linker peptides, and the antigen fragments contain one or more antigenic epitope peptides. In some embodiments, the antigen fragment comprises an amino acid sequence selected from SEQ ID NOs: 108-162; in some embodiments, the polypeptide comprises 20 antigen fragments, each containing an amino acid sequence selected from SEQ ID NOs: 108-127; in some embodiments, the polypeptide comprises 20 antigen fragments, each containing an amino acid sequence selected from SEQ ID NOs: 128-147; in some embodiments, the polypeptide comprises 15 antigen fragments, each containing an amino acid sequence selected from SEQ ID NOs: 148-162. Furthermore, those skilled in the art will understand that antigens do not necessarily need to be encoded by "genes," and antigens can be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

As used herein, the term "antigen epitope peptide" refers to a polypeptide molecule in an antigen molecule that determines the antigen's specificity. Antigens bind to antigen receptors on the surface of corresponding lymphocytes via epitopes, thereby activating lymphocytes and inducing an immune response; antigens also exert their immune effect by specifically binding to corresponding antibodies or sensitized lymphocytes via epitopes. The size of the antigen epitope is adapted to the antigen-binding site of the corresponding antibody. The specificity of an antigen epitope is determined by all the residues that make it up, but some residues play a greater role than others in antibody binding. The "antigen epitope peptide" of this invention refers to a polypeptide molecule containing 6-13 amino acid residues, such as 8-13 or 8-9 amino acid residues, that targets the antigens ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA11. In some embodiments, the antigenic epitope peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-107 or SEQ ID NOs: 163-225.

Unless otherwise specified, the nucleotide sequence of a nucleic acid molecule or amino acid sequence that “encodes” a protein, as used herein, includes all nucleotide sequences in degenerate form that encode the same amino acid sequence. The nucleotide sequence may also include one or more introns.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, rats, mice, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" and "subject" are used interchangeably. In this disclosure, the preferred subject is a human.

As used herein, the term "treatment" refers to the administration of an effective amount of a polypeptide or vaccine, as described herein, to a subject to a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical outcome. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, the reduction of one or more symptoms, a decrease in disease severity, stabilization of the disease state (i.e., no worsening), or a delay in disease progression. A slowing, improvement, or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. Treatment may refer to an extension of survival compared to the expected survival without treatment. Therefore, those skilled in the art will recognize that treatment can improve the disease state but may not be a complete cure. As used herein, the term "treatment" includes prevention. Alternatively, treatment is "effective" when the progression of the disease is reduced or stopped. "Treatment" may also mean an extension of survival compared to the expected survival without treatment. Patients requiring treatment include those already diagnosed with a condition associated with the expression of a polynucleotide sequence, and those who may develop such a condition due to genetic susceptibility or other factors.

The term "disease" includes breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, liver cancer, stomach cancer, non-small cell lung cancer, squamous cell carcinoma, adrenal cancer, melanoma, and ovarian cancer. Preferably, the disease is selected from esophageal squamous cell carcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, hepatocellular carcinoma, stomach cancer, lung adenocarcinoma, colon cancer and rectal cancer, and oral squamous cell carcinoma.

Example

To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

Example 1: Antigen Target Screening

The screening process for esophageal squamous cell carcinoma antigen targets and epitopes is shown in Figure 1. To screen for tumor antigen targets that match the expression characteristics of the esophageal squamous cell carcinoma (ESCC) population, this publication analyzed RNA-seq data from tumor tissues of 80 ESCC patients (from TCGA) and RNA-seq data from 555 esophageal mucosal tissues (from GTEx), and set the following antigen screening rules:

(1) The selected genes showed significantly upregulated expression in esophageal squamous cell carcinoma compared to normal esophageal mucosa. Differential gene expression analysis between the tumor and normal tissue groups was performed using DESeq2 (R package v1.38.3). The Benjamini-Hochberg method was used to adjust the p-value and reduce the false discovery rate. Differential gene screening criteria: adjusted p-value less than 0.01, and |log2(fold-change)|>1);

(2) The average TPM was less than 0.25 in 50 normal human tissues excluding the testes and uterus;

(3) In the TCGA esophageal squamous cell carcinoma cohort, the number of samples with TPM greater than 1 exceeded 20%.

The bioinformatics analysis results are shown in Figure 2. Through differential gene expression screening, this publication identified 4478 genes upregulated in esophageal squamous cell carcinoma. After excluding genes expressed in normal tissues and ensuring sufficient coverage in tumor patients, 27 genes were selected. Following a functional study of the proteins translated by these 27 genes, 12 candidate antigens deemed to have the best therapeutic potential were selected for the experimental validation phase: ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA11.

Example 2: Expression analysis of candidate antigens in other tumor types

Based on patient RNAseq sequencing data from the TCGA database, bioinformatics analysis was performed on the expression of 12 candidate antigens selected in Example 1 in hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, and rectal cancer. The median TPM (transcripts per million) of the gene in normal tissues from the GTEx database was used as the cutoff value. If the TPM of the gene in a tumor sample was greater than the cutoff value, the patient was considered to express the gene. The number of patients expressing the gene was calculated and then compared with the total number of patients with that type of cancer. The ratio of the numbers is shown in Figure 3. The results indicate that the 12 selected candidate antigens were expressed at different proportions in the different tumor types mentioned above.

Example 3: Coverage validation of candidate antigens in clinical samples

To verify the coverage of the selected antigen in patients, we collected and sequenced samples from 132 tumor tissues and 96 adjacent normal tissues provided by Peking University Cancer Hospital. After the samples passed the testing, 1–3 μg of total RNA from each sample was used as starting material to construct a transcriptome sequencing library. The VAHTS Universal V6 RNA-seq Library Prep Kit was used for this purpose. The (NR604-01/02) instructions specify the selection of different index tags for library construction. For qualified total RNA samples, mRNA with polyA tails is enriched using Oligo(dT) magnetic beads. Fragmentation buffer is then added to break the mRNA into short fragments. Using the mRNA as a template, the first strand of cDNA is synthesized using six-base random hexamers. The RNA template strand is then degraded with RNase H, and the second strand of cDNA is synthesized using dNTPs in a DNA polymerase I system. The double-stranded cDNA is then purified using AMPure P beads or a QiaQuick PCR kit. The purified double-stranded cDNA undergoes end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection is then performed, followed by PCR amplification to obtain the final cDNA library. After the library passed inspection, samples were pooled according to the effective library concentration and the target data volume. Sequencing was performed using the Illumina platform, running the PE150 sequencing strategy to obtain 150bp paired-end reads. The basic principle of sequencing is sequencing by synthesis. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers were added to the flow cell for amplification. During the extension of the complementary strand of each sequencing cluster, each added fluorescently labeled dNTP released corresponding fluorescence. The sequencer captured the fluorescence signal and converted it into sequencing peaks using the bcl2fastq software, thus obtaining the sequence information of the target fragment, which was stored in FASTQ file format. Next, the raw data was filtered using fast-0.22.0 software (https://github.com/OpenGene/fastp) to remove adapter sequences, reads with a high proportion of N (where N indicates undetermined base information), and low-quality sequences, resulting in clean data. The rRNA-removed Clean Data was aligned to the reference genome (GRCh38) using STAR-2.7.10b (https://github.com/alexdobin/STAR/). Next, featureCounts v2.0.3 software was used to quantify gene expression at the sample level. The criterion for high antigen expression was that the total molecular weight (TPM) of the gene in the tumor sample was greater than the mean plus the standard deviation of its TPM in 96 adjacent normal tissue samples. Each patient's tumor tissue carried high expression of at least two antigen targets, nearly 90% of patients carried high expression of at least five antigen targets, and the proportion of patients carrying eight or more antigen targets was as high as 53.79%, as shown in Figure 4.

Example 4: Epitope Screening of Candidate Antigens

HLA-A*02:01 and HLA-A*11:01 are the two most frequently represented HLA subtypes in the world. The HLA-A*02:01 subtype is predominant in European and American populations, while the HLA-A*11:01 subtype is predominant in the Chinese population. Considering the influence of HLA biallelic genes, these two HLA subtypes cover more than half of the total population. Therefore, this disclosure targets these two HLA subtypes using K562 cell lines expressing only HLA-A2.1 or HLA-A11.1 as genetically engineered antigen-presenting cells. Plasmids expressing the aforementioned 12 TAAs were electroporated, and after cell lysis, the HLA-presented peptides were separated by immunoprecipitation and analyzed by mass spectrometry. The experimental procedure is as follows:

1. Cell preparation

K562 cells (purchased from Zhejiang Meisen) were revived and cultured according to the instructions. K562-HLA-A2.1 and K562-HLA-A11.1 cells were prepared by transfecting HLA-A2.1 and HLA-A11.1 genes (method referred to Eichmann, Me et al. Tissue antigens vol. 84, 4(2014):378-88).

2. Electroporation of overexpression plasmids

Based on the information of 12 antigens (Uniprot ID: Q9H568, Q58F21, A8MTJ6, P63211, P43363, P43364, P43355, P43357, P43358, P43360, Q9HBJ0, Q8NDV3), an overexpression vector (synthesized by Nanjing GenScript) was constructed using pcDNA3.1(+) as the backbone. Simultaneously, 2 × ^10⁷ cells from each group were aliquoted into 1.5 mL centrifuge tubes. After centrifugation, the cell pellet was washed twice with PBS. After washing, the supernatant was thoroughly removed before preparing the electroporation system. The electroporation procedure was performed according to the "Neon ^™ 100μL Electroporation Kit Instructions". The electroporated cells were quickly transferred to a T175 flask and incubated statically at 37°C and 5% _CO₂ for 24 hours before being harvested for mass spectrometry sample preparation.

3. Mass spectrometry sample preparation

Mass spectrometry sample preparation was performed according to the experimental protocol (Purcell, Anthony W et al. Nature protocols vol.14,6(2019):1687-1707.). First, K562-HLA cells from each group were transferred to 50 mL centrifuge tubes, centrifuged at 1500 rpm for 10 minutes at room temperature, resuspended in 5 mL PBS, and counted. Based on the count results, the cells were aliquoted into 1.5 mL centrifuge tubes, with 2 × ^10⁷ cells per tube. The cells were collected by centrifugation, and the supernatant was discarded. 1 mL of pre-chilled PBS was added to each tube, and the cells were centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and the washing was repeated once. Then, cell lysis buffer was prepared in advance. 1 mL of cell lysis buffer was added to each cell pellet, and the cell pellet was fully resuspended. The EP tubes were placed on a rotating shaker and lysed at 4 °C for 30 minutes. After lysis, the cells were centrifuged at 4 °C and 12000 rpm for 20 minutes, and the supernatant was collected. Next, add 40 μL of affinity gel to the cell lysate supernatant collected from each tube in the previous step and mix thoroughly. Place the tube on a rotary shaker and incubate at 4°C for 3 hours. After incubation, centrifuge at 4°C and 14,000 rpm for 1 minute, and collect the supernatant. Finally, add 1 mL of ice-chilled washing buffer to the pellet, mix thoroughly, and centrifuge at 4°C and 14,000 rpm for 1 minute. Repeat the washing process 4 times. After the final wash, thoroughly remove the supernatant, add 40 μL of acetic acid elution buffer to the pellet tube, mix thoroughly, incubate at 65°C for 15 minutes, and then allow to return to room temperature. Balance the sample and place it in a centrifuge. Centrifuge at 4°C and 14,000 rpm for 1 minute, and carefully collect all sample supernatant. Transfer the collected supernatant to a new 1.5 mL EP tube for later use.

4. Mass spectrometry detection

The prepared samples were sent to Baizhen Biotechnology Co., Ltd. for mass spectrometry detection. The mass spectrometry detection results obtained in this experiment were statistically analyzed to compare the differences in the number of immune peptides that matched the target protein.

Based on mass spectrometry detection, combined with NetMHCPan 4.1BA prediction, IEDB database and literature search, the epitope library of the above 12 candidate antigens was obtained. Among them, the newly discovered HLA-A2.1 and HLA-A11.1 epitopes are shown in Table 1 and Table 2.

Table 1. Epitopes of antigen HLA-A2.1

Table 2. Epitopes of antigen HLA-A11.1

Example 5: Verification of antigen epitope affinity

To compare the relative affinities of various antigenic peptides in the epitope peptide library, two methods, cELISA and cell-loaded peptide assays, were used to identify the affinity of each antigenic peptide. The cELISA assay procedure is as follows:

Preparation before the experiment: 1) Peptide Flex-T ^™ monomer UVX cannot be repeatedly frozen and thawed. Therefore, for the first use, it should be aliquoted and labeled with the name and aliquoting date and stored at -20℃. For subsequent uses, take the aliquoted tube directly; 2) Take the LEGEND MAX ^™ Flex-T ^™ Human Class IPeptide Exchange ELISA Kit out in advance and let it return to room temperature.

Antigen peptide replacement: Take out the reagents required for the experiment and place them on ice; dilute the peptide to 400 μM with PBS (if DMSO is needed for dissolution, the concentration of DMSO should not exceed 10% (v/v)), and place on ice for later use; take a 96-well plate with V-shaped wells, and add 20 μL of diluted antigen peptide and 20 μL of peptide Flex-T ^™ monomer UVX (200 μg/mL) to the wells with a pipette. The blank control (UV only) group consists of 20 μL of PBS and 20 μL of peptide Flex-T ^™ monomer UVX (200 μg/mL). Mix well by pipetting; seal the plate and centrifuge at 2500g for 2 minutes at 4°C to allow the liquid to settle to the bottom of the wells; remove the cap and place the 96-well plate upright under a UV lamp (366nm) while on ice: irradiate for 30 minutes, with a sample-lamp distance of 2-5 cm. #Biolegend recommends using 8W, 366nm UV light; cap the plate and incubate it at 37°C in the dark for 30 minutes to allow the antigen peptides to fully bind; centrifuge and collect the liquid in the wells for later use. (The liquid contains the prepared pMHC monomer complex, with a concentration of 100 μg/mL per well).

Peptide displacement activity assay: Dilute 20×Wash Buffer to 1×Wash Buffer with _ddH₂O ; dilute the prepared pMHC complex sample to 5 ng/mL with Assay Buffer A, this is the working solution for the monomer complex sample; remove the required reagents and allow them to return to room temperature, then place the required ELISA strips into the ELISA plate; add 50 μL of Assay Buffer A to each well of the ELISA plate, followed by 50 μL of sample solution (the sample concentration after this step is 2.5 ng/mL, with two replicates per sample) or control solution (Assay Buffer A) to the corresponding wells. Seal the ELISA plate with sealing film and shake on a shaker at room temperature for 30 minutes (220 rpm is an option); discard the liquid in the wells and wash the plate four times with 200 μL of 1×Wash Buffer per well, ensuring the liquid is as dry as possible each time, by inverting the plate onto absorbent paper laid flat on the table to absorb the liquid.

After draining the liquid, add 100 μL of avidin-HRP reagent to each well, seal with sealing film, and incubate at room temperature on a shaker for 30 minutes (220 rpm is an option); discard the liquid in the wells, and wash 5 times as described above. After the last wash, add Wash Buffer and let the liquid soak in the wells for 30 seconds to 1 minute to minimize background interference.

After draining the liquid in the final step, add 100 μL of substrate solution F to each well and incubate at room temperature in the dark for 10 minutes; (the liquid color in the experimental wells should turn blue after the substrate is added, and the blue color should gradually deepen as the sample concentration increases; this step can be selectively sealed for incubation); after incubation, add 100 μL of stop solution to each well (the liquid color will change from blue to yellow after the stop solution is added); after stopping the color development, use a microplate reader to detect the absorbance values of the OD450 and OD570 bands within 30 minutes.

Data processing and calculation formulas are as follows:

The method for loading peptides into cells is as follows (refer to patent application number 202410069100.3):

Cell seeding: Take an appropriate amount of K562-HLA-A*11:01-TAP_KO/K562-HLA-A*02:01-TAP_KO- cells, resuspend the cells in 1 ml of serum-free RPMI 1640 medium, and count them. Adjust the cell concentration to 1× ^10⁶ /ml, and seed 100 μl/well in a 48-well plate, i.e., 1× ^10⁵ cells per well. Add β2M protein (final concentration 5 μg/ml) and antigenic peptide (final concentration 60 μM), and incubate overnight in a CO₂ incubator.

Flow cytometry: The following day, cells were collected into flow cytometry tubes, centrifuged at 350×g for 5 minutes, and the supernatant was discarded. Cells were washed twice with 2 ml PBS, centrifuged at 350×g at room temperature for 5 minutes, and the supernatant was discarded. Cells were resuspended in 100 μl PBS. 2 μl of BV510 anti-human HLA-ABC was added to each tube, and the cells were incubated at room temperature in the dark for 20 minutes. Cells were washed once with 2 ml PBS, centrifuged at 350×g at room temperature for 5 minutes, and the supernatant was discarded. Cells were resuspended in 200 μl PBS, and the expression level of HLA-ABC molecules on the surface of K562 cells was detected by flow cytometry.

Data processing: MFI Ratio = (MFI test group - MFI control group) / (MFI positive peptide group - MFI control group)

For the epitopes with relatively high mass spectrometry detection intensity in Example 4, corresponding peptides were synthesized (Nanjing Genscript) and affinity tests were performed. The cELISA results for the HLA-A2.1 epitope peptide are shown in Figure 5, and the results for the cell-loaded peptide are shown in Figure 6; the cELISA results for the HLA-A11.1 epitope peptide are shown in Figure 7, and the results for the cell-loaded peptide are shown in Figure 8. The criteria for determining affinity results based on cELISA and cell-loaded peptide tests are as follows: Strong positive: The ratio of test/positive (cELISA) or MFI ratio (cell-loaded peptide) is greater than 1. Moderate positive: The ratio of test/positive (cELISA) or MFI ratio (cell-loaded peptide) is greater than or equal to the median value between the positive and negative control peptides, and less than 1. Weak positive: The ratio of test/positive (cELISA) or MFI ratio (cell-loaded peptide) is less than the median value between the positive and negative control peptides, greater than the negative control peptide, and less than the negative control peptide is considered negative. Detailed results are shown in Tables 3, 4, 5, and 6.

Table 3. HLA-A2.1 epitope peptide cELISA detection results

Table 4. Results of HLA-A2.1 epitope peptide cell-loaded peptide detection

Table 5. HLA-A11.1 epitope peptide cELISA detection results

Table 6. Results of HLA-A11.1 epitope peptide cell-loaded peptide detection

Example 6: ECVAC Vaccine Design

Three vaccines were designed for different HLA populations based on epitope information of 12 antigens. ECVAC-A2 was designed for the HLA-A*02:01 population, selecting 18 fragments of 25 amino acids each from the aforementioned 12 antigens and combining them with two neoantigen fragments commonly found in cancer patients, TP53.175R/H and TP53.220Y/C (Table 7), to form a tandem sequence of 20 antigen fragments. Each fragment contains at least one HLA-A*02:01 epitope, and some fragments may contain multiple epitopes simultaneously, totaling 50 identified HLA-A*02:01 epitopes (see Table 8 for details). ECVAC-A11 was designed for the HLA-A*11:01 population, selecting 18 fragments of 25 amino acids each from the aforementioned 11 antigens (excluding PLAC1) and combining them with two neoantigen fragments commonly found in cancer patients, PIK3CA.545E/K and KRAS.12G/D, to form a tandem sequence of 20 antigen fragments. Each fragment contains at least one HLA-A*11:01 epitope, totaling 30 identified HLA-A*11:01 epitopes (see Table 9 for details). ECVAC-A2/11 is designed to accommodate both HLA-A*02:01 and HLA-A*11:01 populations, selecting 15 fragments of 30 amino acids each from 9 antigens (excluding PLAC1, FOXI3, and MAGEA10). Each fragment contains at least one HLA-A*02:01 and one HLA-A*11:01 epitope. These 15 antigen fragments contain a total of 46 identified HLA-A*02:01 epitopes and 37 HLA-A*11:01 epitopes (see Tables 10 and 11 for details).

To avoid the formation of new epitopes from the linking of different antigen fragments, a non-immunogenic GS linker was used to link different fragments, followed by the universal Th cell epitope PADRE to ensure high population response rate and sufficient immunogenicity. Simultaneously, an HLA-I signal peptide (SP) and a transmembrane/intracellular MITD (MHC class Itrafficking signal domain) structure were added to the N-terminus and C-terminus, respectively, to enhance the overall antigen presentation efficiency. The final amino acid sequences of each vaccine were obtained, as shown in the sequence listing. For example, the structure of ECVAC-A2/11 is shown in Figure 9.

Table 7. Antigen fragments corresponding to ECVAC-A2, ECVAC-A11, and ECVAC-A2/11

Table 8. HLA-A*02:01 epitope of ECVAC-A2

Table 9. HLA-A*11:01 epitope of ECVAC-A11

Table 10. HLA-A*02:01 epitope of ECVAC-A2/11

Table 11. HLA-A*11:01 epitope of ECVAC-A2/11

Table 12. Antigen polypeptide sequences

Table 13. Amino acid sequences

Example 7: mRNA sequence optimization

Sequence optimization of the CDS region of vaccine mRNA can improve its translation efficiency, while structural optimization can increase its half-life. The general principle of CDS sequence optimization is to utilize codon degeneracy to find stable and high-yield mRNA sequences, thereby increasing the expression levels of functional proteins. The codon fitness index (CAI) refers to the degree of similarity between the frequency of codons in a foreign mRNA sequence and the optimal codon usage frequency in the host cell. Replacing codons in the foreign mRNA sequence with synonyms that are frequently used in the host cell ensures a better match between the codon usage bias in the foreign mRNA sequence and the host cell, significantly improving mRNA translation efficiency. Furthermore, GC-rich mRNAs can be transcribed or processed more efficiently, resulting in more stable mRNA. Therefore, when selecting codons, it is also necessary to maximize the GC content to further improve mRNA stability and translation efficiency. In addition, CDS optimization also needs to consider the stability of the mRNA secondary structure, reducing its minimum folding free energy (MFE) and increasing the half-life of the mRNA in vivo, aiming to express more functional proteins. Of course, the standards for processing different functional regions are also different. For example, the fewer secondary structures formed by the first ten codons of the mRNA CDS region, the higher the expression level of the encoded protein; the more secondary structures formed by the remaining CDS region, the higher the expression level of the encoded protein.

Therefore, this disclosure optimizes the sequences of ECVAC-A2, ECVAC-A11 and ECVAC-A2/11 according to the above principles. Taking into account the influence of various factors, a total of 6-7 sequences for each vaccine were selected for experimental verification. The sequences are shown in Table 14.

Table 14. Optimized Nucleic Acid Sequences

Example 8: In vitro screening of optimized mRNA sequences

To identify three more functional ECVAC mRNA sequences targeting different HLA versions, optimizations were made based on GC content, CAI, and MFE. Six-seven sequences were designed for ECVAC-A2, ECVAC-A11, and ECVAC-A2/11, respectively. Nine mRNA sequences were selected. In this embodiment, the optimal sequences were screened from multiple dimensions based on experimental results such as mRNA electrophoretic mobility, mRNA integrity, mRNA intracellular degradation capacity, and mRNA in vitro protein translation level. Three optimal sequences from each version were selected, totaling nine mRNAs, for subsequent immunogenicity verification in HLA transgene (Tg) mice.

The mRNA sequences of ECVAC-A2, ECVAC-A11, and ECVAC-A2/11 are shown in Table 14. The mRNA production was carried out in accordance with the instructions of T7 High Yield RNA Transcription Kit (Novizan, TR101-02).

1. Detection of mRNA migration by non-denaturing gel electrophoresis

Electrophoretic migration assays were performed on the prepared mRNAs using 1% non-denaturing agarose gel. Theoretically, mRNA molecules with lower MFE (Minimum Free Energy) values contain a more compact shape and smaller kinetic dimensions. Since more stable mRNA secondary structures migrate faster electrophoretically, this experiment can be used to screen and validate different mRNA sequences designed based on theoretical MFE values. The results are shown in Figure 10: the migration rates of each mRNA version differ to some extent, all showing a correlation with their theoretical MFE values. Statistical analysis of the electrophoretic migration results shows that: among the 7 mRNAs in the ECVAC-A2/11 group, ECVAC-A2/11-V1.2 migrated the fastest; among the 7 mRNAs in the ECVAC-A2 group, ECVAC-A2-V1.1 migrated relatively quickly; and among the 6 mRNAs in the ECVAC-A11 group, ECVAC-A2-V1.2 migrated relatively quickly, consistent with theoretical values. In summary, the non-denaturing gel electrophoresis results provide a preliminary indication of relatively stable mRNA versions.

2. qPCR detection of intracellular mRNA degradation

Intracellular mRNA expression level (mRNA half-life) was used as another indicator for screening mRNA stability. K562 cells (purchased from Zhejiang Meisen) were selected as experimental cells and transfected with 6-7 mRNAs of each of the three types: A2, A11, and A2/11. K562 cell samples after electroporation were collected at different time points of 0, 3, 6, 12, 24, and 48 hours. The amplification primers were set as UTR-F3 (GTTCCAGACACCTCCCAAGC) and UTR-R3 (TGTGGCTGGCACGAAATTGA). RNA was extracted and qPCR was performed. The qPCR results were analyzed. The intracellular degradation of different versions of mRNA within 48 hours was compared with 2 ^{- ΔCT} as the ordinate and transfection time as the abscissa. As shown in Figure 11, within a certain time range, different versions of mRNA exhibited different degradation patterns within the cell. By comparing the Half-life values obtained from the three groups of mRNAs ECVAC-A2/11, ECVAC-A2, and ECVAC-A11, the three mRNA versions with longer half-lives can be identified as: ECVAC-A2/11 (V1.2, V1.3, V1.4); ECVAC-A2 (V1.2, V1.3, V1.6); and ECVAC-A11 (V1.2, V1.3, V1.5). These can serve as reference indicators for determining the optimal sequence in the future.

3. Cell-free system for detecting mRNA translation levels

ECVAC mRNA consists of a series of tandem antigenic epitopes, lacking corresponding detection antibodies, making it impossible to detect using traditional cell transfection methods via Western blotting. The application of a cell-free reaction system (Rabbit Reticulocyte Lysate System, Promega, L4960) enables the translation of mRNA into proteins in vitro without a cell. This experiment utilized rabbit reticulocytes and an optimized biotin-conjugated tRNA (Transcend ^™ Non-Radioactive Translation Detection System, Promega, L5061) labeled with lysine residues for direct in vitro detection of the synthesized protein, avoiding the limitations imposed by the lack of antibodies. Therefore, this system was used in subsequent experiments. However, this system also has certain limitations: it is a rabbit-derived translation system, while ECVAC mRNA uses a humanized codon-optimized sequence, potentially leading to lower translation efficiency; theoretically, the detection intensity is related to the proportion of lysine residues in the sequence, but since the lysine content in the ECVAC mRNA sequence is essentially the same, this method was chosen for mRNA translation efficiency detection after comprehensive consideration. Therefore, to compare the in vitro translation levels of different versions of mRNA, a cell-free formulation was prepared. The mRNA was translated in vitro, and the protein expression level was detected by Western blotting. The results are shown in Figure 12: ECVAC-A2 (V1.0, V1.3, V1.5, V1.6), ECVAC-A11 (V1.0, V1.3, V1.5), and ECVAC-A2/11 (V1.1, V1.2, V1.4, V1.5, V1.6) were all detected to be expressed in vitro. After quantitative analysis of the obtained bands, the three versions with higher mRNA expression levels were identified as: ECVAC-A2 (V1.0, V1.3, V1.5), ECVAC-A11 (V1.0, V1.3, V1.5), and ECVAC-A2/11 (V1.2, V1.4, V1.5).

4. Determination of ECVAC Optimization Sequence

Through optimization of GC content, CAI, and MFE, certain functional differences were observed in the different versions of ECVAC mRNA. The statistical results are shown in Table 15 below. This experiment focused on screening mRNA stability from three aspects: intracellular degradation level, in vitro expression level, and migration rate. The version with the best stability was selected from each group of mRNAs. Specifically, the following optimal mRNA sequences (ECVAC-A2-1.0, ECVAC-A2-1.3, ECVAC-A2-1.5, ECVAC-A2/11-1.2, ECVAC-A2/11-1.4, ECVAC-A2/11-1.5, ECVAC-A11-1.0, ECVAC-A11-1.3, ECVAC-A11-1.5) were determined from the ECVAC-A2, ECVAC-A2/11, and ECVAC-A11 groups of mRNAs, respectively, as the subjects for subsequent in vivo immunogenicity verification in Tg mice.

Table 15. Statistics on intracellular half-life, in vitro expression level and migration rate of mRNA

Example 9: In vivo screening of optimized mRNA sequences

In vivo experiments in animals can better simulate the actual effects of drugs in clinical practice. Therefore, this disclosure further compares the immunogenicity of three optimized sequences selected from the ECVAC-A2, ECVAC-A11 and ECVAC-A2/11 groups in vitro in HLA transgenic mice to determine the one with the strongest immunogenicity as the final drug candidate molecule.

This experiment used two types of HLA transgenic mice, HLA-A2.1 and HLA-A11.1 (purchased from Biocytogen, catalog numbers 110110 and 112803), with 5 mice in each group. Mice were immunized intramuscularly with the selected mRNA-LNP vaccine, administered once every 7 days for a total of 3 doses. Three days after the third immunization, the spleens of the mice were harvested and the ELISPOT method was used to screen for drug candidate molecules with the best immunogenicity.

mRNA LNP preparation: The preparation equipment used was a microfluidic nanoparticle preparation instrument (Ignite, PNI). The preparation parameters were in accordance with the equipment manual. The lipids (LNPs) used were the same as those used in the COVID-19 vaccine BNT162b2 produced by Pfizer/BioNtech, namely ALC-0315, ALC-0159, DSPC and cholesterol.

Dosing regimen

In the two different HLA transgenic animals, the group design principle was consistent. G1 was the solvent control; G2, G3, and G4 were tumor vaccines with a single HLA epitope of HLA-A2.1 or HLA-A11.1; and G5, G6, and G7 were tumor vaccines with dual HLA epitopes of HLA-A2.1 and HLA-A11.1. Specific dosing regimens are shown in Tables 16 and 17.

Table 16. HLA-A2.1 Animal Dosing Regimen

Note: i.m.: intramuscular injection, injection site is bilateral thigh muscles; the test sample is prepared at a concentration of 40 μg/mL for administration.

Table 17. HLA-A11.1 Animal Dosing Regimen

Note: IM: Intramuscular injection, the injection site is the muscles of both thighs; the test sample is prepared at a concentration of 40 μg/mL for administration.

In ELISPOT, two peptide libraries of different lengths are used to stimulate T cells: an epitope (short) peptide library and a scan (long) peptide library. The epitope peptides selected are the most likely epitope peptides to be presented by HLA for each antigen fragment of ECVAC-A2, ECVAC-A11, and ECVAC-A2/11. Each library consists of five epitope peptides (P1-P4). The long peptide library consists of 15-amino acid scan peptides, each covering the entire sequence of an antigen fragment with a 10-amino acid overlap between adjacent scan peptides. Each library consists of five scan peptides for each antigen fragment (LP1-LP4). The scan peptide library avoids missing immune responses other than those for the selected epitopes during detection.

1. ECVAC-A2 optimized sequence in vivo screening

Table 18 summarizes the frequencies of antigen-specific T cells from the HLA-A2.1 mouse single HLA epitope vaccine. Data were analyzed using IBM SPSS Statistics 25.0 software. One-way ANOVA was used to compare the mean values between groups for different peptide libraries. Two-way ANOVA was used with Graphpad Prism 8 software for overall comparisons between groups. A p-value < 0.05 was considered statistically significant. Detailed statistical analysis results are shown in Table 19. Figure 13 illustrates the data presentation.

Table 18. Summary of frequencies of antigen-specific T cells for HLA-A2.1 single HLA epitope vaccine in mice (Mean ± SD)

Unit: SFU/2*10^5 cells

Table 19. Summary of p-values for statistical analysis of HLA-A2.1 mouse single HLA epitope vaccine antigen-specific T cell frequencies

Note: In the p-values, p ^_a represents the comparison with group G2, and p ^_b represents the comparison with group G3; ^* , p<0.05; ^** , p<0.01; ^*** , p<0.001.

The experimental results showed that the antigenic epitopes in peptide library No. 4 (including short peptides and/or long peptides) contained the most dominant antigenic epitopes produced by HLA-A2.1 transgenic mice stimulated after vaccine immunization. Positive reactions were also detected in peptide libraries No. 1, 2, and 3 (including short peptides and/or long peptides). After stimulation with peptide library No. 1, the frequency of antigen-specific T cells in the spleen cells of G2 group mice was higher than that in G3 and G4 groups. The G2 group was significantly higher than the G4 group after stimulation with P1 and LP1 (p<0.05). Group G2 showed significantly higher levels of antigen-specific T cells than Group G3 after P1 stimulation. Following stimulation with peptide library 4, the frequency of antigen-specific T cells in the spleen cells of Group G3 mice was significantly higher than that in Groups G2 and G4 after both P4 and LP4 stimulation. There were no significant differences among the three groups in stimulation with peptide libraries 2 and 3. Using different peptide libraries as analytical factors, overall inter-group comparisons among the three groups showed that Group G3 (ECVAC-A2-1.3) was significantly superior to Groups G2 and G4, while there was no significant difference between Groups G2 and G4.

2. ECVAC-A11 optimized in vivo sequence screening

Table 20 summarizes the frequencies of antigen-specific T cells from the HLA-A11.1 mouse single HLA epitope vaccine. Data were analyzed using IBM SPSS Statistics 25.0 software. One-way ANOVA was used to compare the mean values between groups of different peptide libraries. Two-way ANOVA was used with Graphpad Prism 8 software for overall comparisons between groups. A p-value < 0.05 was considered statistically significant. Detailed statistical analysis results are shown in Table 21. Figure 14 illustrates the data presentation.

Table 20. Summary of frequencies of antigen-specific T cells for HLA-A11.1 single HLA epitope vaccine in mice (Mean ± SD)

Unit: SFU/2*10^5 cells

Table 21. Summary of p-values for statistical analysis of HLA-A11.1 mouse single HLA epitope vaccine antigen-specific T cell frequencies

Note: In the p-values, p ^_a represents the comparison with group G2, and p ^_b represents the comparison with group G3; ^* , p<0.05; ^** , p<0.01; ^*** , p<0.001.

The experimental results showed that the antigenic epitopes in the second long peptide library contained the most dominant antigenic epitopes produced by HLA-A11.1 transgenic mice stimulated after vaccine immunization. Positive reactions were also detected in the first and third long peptide libraries. The frequencies of reactive T cells in all four short peptide libraries and the fourth long peptide library were relatively low. After stimulation with the LP1 peptide library, the frequency of antigen-specific T cells in the spleen cells of mice in the G3 group was significantly higher than that in the G2 and G4 groups (p<0.05). After stimulation with the P4 and LP4 peptide libraries, the frequency of antigen-specific T cells in the spleen cells of mice in the G2 group was significantly higher than that in the G3 and G4 groups. Using different peptide libraries as analytical factors, the overall inter-group comparisons among the three groups showed that the G3 (ECVAC-A11-1.3) group had the highest mean, but there was no statistically significant difference between the groups.

3. ECVAC-A2/11 optimized sequence in vivo screening

The statistical analysis results of HLA-A2.1 vaccine antigen-specific T cell frequencies are detailed in Table 22, and the data are shown in Figure 15. Data were analyzed using IBM SPSS Statistics 25.0 software. One-way ANOVA was used to compare the mean values between groups of different peptide libraries. Two-way ANOVA was used with Graphpad Prism 8 software for overall comparisons between groups. A p-value < 0.05 was considered statistically significant.

The results showed that the epitopes in peptide library 1 (including short peptide P1 and/or long peptide LP1) contained the most dominant epitopes produced by HLA-A2.1 transgenic mice after vaccine immunization. Positive reactions were also detected in peptide libraries 2 and 3 (including short and/or long peptides), with the weakest positive reaction observed in A2/11-P2. After stimulation with peptide library 1 (including P1 and LP1), the frequency of antigen-specific T cells in the spleen cells of mice in the ECVAC-A2/11-1.2 group was significantly higher than that in the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups (p<0.05). After stimulation with peptide library A2/11-P2, the frequency of antigen-specific T cells in the spleen cells of mice in the ECVAC-A2/11-1.5 group was significantly higher than that in the ECVAC-A2/11-1.4 group. Using different peptide libraries as analytical factors, an overall inter-group comparison was conducted among the three groups. The ECVAC-A2/11-1.2 group was significantly better than the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups, while there was no significant difference between the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups.

Table 22. Summary of p-values for statistical analysis of HLA-A2.1 mouse vaccine antigen-specific T cell frequencies

Note: ^* , p<0.05; ^** , p<0.01; ^*** , p<0.001.

The statistical analysis results of HLA-A11.1 vaccine antigen-specific T cell frequencies are detailed in Table 23, and the data are shown in Figure 16. Data were analyzed using IBM SPSS Statistics 25.0 software. One-way ANOVA was used to compare the mean values between groups of different peptide libraries. Two-way ANOVA was used with Graphpad Prism 8 software for overall comparisons between groups. A p-value < 0.05 was considered statistically significant.

The results showed that positive reactions were detected in peptide libraries No. 4, 5, and 6 (including short and/or long peptides), with the weakest positive reaction observed in A2/11-P5. Due to significant individual variability within the group, the peptide library containing the dominant epitope was not yet clearly defined. After stimulation with the A2/11-LP5 peptide library, the frequency of antigen-specific T cells in the spleen cells of mice in the ECVAC-A2/11-1.4 group was significantly higher than that in the ECVAC-A2/11-1.5 group (p<0.05). Using different peptide libraries as analytical factors, overall inter-group comparisons were performed among the three groups. The ECVAC-A2/11-1.2 group had the highest mean, but there was no statistically significant difference compared to the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups.

Table 23. Summary of p-values for statistical analysis of HLA-A11.1 mouse vaccine antigen-specific T cell frequencies

Note: ^* , p<0.05; ^** , p<0.01; ^*** , p<0.001.

Based on the above animal experiment results, among the three optimized ECVAC-A2/11 vaccine sequences, the ECVAC-A2/11-1.2 group showed the strongest immunogenicity in HLA-A2.1 mice, significantly superior to the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups. In HLA-A11.1 mice, it was slightly stronger than the ECVAC-A2/11-1.4 and ECVAC-A2/11-1.5 groups, but there was no statistically significant difference.

In summary, ECVAC-A2/11-1.2 performed best in both in vitro and in vivo screening studies. Therefore, ECVAC-A2/11-1.2 was selected as the final candidate drug molecule, and will be referred to as ECVAC from now on.

Example 10: In vitro pharmacodynamic study of ECVAC

Because ECVAC requires specific human HLA and immune system responses to function, existing animal models have limitations in evaluating its efficacy. To simulate the biological activity of ECVAC in the human body, this study used in vitro methods to verify the expansion and cytotoxic activity of antigen-specific T cells in human PBMCs under the influence of ECVAC. This study selected three participants (purchased from Miaoshun Biotechnology) with HLA subtypes of HLA-A*02:01 or HLA-A*11:01. CD14+ monocytes (EasySep ^™ Human CD14 Positive Selection Kit II, Stemcell, 17858) and T cells (EasySep ^™ Human TCell Iso Kit, Stemcell, 17951) were obtained through magnetic bead sorting of PBMCs. Following standard procedures (Ali, Muhammad et al. Nature protocols vol. 14, 6(2019): 1926-1943.), the CD14+ monocytes were induced into dendritic cells (DCs) and co-incubated with ECVAC-mRNA-LNP to enable the DCs to express and present the ECVAC antigen. These DCs were then co-cultured with homologous T cells at a specific ratio (between 1:1 and 1:10) to achieve in vitro induction and expansion of antigen-specific T cells. KYSE-410 is a human esophageal squamous cell carcinoma cell line with HLA-A*24:02. To evaluate its HLA-dependent tumor-killing effect, this study overexpressed HLA-A*02:01 and HLA-A*11:01 in the KYSE-410 cell line, abbreviated as KYSE-410-A2.1 and KYSE-410-A11.1, respectively. The expanded antigen-specific T cells were co-incubated with KYSE-410 cells of different HLA types at different effector-target ratios, and the tumor-killing effect of the ECVAC-amplified antigen-specific T cells on tumor cells was detected.

The experimental results are shown in Figure 17. One of the three subjects had HLA-A*02:01 homozygous PBMCs, which showed strong T cell activation signals after in vitro expansion with ECVAC. DCs without antigen treatment served as the control group (DC+T), while DCs co-incubated with ECVAC served as the experimental group (DC+ECVAC-mRNA-LNP). Compared to the control group, the experimental group showed significant upregulation of intracellular IFN-γ, TNF-α, CD107a, and Granzyme-B in CD8+ T cells after overnight stimulation with the ECVAC antigen scanning peptide library (Figure 3A). The IFN-γ positive cell population increased from 0.59% to 4.92%, and TNF-α... The percentage of positive cells increased from 0.19% to 3.12%, the percentage of CD107a positive cells increased from 54.44% to 81.30%, and the percentage of Granzyme-B positive cells increased from 28.46% to 61.04%.

Cell killing results (Figures 17B and C) showed that the antigen-specific T cells expanded by ECVAC had a significant killing effect on KYSE-410-A2.1, which increased with the increase of the effector-to-target ratio. At a high-efficiency effector-to-target ratio of 100:1, the killing rate of tumor cells reached 61.76%. In contrast, the control group without ECVAC showed no killing effect, and neither group of cells killed KYSE-410. This indicates that the killing effect is a specific HLA-restricted and antigen-specific T cell-mediated killing response.

In summary, ECVAC can effectively expand antigen-specific T cells in human PBMCs in vitro and exert a strong anti-tumor effect. In the absence of an ideal animal model, the biological activity of ECVAC in humans was explored at the in vitro level.

Example 11: ECVAC Ex vivo pharmacodynamic study

HLA-A2.1 transgenic mice express the interspecies heterozygous class I MHC gene HHD, which contains alleles of the α-1 and α-2 domains of the human HLA-A2.1 gene and the α-3 transmembrane and cytoplasmic domains of the mouse H-2Db gene. These transgenic mice mimic the T-cell immune response to HLA-A2.1-presented antigens and can be used to study the immunogenicity of HLA-restricted vaccines. HLA-A11.1 transgenic mice are identical to HLA-A2.1 transgenic mice except for their HLA type. MC38 is a mouse colorectal cancer cell line commonly used in studies of the immunogenicity of antitumor drugs. In this study, endogenous MHC was knocked out in MC38 cells, and the HLA-A*02:01/H-2Db or HLA-A*11:01/H-2Db chimeric genes were overexpressed, forming MC38-A2.1-HHD and MC38-A11.1-HHD cell lines. Based on these two cell lines, the antigen fragments in ECVAC were overexpressed to form MC38-A2.1-HHD-EC and MC38-A11.1-HHD-EC cell lines for in vitro killing activity studies.

HLA-A2.1 and HLA-A11.1 transgenic mice (purchased from Biocytogen, catalog numbers 110110 and 112803) were intramuscularly injected with ECVAC LNP three times at 7-day intervals. A control group was also administered the same immunization program, with mice receiving PBS. Mice were sacrificed on day 3 after the last immunization, and spleens were harvested to prepare single-cell suspensions. T cells were then sorted using magnetic beads. The modified MC38 cell lines were transfected with luciferase mRNA to indicate target cells. The sorted T cells were co-incubated with different target cells at varying effector-to-target ratios for 24 hours. Luciferase activity was used to assess the killing effector cells' cytotoxicity against target cells after immunization.

The experimental results are shown in Figure 18. After ECVAC immunization in HLA-A2.1 transgenic mice, T cells in the spleen effectively killed MC38-A2.1-HHD-EC cells, achieving a killing efficiency of 79.65% at an effector-to-target ratio of 40:1, but showing no killing effect on MC38-A2.1-HHD. Similarly, after ECVAC immunization in HLA-A11.1 transgenic mice, T cells in the spleen effectively killed MC38-A11.1-HHD-EC cells, achieving a killing efficiency of 73.28% at an effector-to-target ratio of 10:1 and 93.24% at an effector-to-target ratio of 40:1, but again showing no killing effect on MC38-A11.1-HHD. In both the HLA-A2.1 and HLA-A11.1 mouse PBS groups, T cells showed no killing effect on any of the four target cell types, indicating that this killing effect is an antigen-specific T cell-mediated HLA-dependent killing response.

In summary, both HLA-A2.1 and HLA-A11.1 transgenic mice immunized with ECVAC can produce specific T cells against the ECVAC antigen and exhibit in vitro antitumor activity.

Example 12: In vivo pharmacodynamic study of ECVAC

To evaluate the in vivo efficacy of ECVAC, this study used HLA-A2.1 and HLA-A11.1 transgenic mice (purchased from Biocytogen, catalog numbers 110110 and 112803) as in vivo efficacy models by subcutaneously injecting MC38-A2.1-HHD-EC or MC38-A11.1-HHD-EC tumor cells, respectively, to verify the in vivo tumor-suppressing effect of ECVAC.

On day 4 after tumor implantation, mice were randomly divided into two groups. One group served as the treatment group (Vaccine), receiving intramuscular injections of ECVAC LNP three times at 7-day intervals. The other group received the same immunization procedure but was injected with PBS as the control group. Tumor volume was measured every 3 days after grouping, and the tumor growth inhibition rate (TGITV%) was calculated at the experimental endpoint. TGITV% = 1 - (T/C) × 100%, where T/C = evaluated TV in the treatment group / average TV in the control group (Tumor Volume).

As shown in Figure 19, on day 20 after tumor implantation, the average tumor volume in both HLA-A2.1 and HLA-A11.1 transgenic mice was significantly smaller in the ECVAC vaccine group than in the PBS control group. The TGITV in the ECVAC vaccine group of HLA-A2.1 mice was 79.56% (P = 0.0012), and the TGITV in the ECVAC vaccine group of HLA-A11.1 mice was 83.61% (P = 0.0018).

In summary, ECVAC demonstrated its in vivo tumor-suppressive effect in both HLA-A2.1 and HLA-A11.1 tumor-bearing mouse models, indicating that ECVAC can be used for the treatment of tumors with HLA subtypes HLA-A*02:01 or HLA-A*11:01.

Example 13: In vivo pharmacodynamic study of ECVAC combined with anti-PD-1 antibody

MC38-A2.1-HHD-EC cells were subcutaneously in the lateral flank area of 69 HLA-A2.1 Tg mice (purchased from Biocytogen, catalog number 110110, total number of mice) (the day of inoculation was recorded as D0). Starting on D4, 34 of these animals were randomly immunized with ECVAC LNP (abbreviated as ECVAC), and the remaining 35 animals were immunized with empty vector LNP (abbreviated as LNP). Each animal received 2 μg of ECVAC cells, and the second and third immunizations were administered on D11 and D18, respectively. When the average tumor volume reached approximately 60 ^mm³ (D11), animals immunized with ECVAC and LNP were randomly divided into 6 groups based on tumor volume for treatment (the day of grouping was recorded as PG-D0): LNP + Isotype Ab 1.6 mg/kg (G1), ECVAC + Isotype Ab 1.6 mg/kg (G2), LNP + anti-mPD-1 0.4 mg/kg (G3), LNP + anti-mPD-1 1.6 mg/kg (G4), ECVAC + anti-mPD-1 0.4 mg/kg (G5), and ECVAC + anti-mPD-1 1.6 mg/kg (G6), with 6 animals in each group. Animals in groups G1 to G6 received Isotype Ab or anti-mPD-1 via tail vein, once every 4 days, for a total of 4 doses. Tumor volume and body weight of mice were measured 3 times per week after group administration. The tumor growth inhibition rate (TGITV) of the treatment group was calculated and statistically analyzed.

After administration to the groups, the mice in each group ate and drank normally, with no significant decrease in body weight, no abnormal symptoms, and generally good condition. At the end of the experiment (PG-D19), compared with LNP+Isotype Ab 1.6 mg/kg (G1), the TGI TV (%) of the ECVAC+Isotype Ab 1.6 mg/kg (G2), LNP+anti-mPD-1 0.4 mg/kg (G3), LNP+anti-mPD-1 1.6 mg/kg (G4), ECVAC+anti-mPD-1 0.4 mg/kg (G5), and _ECVAC +anti-mPD-1 1.6 mg/kg (G6) groups were 55%, 55%, 60%, 68%, and 75%, respectively. Statistical analysis showed that at PG-D19, except for LNP+anti-mPD-1 0.4 mg/kg (G3), the tumor volume of all other treatment groups was significantly lower than that of the control group (P<0.05). The combination therapy group showed better tumor-suppressing effects than the single-drug group, suggesting that ECVAC combined with anti-mPD-1 has a better tumor-suppressing effect than ECVAC or anti-mPD-1 alone. Tumor growth curves are shown in Figure 20, and the summary of tumor growth inhibition effects is shown in Table 24.

Table 24. Antitumor effect of test substances

Note: a. Mean ± standard error; b. Comparison with group G1; c. Comparison of G5 with group G3, and G6 with group G4; d. Comparison of G6 and G5 with group G2.

In summary, in the MC38-A2.1-HHD-ECVAC tumor-bearing model, both the single-drug and combination therapy groups showed significant tumor-suppressive effects; the combination of ECVAC and anti-mPD-1 showed better tumor-suppressive effects compared to ECVAC or anti-mPD-1 alone. Furthermore, the tumor-bearing animals exhibited good tolerance to all tested drugs.

By incorporating via reference

The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

Equivalence

This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims (19)

A polypeptide comprising one or more targeting antigen fragments, said antigen fragments comprising one or more antigenic epitope peptides, and said one or more antigen fragments being linked by linker peptides, wherein said antigen is selected from one or more of ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10, and MAGEA11; preferably, said antigen further comprises one or more of TP53.175R/H, TP53.220Y/C, PIK3CA.545E/K, and KRAS.12G/D. The polypeptide of claim 1, wherein the antigen fragment comprises an amino acid sequence selected from SEQ ID NOs: 108-162. The polypeptide of claim 1, wherein the polypeptide comprises: 1) 20 antigen fragments, wherein the antigen fragments are selected from the amino acid sequences shown in SEQ ID NOs: 108-127; 2) 20 antigen fragments, wherein the antigen fragments are selected from the amino acid sequences shown in SEQ ID NOs: 128-147; or 3) 15 antigen fragments, wherein the antigen fragments are selected from the amino acid sequences shown in SEQ ID NOs: 148-162. The polypeptide of claim 1, wherein the antigenic epitope peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-107 or SEQ ID NOs: 163-225. The polypeptide of claim 1, comprising: (1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NOs: 3 or 7; (2) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 28; (3) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 12; (4) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 13; (5) An antigenic epitope peptide targeting PLAC1, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 163; (ii) The amino acid sequence shown in SEQ ID NO: 164; and (iii) The amino acid sequence shown in SEQ ID NO: 165; (6) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 9; (7) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 or SEQ ID NO: 169; (8) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises: (i) The amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 70, SEQ ID NO: 170, SEQ ID NO: SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and (ii) The amino acid sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 176; (9) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 177, SEQ ID NO: 178, or SEQ ID NO: 179; and (ii) The amino acid sequence shown in SEQ ID NO: 180; (10) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 181, SEQ ID NO: 182 or SEQ ID NO: 183; and (ii) The amino acid sequence shown in SEQ ID NO: 184; (11) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 185, SEQ ID NO: 186 or SEQ ID NO: 187; and (ii) The amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188 or SEQ ID NO: 189; (12) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 14, SEQ ID NO: 190, SEQ ID NO: 191 or SEQ ID NO: 192; and (ii) The amino acid sequence shown in SEQ ID NO: 193 or SEQ ID NO: 194; (13) An antigenic epitope peptide targeting TP53.175R/H, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 195; and (14) An antigenic epitope peptide targeting TP53.220Y/C, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 196. The polypeptide of claim 1, comprising: (1) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NOs: 93, SEQ ID NOs: 101, or SEQ ID NOs: 106; and (ii) The amino acid sequence shown in SEQ ID NOs: 104; (2) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 42 or SEQ ID NO: 46; and (ii) The amino acid sequence shown in SEQ ID NO: 50; (3) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 54 and SEQ ID NO: 55; (4) An antigenic epitope peptide targeting FOXI3, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 53; (5) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 40 and SEQ ID NO: 31; (6) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 73 or SEQ ID NO: 78; and (ii) The amino acid sequence shown in SEQ ID NO: 74; (7) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 59 and SEQ ID NO: 60; (8) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 197 and SEQ ID NO: 65; (9) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises the amino acid sequences shown in SEQ ID NO: 71 and SEQ ID NO: 72; (10) An antigenic epitope peptide targeting MAGEA10, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 57; (11) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 198; and (ii) The amino acid sequence shown in SEQ ID NO: 199, SEQ ID NO: 200 or SEQ ID NO: 201; (12) An antigenic epitope peptide targeting PIK3CA.545E/K, said antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 202; and (13) An antigenic epitope peptide targeting KRAS.12G/D, the antigenic epitope peptide comprising the amino acid sequence shown in SEQ ID NO: 203. The polypeptide of claim 1, comprising: (1) An antigenic epitope peptide targeting ACTL8, wherein the antigenic epitope peptide comprises: (i) The amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 38; and (ii) The amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 34 or SEQ ID NO: 40; (2) An antigenic epitope peptide targeting GNGT1, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 54; and (ii) The amino acid sequence shown in SEQ ID NO: 55; (3) An antigenic epitope peptide targeting MAGEA1, wherein the antigenic epitope peptide comprises: (i) The amino acid sequences shown in SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218 or SEQ ID NO: 219; and (ii) The amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 63, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 220, SEQ ID NO: 221 or SEQ ID NO: 222; (4) An antigenic epitope peptide targeting MAGEA6, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 208 or SEQ ID NO: 77; (ii) The amino acid sequence shown in SEQ ID NO: 180 or SEQ ID NO: 223; and (iii) The amino acid sequence shown in SEQ ID NO: 209; (5) An antigenic epitope peptide targeting MAGEA3, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 67; (ii) The amino acid sequence shown in SEQ ID NO: 210, SEQ ID NO: 211 or SEQ ID NO: 212; (iii) The amino acid sequence shown in SEQ ID NO: 213; and (iv) The amino acid sequence shown in SEQ ID NO: 184 or SEQ ID NO: 65; (6) An antigenic epitope peptide targeting SMC1B, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 185, SEQ ID NO: 94, SEQ ID NO: 105, SEQ ID NO: 95 or SEQ ID NO: 89; (ii) The amino acid sequence shown in SEQ ID NO: 81 or SEQ ID NO: 99; and (iii) The amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 80; (7) An antigenic epitope peptide targeting MAGEA4, wherein the antigenic epitope peptide comprises: (i) The amino acid sequence shown in SEQ ID NO: 70, SEQ ID NO: 19, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173; and (ii) The amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176 or SEQ ID NO: 214; (8) An antigenic epitope peptide targeting MAGEA11, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 58 or SEQ ID NO: 60; (9) An antigenic epitope peptide targeting BRDT, wherein the antigenic epitope peptide comprises the amino acid sequence shown in SEQ ID NO: 11, SEQ ID NO: 43 or SEQ ID NO: 45. The polypeptide according to any one of claims 1 to 7, comprising an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 226-228. The polypeptide of claim 1, wherein the C-terminus of the polypeptide further comprises the Th cell epitope PADRE and the major histocompatibility complex (MHC) class I transmembrane and transport domain (MITD). The polypeptide of claim 1, wherein the N-terminus of the polypeptide comprises a signal peptide. The polypeptide of claim 1, wherein the linker peptide has the sequence shown in SEQ ID NO: 235-236. The polypeptide according to any one of claims 1 to 11, wherein the polypeptide is selected from the amino acid sequences shown in SEQ ID NO: 229-231. A linear epitope peptide comprising at least one amino acid sequence selected from SEQ ID NOs: 1-107 and SEQ ID NOs: 163-225; preferably, the epitope peptide targets one or more antigens selected from the following: ACTL8, BRDT, FOXI3, GNGT1, SMC1B, PLAC1, MAGEA1, MAGEA3, MAGEA4, MAGEA6, MAGEA10 and/or MAGEA11. The linear epitope peptide of claim 13, wherein the linear epitope peptide is selected from one or more of the following: (1) A linear epitope peptide targeting ACTL8, comprising an amino acid sequence selected from SEQ ID NOs: 1-7 and SEQ ID NOs: 29-41; (2) A linear epitope peptide targeting BRDT, comprising an amino acid sequence selected from SEQ ID NOs: 8-11 and SEQ ID NOs: 42-52; (3) A linear epitope peptide targeting FOXI3, comprising an amino acid sequence selected from SEQ ID NOs: 12 and SEQ ID NO: 53; (4) A linear epitope peptide targeting GNGT1, comprising an amino acid sequence selected from SEQ ID NOs: 13 and SEQ ID NOs: 54-56; (5) Linear epitope peptides targeting SMC1B, comprising those selected from SEQ ID NOs: 24-28, SEQ ID NOs: 79- The amino acid sequences of SEQ ID NO: 107 and SEQ ID NO: 185; (6) A linear epitope peptide targeting PLAC1, comprising an amino acid sequence selected from SEQ ID NO: 23 and SEQ ID NOs: 163-165; (7) A linear epitope peptide targeting MAGEA1, comprising an amino acid sequence selected from SEQ ID NOs: 16-17, SEQ ID NOs: 61-63, SEQ ID NOs: 166-169, SEQ ID NOs: 198-201, and SEQ ID NOs: 204-207; (8) A linear epitope peptide targeting MAGEA3, comprising an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NOs: 64-68, SEQ ID NOs: 181-184, SEQ ID NO: 197 and SEQ ID NOs: 210-213; (9) A linear epitope peptide targeting MAGEA4, comprising an amino acid sequence selected from SEQ ID NOs: 19-21, SEQ ID NOs: 69-72, SEQ ID NOs: 170-176, SEQ ID NO: 214 and SEQ ID NOs: 224-225; (10) A linear epitope peptide targeting MAGEA6, comprising an amino acid sequence selected from SEQ ID NO: 22, SEQ ID NOs: 73-78, SEQ ID NOs: 177-180, SEQ ID NOs: 208-209 and SEQ ID NO: 223; (11) A linear epitope peptide targeting MAGEA10, comprising an amino acid sequence selected from SEQ ID NO: 14, SEQ ID NO: 57 and SEQ ID NOs: 190-194; and (12) A linear epitope peptide targeting MAGEA11, comprising an amino acid sequence selected from SEQ ID NO: 15, SEQ ID NOs: 58-60 and SEQ ID NOs: 185-189. A nucleic acid comprising a nucleotide sequence encoding a polypeptide as described in any one of claims 1-12 or a linear epitope peptide as described in any one of claims 13-14. An RNA nucleic acid molecule comprising an open reading frame encoding a polypeptide as described in any one of claims 1-12; preferably, the RNA nucleic acid molecule comprises a nucleic acid sequence selected from those shown in SEQ ID NOs: 237-256. A tumor-associated antigen vaccine comprising an RNA nucleic acid molecule as described in claim 16; preferably, the RNA nucleic acid molecule is mRNA; preferably, the vaccine is prepared using lipid nanoparticles (LNPs). A pharmaceutical composition comprising a polypeptide as described in any one of claims 1-12, a linear epitope peptide as described in any one of claims 13-14, a nucleic acid as described in claim 15, an RNA nucleic acid molecule as described in claim 16, or a tumor-associated antigen vaccine as described in claim 17. The use of the polypeptide of any one of claims 1-12, the linear epitope peptide of any one of claims 13-14, the nucleic acid of claim 15, the RNA nucleic acid molecule of claim 16, the tumor-associated antigen vaccine of claim 17, or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating and preventing diseases in a subject; preferably, the disease is selected from esophageal squamous cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, colon cancer, and rectal cancer; preferably, the subject is preferably a human.