Method for identifying a shared neoantigen-reactive T cell receptor

Description

FIELD OF THE INVENTION

The present invention relates to the field of cancer immunotherapy. In particular, this invention provides a method for identifying a shared neoantigen-reactive T cell receptor. The invention encompasses a comprehensive workflow that integrates neoantigen identification, TCR prioritization, and functional validation.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of disclosure, a sequence listing in computer-readable form (filename: GENS_005_Seqlisting.xml; 151,525 bytes; created Feb. 3, 2025), which is incorporated by reference in its entirety.

BACKGROUND ART

Cancer immunotherapy has emerged as a promising approach that uses the immune system to target and destroy cancer cells. Among these strategies, therapies based on T-cell receptors (TCRs) have gained significant attention because they can recognize tumor antigens presented by major histocompatibility complex (MHC) molecules. Identifying tumor-specific TCRs and validating their ability to target cancer cells effectively is crucial for advancing personalized cancer treatments.

Neoantigens, generated from somatic mutations found only in tumor cells, are desirable targets for T-cell responses. Unlike tumor-associated antigens, which are also expressed in normal tissues and can cause off-target effects, neoantigens are tumor-specific, reducing the risk of toxicity. This makes them ideal candidates for immunotherapy. As a result, finding and characterizing TCRs that recognize neoantigens has become a key focus in developing next-generation cancer treatments.

Various studies have employed high-throughput DNA and RNA sequencing to detect tumor-specific mutations and predict neoantigen candidates. Computational tools like pVAC-Seq have been widely used to indicate the ability of these neoantigens to bind MHC molecules and trigger immune responses.

According to patent application No. WO 2024197072 A2 refers to a method for identifying a neoantigen-reactive TCR, comprising: (i) obtaining single-cell gene expression profiles from a population of tumor infiltrating lymphocytes (TIL) isolated from a patient sample; (ii) performing bioinformatics analyses on the single cell gene expression data to identify TCR clonotypes of interests; (iii) creating recombinant TCR sequences; (iv) preparing a reporter T cell comprising a TCR expression cassette encoding a TCR sequence reconstructed from paired TCR α and β chain sequences identified from the clonotype of interest in step ii; (v) preparing a tandem minigene (TMG) expression vector; (vi) analyzing the patient sequencing data to identify class I and class IIHLA alleles and preparing HLA expression vectors comprising the class IHLA and class IIHLA allele sequences; (vii) preparing an antigen presenting cell (APC) comprising transecting the TMG expression vector and one or more HLA expression vectors into a cell wherein each transection condition comprises a TMG and one or two HLA types; (viii) co-culturing the reporter T cell in step iii with the APC of step vii; and (ix) identifying a positive reporter activity in the reporter T cell to identify a neoantigen-reactive TCR.

According to patent No. U.S. Ser. No. 11/898,207 B2 refers to a method of isolating paired T cell receptor (TCR) alpha and beta chain sequences or an antigen-binding portion thereof. Also disclosed are methods of automatically identifying the TCR alpha and beta chain V segment sequences and CDR3 sequences of a TCR having antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation. Methods of preparing a population of cells expressing paired TCR alpha and beta chain sequences, or an antigen-binding portion, are also disclosed. Isolated pairs of TCR alpha and beta chain sequences and isolated populations of cells prepared by the methods are also disclosed.

According to patent application No. US 20240052010 A1 refers to a method of obtaining a plurality of T cell receptors (TCRs) specifically recognizing a target tumor antigen peptide, comprising: (a) a first co-culturing step comprising co-culturing a first population of dendritic cells (DCs) loaded with the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; (b) an enrichment step comprising subjecting the first co-culture to an enrichment process to obtain enriched activated T cells; (c) a second co-culturing step comprising co-culturing the enriched activated T cells with a second population of DCs loaded with the target tumor antigen peptide to obtain a population of tumor antigen-specific T cells, wherein at least about 10% of the tumor antigen-specific T cells specifically responds to the target tumor antigen peptide; and (d) a sequencing step, comprising subjecting the tumor antigen-specific T cells to next-generation sequencing to identify a plurality of pairs of genes encoding TCRα and TCRβ, thereby providing the plurality of T cell receptors based on paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefitted from a Multiple Antigen Specific Cell Therapy (MASCT) comprising administering to the individual an effective amount of activated T cells prepared by co-culturing a population of T cells with a population of dendritic cells loaded with a plurality of tumor antigen peptides comprising the target tumor antigen peptide.

The above inventions meet the specific purposes and requirements of a technical solution. However, existing approaches often fail to provide integrated workflows seamlessly combining neoantigen discovery, TCR prioritization, and functional validation within a unified process. Furthermore, many current methods prioritize sequence identification while placing insufficient emphasis on functional testing.

It is necessary to create a more comprehensive workflow that combines neoantigen identification, TCR prioritization, and functional validation.

Therefore, the invention provides isolated and purified T cell receptors (TCRs) with antigenic specificity for a panel of mutated human shared neoantigen (previously identified public neoantigen).

Furthermore, further embodiments of the invention provide a strategy for priming shared neoantigen-specific TCRs, isolating TCRs, and identifying neoantigen-specific TCRs.

Finally, still further embodiments of the invention provide related polypeptides and proteins, along with associated nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions linked to the TCRs described in the invention.

This invention provides solutions to achieve the above goals.

SUMMARY OF THE INVENTION

Accordingly, an objective of the present invention is to provide a method for identifying a shared neoantigen-reactive T cell receptor (TCR), comprising steps performed in the following specific order:

• • (A) collecting and processing sample of a subject with a cancer, comprising:
    • isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood sample from the subject with cancer; and
    • employing next-generation genomic and transcriptomic sequencing on this sample of tumor tissue and white blood cells, and using bioinformatic analysis to extract subject's profile including a plurality of mutation sequences and its corresponding a plurality of wild-type sequences;
  • (B) obtaining a shared neoantigen by filtered the mutation sequences at step (A) based on a collection of 67 off-the-shelf peptides consists of KRAS_p.G13D, KRAS_p.G12V, KRAS_p.G12A, KRAS_p.G12D, KRAS_p.G12C, CDX2_p.V306X, RNF43_p.G659X, TP53_p.R282W, TP53_p.R273H, TP53_p.R248Q, TP53_p.R175H, GNAS_p.R201H, PIK3CA_p.E545K, BRAF_p.V640E, TCF7L2_p.R471C, ATM_p.A2301X, POU2AF1_p.A226V, KRAS_p.G12S, CHD4_p.K73X, TP53_p.E286K, TP53_p.Y220C, TP53_p.C176F, TP53_p.A159P, TP53_p.V157F, CIC_p.T1740M, ELK4_p.S359X, ARID1A_p.K1071X, BARD1_p.K171X, PIK3CA_p.V344G, PIK3CA_p.E542K, AKAP9_p.SE1650-1651 SX, TCF7L2_p.H198X, ATM_p.V60X, BCL9L_p.Q452X, NCOR2_p.P975X, KRAS_p.A146T, BRCA2_p.Q1782X, CDK12_p.R663C, TP53_p.R273C, SMAD4_p.G30X, SMAD4_p.R361H, MTOR_p.S2215F, ATP1A1_p.G98X, ARID1A_p.S764SX, ARID1A_p.G1848X, ASXL1_p.G643X, GNAS_p.R201C, ERG_p.446-447X, AMER1_p.F173X, DCTN1_p.R1173H, PIK3CA_p.R88Q, PIK3CA_p.R357Q, PIK3CA_p.E545A, PIK3CA_p.E970K, FAT4_p.L3V, FBXW7_p.S582L, FBXW7_p.R465H, PDGFRA_p.R151H, APC_p.M1413X, APC_p.KR1462-1463X, IL7R_p.K119X, IL6ST_p.K529X, BRAF_p.D634N, BRAF_p.G509V, EGFR_p.L858R, AKAP9_p.K37X, and UBR5_p.R1331C;
  • (C) synthesizing a long peptide corresponding to a panel of shared neoantigen and its corresponding of wild type peptides;
  • (D) stimulating the PBMCs with the long synthetic peptides to obtain a stimulated PBMC, comprising the following steps:
    • (i) thawing frozen PBMCs in AIM-V media supplemented with 10% fetal bovine serum (FBS) and 1 μg/mL deoxyribonuclease I (DNase I) solution;
    • (ii) allowing 10⁵ PBMCs to rest in 96-round bottom well-plate containing AIM-V media supplemented with 10% FBS, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and 50 μM β-mercaptoethanol overnight before stimulation with synthesized long peptide at a concentration of 5 μM in a humidified incubator at 37° C. with 5% CO₂;
    • (iii) further stimulating PBMCs with 2000 IU/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 IU/mL interleukin-4 (IL-4) for 24 hours;
    • (iv) adding 100 ng/mL LPS and 10 ng/mL IFN-γ to the PBMCs along with the peptides for an additional 12 hours; and
    • (v) restimulating PBMCs by adding 10 ng/mL interleukin-7 (IL-7), 10 ng/mL interleukin-15 (IL-15), and 10 ng/mL interleukin-21 (IL-21) to the PBMCs on the following day, in which a restimulation frequency of 3 days/time and the number of restimulations is three times;
  • (E) screening the stimulated PBMC based on response of T cells is measured by interferon-γ (IFN-γ) secretion to mutant peptides and wild type peptides, which is more than twofold higher compared to its corresponding wild-type peptides;
  • (F) isolating a neoantigen-specific T cell from the screened stimulated PBMC to identify a clonotype-purified cell, comprising steps (a1) to (a7):
    • (a1) determining the viability of the stimulated PBMC using a hemocytometer to ensure viability above 90%, and adjusting the cell concentration to between 700-1,200 cells per microliter to obtain a uniform PBMC suspension;
    • (a2) mixing the uniform PBMC suspension at step (a1) with a reverse transcription (RT) master mix to obtain a cell-master mix solution, then loading the cell-master mix solution onto a microfluidic device configured to partition individual cells into emulsions for unique nucleic acid barcoding, wherein the loading is performed along with barcoded 5′ gel beads and partitioning oil to obtain a single-cell gel bead in emulsion (GEMs); and
    • (a3) performing cell lysis and barcoded reverse transcription of RNA within each the GEMs to obtain a barcoded complementary DNA (cDNA);
    • (a4) producing and validating cDNA of gene expression library and VDJ library, comprising:
      • recovering the barcoded cDNA from the GEMs at step (a4) to obtain a cDNA sample;
      • amplifying the cDNA sample using polymerase chain reaction (PCR) to obtain an amplified cDNA; and
      • assessing the quality of the amplified cDNA using sensitivity-based screening systems to obtain a validated cDNA;
    • (a5) constructing sequencing libraries, comprising:
      • utilizing the validated cDNA at step (a5) to prepare 5′ gene expression libraries;
      • indexing each library with a sample indexing system to obtain an indexed gene expression library; and
      • sequencing the indexed gene expression library on a sequencing platform to generate at least 30,000 read pairs per cell with paired-end reads of 2×300 base pairs;
    • (a6) enriching and sequencing V(D)J regions, and RNA transcriptomic profile comprising:
      • using the libraries generated in step (a6) to amplify full-length variable (V), diversity (D), and joining (J) segments of T cell receptor (TCR) alpha and beta chains using an enrichment system to obtain an enriched TCR product;
      • quantifying the enriched TCR product obtained from the amplification using sensitivity-based quantification systems to produce a quantified enriched TCR product;
      • preparing sequencing libraries using 50 ng of the quantified enriched TCR product to produce a TCR sequencing library; and
      • sequencing the TCR sequencing library on a sequencing platform to generate paired-end reads of 2×300 base pairs with a depth of 5,000 read pairs per cell; and
    • (a7) performing bioinformatics analyses on the single cell gene expression data to identify the clonotype-purified cell, comprising:
      • retaining cells with available clonotype information; and
      • excluding cells with mitochondrial genome-derived reads exceeding 15%, more than 7,000 detected genes, or more than two TRA (T-cell receptor alpha locus) or TRB (T-cell receptor beta locus) sequences to obtain the clonotype-purified cell;
  • (G) identifying a TCR candidate for shared neoantigen by performing steps (b1) to (b5):
    • (b1) isolating CD3+ T cells from both mutant and wild-type groups, wherein each isolated cell includes a number of genes detected values in each cell between 200 and 6000, and a mitochondrial gene expression percentage below 15%;
    • (b2) defining the T cell activation score based on the average expression of 10 genes associated with T cell activation for each T cell, in which the 10 genes associated consisting of interferon gamma (IFNG), interleukin 2 (IL2), tumor necrosis factor (TNF), interleukin-2 receptor alpha (IL2RA), cluster of differentiation 69 (CD69), TNF receptor superfamily member 9 (TNFRSF9), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK), and perforin 1 (PRF1);
    • (b3) normalizing the size of TCR clonotypes stimulated by mutant sequences relative to the corresponding wild-type sequences; wherein, if any TCR clonotype is stimulated only by mutant sequences and is not found in the sample stimulated by the corresponding wild-type sequences, its size is calculated by taking the smallest size of the TCR clonotype stimulated by the wild-type sequences;
    • (b4) calculating a ratio size of each TCR clonotype from group which is stimulated by mutant sequences compared to the corresponding wild-type sequences; and
    • (b5) ranking the clonotypes based on their IFNG expression and T cell activation score at step (b2), and their ratio size at step (b4) to identify the TCR candidate for shared neoantigen;
  • (H) evaluating antigenic specificity of the TCR candidate for shared neoantigen through T cell activation bioassay using Nuclear Factor of Activated T cells (NFAT) system and using PBMCs or jurkat (JKT) del beta/CD8 to identify a shared neoantigen-reactive TCR, comprising the following steps:
    • (c1) co-culturing a) a reporter T cell comprising the TCR candidate for shared neoantigen expression cassette, and b) an antigen presenting cell (APC) that expresses the shared neoantigen sequence and a human leukocyte antigen (HLA) sequence extracted from subject's profile;
    • wherein the reporter T cell is a jurkat del beta cell; and
    • wherein the TCR candidate for shared neoantigen expression cassette comprises a TCR candidate sequence reconstructed from TCR α and β chain sequences;
    • (c2) identifying a positive reporter signal in the reporter T cell to identify the neoantigen-reactive TCR; wherein the shared neoantigen-reactive TCR comprises a sequence selected from the group consisting of SEQ ID NOs:135 to 142.

Another objective of the present invention is to provide a neoantigen-reactive T cell receptor TCR comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142, wherein the shared neoantigen-reactive TCR bind to a shared neoantigen/HLA complex;

• • in which the shared neoantigen comprises TP53_pR273H, and TP53_p.V157F;
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

Yet another objective of the present invention is to provide a method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • wherein the cancer includes lung cancer, and colorectal cancer;
  • wherein the shared neoantigen-reactive TCR bind to a shared neoantigen/HLA complex;
  • wherein the shared neoantigen comprises TP53_p.R273H, and TP53_p.V157F;
    • TP53_p.R273H comprises a sequence selected from the group consisting of SEQ ID NOs:9, 160, 162, 164, and 166;
    • TP53_p.V157F comprises a sequence selected from the group consisting of SEQ ID NOs:24, 168, 170, 172, and 174;
  • wherein the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

These and other advantages of the present invention will no doubt become obvious to those of ordinary skill in the art after having read the following detailed description of the preferred embodiments, which are illustrated in the various drawing Figures.

BRIEF DESCRIPTION OF THE DRAWING

The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

FIG. 1 is a flowchart illustrating the principle of a method for identifying the shared neoantigen-reactive TCR in accordance with an exemplary embodiment of the present invention;

FIG. 2 is a flowchart illustrating the principle of a method for isolating the neoantigen-specific T cell from the screened stimulated PBMC to identify the clonotype-purified cell in accordance with an exemplary embodiment of the present invention;

FIG. 3 is a flowchart illustrating the principle of a method for identifying the TCR candidates for shared neoantigen in accordance with an exemplary embodiment of the present invention;

FIG. 4A is a graph showing the fold change of spots for mutant peptides compared to the corresponding wild-type peptides in patients with IDs ZNC34, ZNC35, ZNC39, ZNC41, ZNC42, ZNC46, ZNC47, ZNL49, ZNL50, and ZNL64;

FIG. 4B is an image of spots from PBMCs of patients with IDs ZNC35 and ZNL49, which are determined to be immunogenic peptides;

FIG. 5 is an image showing the results of cDNA length was measured using the Tapestation for the Gene Expression library;

FIG. 6 is Tapestation for the VDJ library;

FIG. 7 is an image showing single-cell TCR and gene expression sequencing of the experimental scheme for neoantigen-specific TCR from Patient ID ZNC35;

FIG. 8A is an image showing the quality control (QC) results for the gene expression library following sequencing. Metrics such as library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data from Patient ID ZNC35;

FIG. 8B is an image showing the quality control (QC) results for the VDJ library following sequencing. Metrics such as library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data from Patient ID ZNC35;

FIG. 9 is an image showing single-cell TCR and gene expression sequencing of the experimental scheme for neoantigen-specific TCR from Patient ID ZNL49;

FIG. 10A is an image showing the quality control (QC) results for the gene expression library following sequencing. Metrics such as library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data from Patient ID ZNL49;

FIG. 10B is an image showing the quality control (QC) results for the VDJ library following sequencing. Metrics such as library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data from Patient ID ZNL49;

FIG. 11A is an image showing quality control of selected clonotypes for further analysis of patient ID ZNC35;

FIG. 11B is an image showing quality control of selected clonotypes for further analysis of patient ID ZNL49;

FIG. 12 is an image showing UMAP of clusters of T cell subsets of patient ID ZNC35;

FIG. 13 is an image showing UMAP of clusters of T cells subsets of patient ID ZNL49;

FIG. 14 is an image showing expression of top −20 genes in patient ID ZNC35;

FIG. 15 is an image showing expression of top 20 genes in patient ID ZNL49;

FIG. 16 is an image showing the fold change of clusters in the mutant group compared to clusters in the wild type group of patient ID ZNC35;

FIG. 17 is an image showing the fold change of clusters in the mutant group compared to clusters in the wild type group of patient ID ZNL49;

FIG. 18 is an image showing the TCR clonotypes based on their fraction, expansion, their IFNG expression and T cell activation score of TP53_R273H in patient ID ZNC35;

FIG. 19 is an image showing the TCR clonotypes based on their fraction, expansion, their IFNG expression and T cell activation score of TP53_V157F in patient ID ZNL49;

FIG. 20 is an image showing of the killing ability of TCR-transduced JKT/CD8 cells assessed at effector-to-target (E:T) ratios respectively 0.5:1 and 2:1, in which target cells (K562) expressing different HLA-I molecules, (HLA-A1101, HLA-A0206, HLA-B5401, and HLA-C0102);

FIG. 21 is an image showing of the killing ability of TCR-transduced JKT/CD8 cells represents target cells (K562) not expressing HLA-I molecules and expressing HLA-I molecules (HLA-A1101 and HLA-C0102) assessed at different target-to-effector (T:E) ratios (1:1 and 1:10);

FIG. 22A is an image showing expression of the transduced TCR in PBMCs from healthy donor 1 before transduction by flow cytometry using multimer technologies and CD8, CD4, and mouse constant TCR beta staining;

FIG. 22B is an image showing expression of the transduced TCR in PBMCs from healthy donor 1 after transduction by flow cytometry CD8 and CD4, mouse constant TCR beta staining;

FIG. 23 is an image showing of the killing ability of TCR-transduced T cells assessed at effector-to-target (E:T) ratios respectively 0.5:1 and 2:1, in which target cells (K562) expressing different HLA-I molecules (HLA-A1101, HLA-A0206, HLA-B5401, and HLA-C0102);

FIG. 24 is an image showing of capacity to secrete IFN-γ of the TCR-transduced T cells which cocultured with K562 expressing monoallelic HLA-A1101 or HLA-C0102 pulsed with DMSO, wild type peptides, mutant peptides with different concentration of peptides by Elispot;

FIG. 25 is an image showing a representative ELISpot image for IFN-γ secretion, highlighting the immunogenic activity of the TCR-transduced T cells of healthy donor 1;

FIG. 26 is an image showing of the killing ability of TCR-transduced T cells of healthy donor 1 assessed at different target-to-effector (T:E) ratios 1:1 and 1:10, in which target cells (K562) expressing different HLA-I molecules, including HLA-I is HLA-A1101 or HLA-C0102 pulsed with DMSO, wild type peptides, mutant peptides;

FIG. 27A is an image showing the killing ability of TCR-transduced T cells from healthy donor 2, assessed at different target-to-effector (T:E) ratios (1:1 and 1:10). The targets express monoallelic HLA-A11:01 or HLA-C01:02 and were pulsed with the mutant peptide, the wild-type peptide; and

FIG. 27B is an image showing CD69 expression of the TCR-transduced T cells of healthy donor 2. The targets express monoallelic HLA-A1101 or HLA-C0102 and were pulsed with the mutant peptide, the wild type peptide.

Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of embodiments of the present invention.

The method components have been represented where appropriate by conventional symbols in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with these preferred embodiments, it should be understood that they are not intended to limit the invention. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, numerous specific details are set forth to provide a thorough understanding. However, it will be obvious to one of ordinary skills in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present invention.

It should be noted that the terms “comprises” and “comprising”, as well as “the” and “these”, are intended to cover a non-exclusive inclusion. For example, a process, method, system, product, or device that comprises a series of steps or units is not necessarily limited to those explicitly listed and may include other steps or units not explicitly mentioned or inherent to such processes, methods, products, or devices.

In the following, to facilitate the understanding of the present solution, some proper nouns appearing in the subsequent embodiments of the present application are explained.

“Subject” and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.

Within the scope of the present invention, the term “T-cell receptor (TCR)” meaning is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The binding between TCR and antigen peptides is of relatively low affinity and is degenerate: that is, many TCRs recognize the same antigen peptide, and many antigen peptides are recognized by the same TCR.

Within the scope of the present invention, the term “human leukocyte antigen (HLA)” refers to the expression product of the Major Histocompatibility Complex (MHC) in humans.

The term “Peripheral blood mononuclear cells (PBMC)” refers to the group of mononuclear cells retrievable from a subject's blood, including T cells.

Here, the term “peptide” refers to a polypeptide composed of multiple amino acids. An immune system exists in a living organism to remove foreign materials that are not derived from the living organism itself, and particularly, there exist immunogenic peptides among exogenously derived peptides triggering responses from the immune system. Alternations in the subject's genetic materials (so-called mutations) during cancer development also provide a source of immunogenic peptides (shared neoantigen) triggering immune response against the tumor. More specifically, the shared neoantigen are eluted and presented by the subject's HLA (either HLA-1 or HLA-II), attract and trigger T cells (including CD8+ T cells and/or CD4+ T cells), thereafter lead to immune response through a complex intracellular and intercellular cascade process. The shared neoantigen contains at least one mutated amino acid, and its length depends on specific peptide-presenting HLA, usually 8-11 amino acid long for HLA-1 and 25 amino acid long for HLA-II, but is not limited thereto and may be of various lengths.

The term “mutant peptide” or “MT” refers to a peptide that comprises at least one mutated amino acid exclusively present in a tumor and not in a normal tissue.

By “wild-type” or “WT” and grammatical equivalents thereof herein is meant the typical form of an amino acid sequence or a nucleotide sequence including normal allelic variations; that is, an amino acid sequence or a nucleotide sequence that has not been modified in cancer cells.

The phrase “immunogenic neoantigens” refers to long peptide carrying shared recurrent mutations that can stimulate a T-cell response to the mutant peptide.

One embodiment of the invention is now described with reference to FIG. 1. FIG. 1 illustrated a rapid, comprehensive and sensitive method for neoantigen screening from recurrent cancer mutations 100 (“method 100”) based on the above principle in accordance with an exemplary embodiment of the present invention. In particular, method 100 includes the following three steps: step 101, step 102, step 103, step 104, step 105, step 106, step 107 and step 108.

At step 101, collecting and processing sample of a subject with a cancer, comprising:

• • isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood sample from the subject with cancer; and
  • employing next-generation genomic and transcriptomic sequencing on this sample of tumor tissue and white blood cells, and using bioinformatic analysis to extract the subject's profile including a plurality of mutation sequences and its corresponding a plurality of wild-type sequences.

At step 102, obtaining a shared neoantigen by filtered the mutation sequences at step 101 based on a collection of 67 off-the-shelf peptides consists of KRAS_p.G13D, KRAS_p.G12V, KRAS_p.G12A, KRAS_p.G12D, KRAS_p.G12C, CDX2_p.V306X, RNF43_p.G659X, TP53_p.R282W, TP53_p.R273H, TP53_p.R248Q, TP53_p.R175H, GNAS_p.R201H, PIK3CA_p.E545K, BRAF_p.V640E, TCF7L2_p.R471C, ATM_p.A2301X, POU2AF1_p.A226V, KRAS_p.G12S, CHD4_p.K73X, TP53_p.E286K, TP53_p.Y220C, TP53_p.C176F, TP53_p.A159P, TP53_p.V157F, CIC_p.T1740M, ELK4_p.S359X, ARID1A_p.K1071X, BARD1_p.K171X, PIK3CA_p.V344G, PIK3CA_p.E542K, AKAP9_p.SE1650-1651 SX, TCF7L2_p.H198X, ATM_p.V60X, BCL9L_p.Q452X, NCOR2_p.P975X, KRAS_p.A146T, BRCA2_p.Q1782X, CDK12_p.R663C, TP53_p.R273C, SMAD4_p.G30X, SMAD4_p.R361H, MTOR_p.S2215F, ATP1A1_p.G98X, ARID1A_p.S764SX, ARID1A_p.G1848X, ASXL1_p.G643X, GNAS_p.R201C, ERG_p.446-447X, AMER1_p.F173X, DCTN1_p.R1173H, PIK3CA_p.R88Q, PIK3CA_p.R357Q, PIK3CA_p.E545A, PIK3CA_p.E970K, FAT4_p.L3V, FBXW7_p.S582L, FBXW7_p.R465H, PDGFRA_p.R151H, APC_p.M1413X, APC_p.KR1462-1463X, IL7R_p.K119X, IL6ST_p.K529X, BRAF_p.D634N, BRAF_p.G509V, EGFR_p.L858R, AKAP9_p.K37X, and UBR5_p.R1331C.

According to the priority embodiment of the invention, the collection of 67 off-the-shelf peptides is referenced in patent application Ser. No. 18/492,794 filed Oct. 24, 2023. The sequences of mutant peptides and its corresponding wild-type peptides presented in the collection of 67 off-the-shelf peptides are listed in Table 1 below.

TABLE 1
The sequences of mutant peptides and its
corresponding wild-type peptides
presented in the collection of 67 off-the-
shelf peptides according to the invention

Off-the-shelf	SEQ
peptide	ID No.

		Mutant sequence
KRAS_p.G13D	1	MTEYKLVVVGAGDVGKSALTIQLIQ
KRAS_p.G12V	2	MTEYKLVVVGAVGVGKSALTIQLI
KRAS_p.G12A	3	MTEYKLVVVGAAGVGKSALTIQLI
KRAS_p.G12D	4	MTEYKLVVVGADGVGKSALTIQLI
KRAS_p.G12C	5	MTEYKLVVVGACGVGKSALTIQLI
CDX2_p.V306X	6	GSVPGVLGPTGGC
RNF43_p.G659X	7	ARHPQRKRRGVPPSPPLALGPRMQL
TP53_p.R282W	8	FEVRVCACPGRDWRTEEENLRKKGE
TP53_p.R273H	9	SGNLLGRNSFEVHVCACPGRDRRTE
TP53_p.R248Q	10	YMCNSSCMGGMNQRPILTIITLEDS
TP53_p.R175H	11	YKQSQHMTEVVRHCPHHERCSDSDG
GNAS_p.R201H	12	DYVPSDQDLLRCHVLTSGIFETKFQ
PIK3CA_p.E545K	13	AISTRDPLSEITKQEKDFLWSHRHY
BRAF_p.V640E	14	LTVKIGDFGLATEKSRWSGSHQFEQ
TCF7L2_p.R471C	15	WCKPCRRKKKCVCYIQGEGSCLSPP
ATM_p.A2301X	16	SEWQLEEAQVFWAKRSRVLP
POU2AF1_p.A226V	17	EPVLQDMEDPRRVASSLTIDKLLLE
KRAS_p.G12S	18	MTEYKLVVVGASGVGKSALTIQLI
CHD4_p.K73X	19	RDPKIPKSKRQKRSVCSYAGSWGTA
TP53_p.E286K	20	VCACPGRDRRTEKENLRKKGEPHHE
TP53_p.Y220C	21	DRNTFRHSVVVPCEPPEVGSDCTTI
TP53_p.C176F	22	KQSQHMTEVVRRFPHHERCSDSDGL
TP53_p.A159P	23	VDSTPPPGTRVRPMAIYKQSQHMTE
TP53_p.V157F	24	LWVDSTPPPGTRFRAMAIYKQSQHM
CIC_p.T1740M	25	AGGITQVQYILPMLPQQLQVAPAPA
ELK4_p.S359X	26	SLPTASLTPAFFHRHPSY
ARID1A_p.K1071X	27	KEIGGLTQVNKNKNGGNLQPTSMWA
BARD1_p.K171X	28	SKASVQTQPAIKKMQVLSKTHMNLF
PIK3CA_p.V344G	29	SALRIKILCATYGNVNIRDIDKIYV
PIK3CA_p.E542K	30	QLKAISTRDPLSKITEQEKDFLWSH
AKAP9_p.SE1650-	31	AQRSSIDNENLVSEREGAFRGAGST
1651SX
TCF7L2_p.H198X	32	NKVPVVQHPHHVHPSRLLSRTAMNT
ATM_p.V60X	33	DSKQGKYLNWDAVLDFYRNIFRKKQ
BCL9L_p.Q452X	34	EGGPPAQAPPPPSSHPRPLPAG
NCOR2_p.P975X	35	LKQLKQRAAAIPPSRSPKSMSPPGR
KRAS_p.A146T	36	ARSYGIPFIETSTKTRQGVDDAFYT
BRCA2_p.Q1782X	37	SGIEPVLKNVEDQKTLVFPK
CDK12_p.R663C	38	SKPVKKEKEQRTCHLLTDLPLPPEL
TP53_p.R273C	39	SGNLLGRNSFEVCVCACPGRDRRTE
SMAD4_p.G30X	40	SIVHSLMCHRQGGE
SMAD4_p.R361H	41	TVDGYVDPSGGDHFCLGQLSNVHRT
MTOR_p.S2215F	42	GLVNTLLANDPTFLRKNLSIQRYAV
ATP1A1_p.G98X	43	TPEWIKFCRQLFGGSQCYCGLERFF
ARID1A_p.S764SX	44	YMQRNPQMPQYSSPPARLSLISASA
ARID1A_p.G1848X	45	EFDSGLLHWRIGGGTPLSISRPTSR
ASXL1_p.G643X	46	HCHREAATTAIGGGVARVEVAAGPP
GNAS_p.R201C	47	DYVPSDQDLLRCCVLTSGIFETKFQ
ERG_p.-446-447X	48	PPALPVTSSSFFCCPKPILEFTNWG
AMER1_p.F173X	49	SMPKPKKGLKGFLAVSAVTGRARSL
DCTN1_p.R1173H	50	QLSTHTHVVDITHTSPAAKSPSAQL
PIK3CA_p.R88Q	51	EAEREEFFDETRQLCDLRLFQPFLK
PIK3CA_p.R357Q	52	NVNIRDIDKIYVQTGIYHGGEPLCD
PIK3CA_p.E545A	53	AISTRDPLSEITAQEKDFLWSHRHY
PIK3CA_p.E970K	54	QDFLIVISKGAQKCTKTREFERFQE
FAT4_p.L3V	55	MDVAPDRATGRPWLP
FBXW7_p.S582L	56	TGNCIHTLTGHQLLTSGMELKDNIL
FBXW7_p.R465H	57	CIHTLYGHTSTVHCMHLHEKRVVSG
PDGFRA_p.R151H	58	IVEDDDSAIIPCHTTDPETPVTLHN
APC_p.M1413X	59	IASSVQSEPCSGM
APC_p.KR1462-	60	AQTKREVPKNKAPTAEKRVDLSKLQ
1463X
IL7R_p.K119X	61	ICVKVGEKSLTCKK
IL6ST_p.K529X	62	APPSKGPTVRTKK
BRAF_p.D634N	63	IFLHEDLTVKIGNFGLATVKSRWSG
BRAF_p.G509V	64	ITVGQRIGSGSFVTVYKGKWHGDVA
EGFR_p.L858R	65	KTPQHVKITDFGRAKLLGAEEKEYH
AKAP9_p.K37X	66	AQSDGQSPSKKQKKREKRQAVNMMC
UBR5_p.R1331C	67	LEPPRFAQLALECVLQDWNALKSMI
		Wild-type peptides sequence
KRAS_p.G13D	68	MTEYKLVVVGAGGVGKSALTIQLIQ
KRAS_p.G12V	69	MTEYKLVVVGAGGVGKSALTIQLI
KRAS_p.G12A	70	MTEYKLVVVGAGGVGKSALTIQLI
KRAS_p.G12D	71	MTEYKLVVVGAGGVGKSALTIQLI
KRAS_p.G12C	72	MTEYKLVVVGAGGVGKSALTIQLI
CDX2_p.V306X	73	GSVPGVLGPTGGVLNPTVTQ
RNF43_p.G659X	74	LSARHPQRRRGGPSEPTPGSRPQD
TP53_p.R282W	75	FEVRVCACPGRDRRTEEENLRKKGE
TP53_p.R273H	76	SGNLLGRNSFEVRVCACPGRDRRTE
TP53_p.R248Q	77	YMCNSSCMGGMNRRPILTIITLEDS
TP53_p.R175H	78	YKQSQHMTEVVRRCPHHERCSDSDG
GNAS_p.R201H	79	DYVPSDQDLLRCRVLTSGIFETKFQ
PIK3CA_p.E545K	80	AISTRDPLSEITEQEKDFLWSHRHY
BRAF_p.V640E	81	LTVKIGDFGLATVKSRWSGSHQFEQ
TCF7L2_p.R471C	82	WCKPCRRKKKCVRYIQGEGSCLSPP
ATM_p.A2301X	83	SEWQLEEAQVFWAKKEQSLALSILK
POU2AF1_p.A226V	84	EPVLQDMEDPRRAASSLTIDKLLLE
KRAS_p.G12S	85	MTEYKLVVVGAGGVGKSALTIQLI
CHD4_p.K73X	86	RDPKIPKSKRQKKERMLLCRQLGDS
TP53_p.E286K	87	VCACPGRDRRTEEENLRKKGEPHHE
TP53_p.Y220C	88	DRNTFRHSVVVPYEPPEVGSDCTTI
TP53_p.C176F	89	KQSQHMTEVVRRCPHHERCSDSDGL
TP53_p.A159P	90	VDSTPPPGTRVRAMAIYKQSQHMTE
TP53_p.V157F	91	LWVDSTPPPGTRVRAMAIYKQSQHM
CIC_p.T1740M	92	AGGITQVQYILPTLPQQLQVAPAPA
ELK4_p.S359X	93	SLPTASLTPAFFSQTPIILTPSPLL
ARID1A_p.K1071X	94	KEIGGLTQVNKNKKWRELATNLNVG
BARD1_p.K171X	95	SKASVQTQPAIKKDASAQQDSYEFV
PIK3CA_p.V344G	96	SALRIKILCATYVNVNIRDIDKIYV
PIK3CA_p.E542K	97	QLKAISTRDPLSEITEQEKDFLWSH
AKAP9_p.SE1650-	98	AQRSSIDNENLVSERERVLLEELEA
1651SX
TCF7L2_p.H198X	99	NKVPVVQHPHHVHPLTPLITYSNEH
ATM_p.V60X	100	DSKQGKYLNWDAVFRFLQKYIQKET
BCL9L_p.Q452X	101	EGGPPAQAPPPPQQPPTAPPSGLKK
NCOR2_p.P975X	102	LKQLKQRAAAIPPIQVTKVHEPPRE
KRAS_p.A146T	103	ARSYGIPFIETSAKTRQGVDDAFYT
BRCA2_p.Q1782X	104	SGIEPVLKNVEDQKNTSFSKVISNV
CDK12_p.R663C	105	SKPVKKEKEQRTRHLLTDLPLPPEL
TP53_p.R273C	106	SGNLLGRNSFEVRVCACPGRDRRTE
SMAD4_p.G30X	107	SIVHSLMCHRQGGESETFAKRAIES
SMAD4_p.R361H	108	TVDGYVDPSGGDRFCLGQLSNVHRT
MTOR_p.S2215F	109	GLVNTLLANDPTSLRKNLSIQRYAV
ATP1A1_p.G98X	110	TPEWIKFCRQLFGGFSMLLWIGAIL
ARID1A_p.S764SX	111	YMQRNPQMPQYSSPQPGSALSPRQP
ARID1A_p.G1848X	112	EFDSGLLHWRIGGGDTTEHIQTHFE
ASXL1_p.G643X	113	HCHREAATTAIGGGGGPGGGGGGAT
GNAS_p.R201C	114	DYVPSDQDLLRCRVLTSGIFETKFQ
ERG_p.-446-447X	115	PPALPVTSSSFFAAPNPYWNSPTGG
AMER1_p.F173X	116	SMPKPKKGLKGFFSSIRRHRKSKVT
DCTN1_p.R1173H	117	QLSTHTHVVDITRTSPAAKSPSAQL
PIK3CA_p.R88Q	118	EAEREEFFDETRRLCDLRLFQPFLK
PIK3CA_p.R357Q	119	NVNIRDIDKIYVRTGIYHGGEPLCD
PIK3CA_p.E545A	120	AISTRDPLSEITEQEKDFLWSHRHY
PIK3CA_p.E970K	121	QDFLIVISKGAQECTKTREFERFQE
FAT4_p.L3V	122	MDLAPDRATGRPWLP
FBXW7_p.S582L	123	TGNCIHTLTGHQSLTSGMELKDNIL
FBXW7_p.R465H	124	CIHTLYGHTSTVRCMHLHEKRVVSG
PDGFRA_p.R151H	125	IVEDDDSAIIPCRTTDPETPVTLHN
APC_p.M1413X	126	IASSVQSEPCSGMVSGIISPSDLPD
APC_p.KR1462-	127	REVPKNKAPTAEKRESGPKQAAVNA
1463X
IL7R_p.K119X	128	ICVKVGEKSLTCKKIDLTTIVKPEA
IL6ST_p.K529X	129	APPSKGPTVRTKKVGKNEAVLEWDQ
BRAF_p.D634N	130	IFLHEDLTVKIGDFGLATVKSRWSG
BRAF_p.G509V	131	ITVGQRIGSGSFGTVYKGKWHGDVA
EGFR_p.L858R	132	KTPQHVKITDFGLAKLLGAEEKEYH
AKAP9_p.K37X	133	AQSDGQSPSKKQKKKRKTSSSKHDV
UBR5_p.R1331C	134	LEPPRFAQLALERVLQDWNALKSMI

At step 103, synthesizing a long peptide corresponding to a panel of shared neoantigen at step 102 and its corresponding of wild type peptides.

At step 104, stimulating the PBMCs with the long synthetic peptides at step 103 to obtain a stimulated PBMC, comprising the following steps:

• • (i) thawing frozen PBMCs in AIM-V media supplemented with 10% fetal bovine serum (FBS) and 1 μg/mL deoxyribonuclease I (DNase I) solution;
  • (ii) allowing 10⁵ PBMCs to rest in 96-round bottom well-plate containing AIM-V media supplemented with 10% FBS, 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and 50 μM β-mercaptoethanol overnight before stimulation with synthesized long peptide at a concentration of 5 μM in a humidified incubator at 37° C. with 5% CO₂;
  • (iii) further stimulating PBMCs with 2000 IU/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) and 1000 IU/mL interleukin-4 (IL-4) for 24 hours;
  • (iv) adding 100 ng/mL LPS and 10 ng/mL IFN-γ to the PBMCs along with the peptides for an additional 12 hours; and
  • (v) restimulating PBMCs by adding 10 ng/mL interleukin-7 (IL-7), 10 ng/mL interleukin-15 (IL-15), and 10 ng/mL interleukin-21 (IL-21) to the PBMCs on the following day, in which a restimulation frequency of 3 days/time and the number of restimulations is three times.

At step 105, screening the stimulated PBMC at step 104 based on response of T cells is measured by interferon-γ (IFN-γ) secretion to mutant peptides and wild type peptides, which is more than twofold higher compared to its corresponding wild-type peptides.

At step 106, isolating a neoantigen-specific T cell from the screened stimulated PBMC at step 105 to identify a clonotype-purified cell.

According to the priority embodiment of the invention, the clonotype-purified cell obtained from a method 200 will be described in detail later below.

At step 107, identifying a TCR candidate for shared neoantigen according to a method 300, which will be described in detail later below.

Finally, at step 108 evaluating antigenic specificity of the TCR candidate for shared neoantigen through T cell activation bioassay using Nuclear Factor of Activated T cells (NFAT) system and using PBMCs or jurkat (JKT) del beta/CD8 to identify a shared neoantigen-reactive TCR, comprising the following steps:

• • (c1) co-culturing a) a reporter T cell comprising the TCR candidate for shared neoantigen expression cassette, and b) an antigen presenting cell (APC) that expresses the shared neoantigen sequence and a human leukocyte antigen (HLA) sequence extracted from subject's profile;
    • wherein the reporter T cell is a jurkat del beta cell; and
    • wherein the TCR candidate for shared neoantigen expression cassette comprises a TCR candidate sequence reconstructed from TCR α and β chain sequences;
  • (c2) identifying a positive reporter signal in the reporter T cell to identify the neoantigen-reactive TCR; wherein the shared neoantigen-reactive TCR comprises a sequence selected from the group consisting of SEQ ID NOs:135 to 142.

According to the preferred embodiment of the present invention, each shared neoantigen-reactive TCR includes a TCR alpha chain and a TCR beta chain, in which the TCR alpha chain is generated by VJ recombination, whereas the beta chain is generated by VDJ recombination, all are listed in Table 2 below.

TABLE 2
The sequences of the TCR alpha and TCR beta chains of each
neoantigen-reactive TCR is identified by method 100
according to the invention

Neoantige		SEQ
n-reactive		ID
TCR name	Chain	No.	CDR3	V_gene	J_gene	D_gene
TCR12.1_	alpha	143	CAASDNDMRF	TRAV29/DV5	TRAJ43	X
ZNC35348	(α)
	beta	144	CATKSSLTYEQ	TRBV6-1	TRBJ2-7	X
	(β)		YF
TCR32_Z	alpha	145	CATDQAGTALIF	TRAV17	TRAJ15	X
NC3548	(α)
	beta	146	CASSVQGGAQ	TRBV9	TRBJ2-5	X
	(β)		ETQYF
TCR179_Z	alpha	147	CAESQGSARQL	TRAV5	TRAJ22	X
NL4901	(α)		TF
	beta	148	CASRALTGNTG	TRBV28	TRBJ2-2	X
	(β)		ELFF
TCR314_Z	alpha	149	CAASSGAGSY	TRAV23/DV6	TRAJ28	X
NL4901	(α)		QLTF
	beta	150	CASSPKISFTGT	TRBV27	TRBJ1-1	TRBD1
	(β)		GKLNTEAFF
TCR61_Z	alpha	151	CAVGALYGNKL	TRAV8-3	TRAJ47	X
NL4901	(α)		VF
	beta	152	CASSYDDRGSS	TRBV6-2	TRBJ2-2	TRBD1
	(β)		NGELFF
TCR6_ZN	alpha	153	CALFSTSGTYK	TRAV6	TRAJ40	X
L4901	(α)		YIF
	beta	154	CAWSGDNYEQ	TRBV30	TRBJ2-7	X
	(β)		YF
TCR1034_	alpha	155	CAVSIRLMNTG	TRAV3	TRAJ8	X
ZNL4901	(α)		FQKLVF
	beta	156	CASSEGAPNSI	TRBV4-2	TRBJ1-1	X
	(β)		DEAFF
TCR1_ZN	alpha	157	CAVEDRNRDD	TRAV2	TRAJ30	X
L4901	(α)		KIIF
	beta	158	CASSLRTASKV	TRBV5-1	TRBJ1-1	X
	(β)		AFF
X: Unknown

According to the embodiment of the invention, step 108, the HLA allele is encoded by any one of the following loci: HLA-A, HLA-B, and HLA-C.

According to the preferred embodiment of the present invention, step 108, the HLA allele is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

According to the embodiment of the invention, step 108, the APC is a K562 cell which expresses a CD80 molecule.

According to the embodiment of the invention, step 108, the reporter T cell and the APC are co-cultured in a ratio of 2:1 or 0.5:1 with 5% CO₂ at 37° C. for 6 hours.

According to another embodiment of the invention, step 108, the reporter T cell and the APC are co-cultured in a ratio of 5:1 with 5% CO₂ at 37° C. for 6 hours.

According to another embodiment of the invention, step 108, the reporter T cell and the APC are co-cultured in a ratio of 5:1 with 5% CO₂ at 37° C. for 24 hours.

The shared neoantigen-reactive TCR is identified by method 100 which bind to a shared neoantigen/HLA complex. The shared neoantigen/HLA complex is the shared neoantigen presented by the HLA molecule, in which the shared neoantigen comprises TP53_pR273H, and TP53_p.V157F, and wherein the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

According to the preferred embodiment of the invention, the shared neoantigen-reactive TCR is identified by method 100 which bind to a shared neoantigen/HLA complex, in which the shared neoantigen is TP53_p.R273H, and the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, and HLA-C0702.

According to the preferred embodiment of the invention, the shared neoantigen-reactive TCR is identified by method 100 which bind to a shared neoantigen/HLA complex, in which the shared neoantigen is TP53_p.R273H, and the HLA is selected from the group consisting of HLA-A1101, and HLA-C0102.

According to the preferred embodiment of the invention, the shared neoantigen-reactive TCR is identified by method 100 which bind to a shared neoantigen/HLA complex, in which the shared neoantigen is TP53_p.V157F, and the HLA is selected from the group consisting of HLA-A1101, HLA-B0705, HLA-C0702, HLA-C1203, and HLA-B11532.

According to the preferred embodiment of the invention, the shared neoantigen-reactive TCR is identified by method 100 which bind to a shared neoantigen/HLA complex, in which the shared neoantigen is TP53_p.V157F, and the HLA is HLA-A1101.

According to the preferred embodiment of the invention, TP53 wild-type p.R273H and mutant p.R273H peptides were designed based on the HLA-A*11:01 and HLA-C0102 bindings predictions to the TP53_p.R273H MUT 10 to 11, and 25mer sequences. The top predicted mutant epitopes and corresponding wild-type sequences are listed in Table 3 below.

TABLE 3
TP53_p.R273H peptides tested

	SEQ
Peptide	ID No.	Sequence

TP53_p.R273H WT 25 mer	76	SGNLLGRNSFEVR
		VCACPGRDRRTE
TP53_p.R273H MT 25 mer	9	SGNLLGRNSFEVH
		VCACPGRDRRTE
TP53_p.R273H WT 10 mer	159	EVRVCACPGR
TP53_p.R273H MT 10 mer	160	EVHVCACPGR
TP53_p.R273H WT 11 mer	161	VRVCACPGRDR
TP53_p.R273H MT 11 mer	162	VHVCACPGRDR
TP53_p.R273H WT 11 mer	163	RVCACPGRDRR
TP53_p.R273H MT 11 mer	164	HVCACPGRDRR

According to the preferred embodiment of the invention, TP53 wild-type p.V157F and mutant p.V157F peptides were designed based on the HLA-A*11:01, bindings predictions to the TP53_p.V157F MUT 25mer sequence. The top predicted mutant epitopes and corresponding wild-type sequences are listed in Table 4 below.

TABLE 4
TP53_p.V157F peptides tested

	SEQ
Peptide	ID No.	Sequence
TP53_p.V157F WT	91	LWVDSTPPPGTRVRAMAIYKQSQHM
25 mer
TP53_p.V157F MT	24	LWVDSTPPPGTRFRAMAIYKQSQHM
25 mer

According to the embodiment of the invention, the method 100 is applied to lung cancer, and colorectal cancer.

Colorectal cancer is a type of gastrointestinal cancer that starts in the colon or the rectum. These cancers are also called colon cancer or rectal cancer, depending on where they start. The colon is the large intestine or large bowel. The rectum is the passageway that connects the colon to the anus. Colon cancer and rectal cancer are often grouped together because they have many features in common. Sometimes abnormal growths, called polyps, form in the colon or rectum. Over time, some polyps may turn into cancer. Screening tests can find polyps so they can be removed before turning into cancer. Screening aids in the detection of colorectal cancer at early stages, when treatment is most successful at treating the cancer. The most common type of colorectal cancer is adenocarcinoma. Adenocarcinomas of the colon and rectum make up 95% of all colorectal cancer cases in the United States. In the gastrointestinal tract, rectal and colon adenocarcinomas develop in the cells of the lining inside the large intestine. These adenocarcinomas typically start as a polyp.

Lung cancer is cancer that forms in tissues of the lung, usually in the cells lining air passages. Lung cancers usually are grouped into two main types, small cell lung cancer and non-small cell lung cancer (including adenocarcinoma and squamous cell carcinoma). Non-small cell lung cancer is more common than small cell lung cancer.

Referring to FIG. 2, the method for isolating a neoantigen-specific T cell from the screened stimulated PBMC to identify the clonotype-purified cell 200 (“method 200”) in accordance with the present invention. In particular, method 200 includes the following steps:

At step 201, determining the viability of the stimulated PBMC using a hemocytometer to ensure viability above 90%, and adjusting the cell concentration to between 700-1,200 cells per microliter to obtain a uniform PBMC suspension.

At step 202, mixing the uniform PBMC suspension at step 201 with a reverse transcription (RT) master mix to obtain a cell-master mix solution, then loading the cell-master mix solution onto a microfluidic device configured to partition individual cells into emulsions for unique nucleic acid barcoding, wherein the loading is performed along with barcoded 5′ gel beads and partitioning oil to obtain a single-cell gel bead in emulsion (GEMs).

At step 203, performing cell lysis and barcoded reverse transcription of RNA within each the GEMs to obtain a barcoded complementary DNA (cDNA).

At step 204, producing and validating cDNA of gene expression library and VDJ library, comprising:

• • recovering the barcoded cDNA from the GEMs at step 203 to obtain a cDNA sample;
  • amplifying the cDNA sample using polymerase chain reaction (PCR) to obtain an amplified cDNA; and
  • assessing the quality of the amplified cDNA using sensitivity-based screening systems to obtain a validated cDNA.

At step 205, constructing sequencing libraries, comprising:

• • utilizing the validated cDNA at step 204 to prepare 5′ gene expression libraries;
  • indexing each library with a sample indexing system to obtain an indexed gene expression library; and
  • sequencing the indexed gene expression library on a sequencing platform to generate at least 30,000 read pairs per cell with paired-end reads of 2×300 base pairs.

At step 206, enriching and sequencing V(D)J regions, and RNA transcriptomic profile comprising:

• • using the libraries generated in step 205 to amplify full-length variable (V), diversity (D), and joining (J) segments of T cell receptor (TCR) alpha and beta chains using an enrichment system to obtain an enriched TCR product;
  • quantifying the enriched TCR product obtained from the amplification using sensitivity-based quantification systems to produce a quantified enriched TCR product; preparing sequencing libraries using 50 ng of the quantified enriched TCR product to produce a TCR sequencing library; and
  • sequencing the TCR sequencing library on a sequencing platform to generate paired-end reads of 2×300 base pairs with a depth of 5,000 read pairs per cell.

Finally, at step 207, performing bioinformatics analyses on the single cell gene expression data to identify a clonotype-purified cell, comprising:

• • retaining cells with available clonotype information; and
  • excluding cells with mitochondrial genome-derived reads exceeding 15%, more than 7,000 detected genes, or more than two TRA (T-cell receptor alpha locus) or TRB (T-cell receptor beta locus) sequences to obtain the clonotype-purified cell.

Referring to FIG. 3, the method for identifying the TCR candidates for shared neoantigen 300 (“method 300”) in accordance with the present invention. In particular, method 300 includes the following steps:

At step 301, isolating CD3+ T cells from both mutant and wild-type groups, wherein each isolated cell based on a number of genes detected values in each cell between 200 and 6000, and a mitochondrial gene expression percentage below 15%.

At step 302, defining the T cell activation score based on the average expression of 10 genes associated with T cell activation for each T cell, in which the 10 genes associated consisting of interferon gamma (IFNG), interleukin-2 (IL-2), tumor necrosis factor (TNF), interleukin-2 receptor alpha (IL2RA), cluster of differentiation 69 (CD69), TNF receptor superfamily member 9 (TNFRSF9), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK), and perforin 1 (PRF1).

At step 303, normalizing the size of TCR clonotypes stimulated by mutant sequences relative to the corresponding wild-type sequences; wherein, if any TCR clonotype is stimulated only by mutant sequences and is not found in the sample stimulated by the corresponding wild-type sequences, its size is calculated by taking the smallest size of the TCR clonotype stimulated by the wild-type sequences.

At step 304, calculating a ratio size of each TCR clonotype from group which is stimulated by mutant sequences compared to the corresponding wild-type sequences.

Finally, at step 305, ranking the clonotypes based on their IFNG expression and T cell activation score at step 302, and their ratio size at step 304 to identify the TCR candidates for shared neoantigen.

The present disclosure provides a shared neoantigen-reactive T cell receptor (TCR) comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142, wherein the shared neoantigen-reactive TCR is identified by the method 100 described above.

According to the priority embodiment of the invention, the shared neoantigen-reactive TCR bind to a shared neoantigen/HLA complex, wherein the neoantigen-reactive TCR bind to a shared neoantigen/HLA complex;

• • in which the shared neoantigen comprises TP53_p.R273H, and TP53_p.V157F;
    • TP53_p.R273H comprises a sequence selected from the group consisting of SEQ ID NOs:9, 160, 162, 164, and 166;
    • TP53_p.V157F comprises a sequence selected from the group consisting of SEQ ID NOs:24, 168, 170, 172, and 174;
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

According to the priority embodiment of the invention, the shared neoantigen-reactive TCR bind to the shared neoantigen/HLA complex;

• • in which the shared neoantigen is TP53_p.R273H; and
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, and HLA-C0702.

According to the priority embodiment of the invention, the shared neoantigen-reactive TCR bind to the shared neoantigen/HLA complex;

• • in which the shared neoantigen is TP53_p.R273H; and
  • in which the HLA is selected from the group consisting of HLA-A1101, and HLA-C0102.

According to the priority embodiment of the invention, the shared neoantigen-reactive TCR bind to the shared neoantigen/HLA complex;

• • in which the shared neoantigen is TP53_p.V157F; and
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-B0705, HLA-C0702, HLA-C1203, and HLA-B1532.

According to the priority embodiment of the invention, the shared neoantigen-reactive TCR bind to the shared neoantigen/HLA complex;

• • in which the shared neoantigen is TP53_p.V157F; and
  • in which the HLA is HLA-A1101.

The present disclosure provides a method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • wherein the cancer includes lung cancer, and colorectal cancer;
  • wherein the shared neoantigen comprises TP53_p.R273H, and TP53_p.V157F;
    • TP53_p.R273H comprises a sequence selected from the group consisting of SEQ ID NOs:9, 160, 162, 164, and 166;
    • TP53_p.V157F comprises a sequence selected from the group consisting of SEQ ID NOs:24, 168, 170, 172, and 174;
  • wherein the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-B0705, HLA-C1203, HLA-B1532, and HLA-C0702.

According to the preferred embodiment of the invention, the method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • in which the shared neoantigen is TP53_p.R273H; and
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, and HLA-C0702.

According to the preferred embodiment of the invention, the method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • in which the shared neoantigen is TP53_p.R273H; and
  • in which the HLA is selected from the group consisting of HLA-A1101, and HLA-C0102.

According to the preferred embodiment of the invention, the method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • in which the shared neoantigen is TP53_p.V157F; and
  • in which the HLA is selected from the group consisting of HLA-A1101, HLA-B0705, HLA-C0702, HLA-C1203, and HLA-B1532.

According to the preferred embodiment of the invention, the method of preparing a medicament for the treatment or prevention of a cancer, the method comprising preparing a population of cells that comprise a recombinant vector expressing the shared neoantigen-reactive T cell receptor comprising a sequence selected from the group consisting of SEQ ID NOs:135 to 142;

• • in which the shared neoantigen is TP53_p.V157F; and
  • in which the HLA is HLA-A1101.

According to the preferred embodiment of the invention, the method of preparing a medicament for the treatment or prevention of cancer includes lung cancer and colorectal cancer.

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Tissue samples from clinical patients include seven patients with colorectal cancer samples (ZNC34, ZNC35, ZNC39, ZNC41, ZNC42, ZNC46, and ZNC47) listed in Table 5 below and three patients with lung cancer (ZNL49, ZNL50, and ZNL64). All the human tissue samples were stored at −80° C. before RNA extraction.

TABLE 5
Information of the ten samples for determine the neoantigen-reactive
TCR

	Sample			Cancer
No.	ID	Sex	Age	status	Histologic type
1	ZNC34	Female	69	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the serosal layer. Metastasis in
					0/33 lymph nodes pT4aN0M0
2	ZNC35	Female	71	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the serosal layer, vascular and
					nerve invasion present. Metastasis in 1/10
					lymph nodes pT4aN1M0
3	ZNC39	Male	67	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the stomach. No vascular or nerve
					invasion. Metastasis in 0/9 lymph nodes
					pT4bN0M0
4	ZNC41	Male	69	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the muscle layer. Metastasis in
					0/12 lymph nodes pT2N0M0
5	ZNC42	Male	60	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the serosal layer. No vascular or
					nerve invasion. Metastasis in 4/16 lymph
					nodes pT4aN2M0
6	ZNC46	Male	78	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading the subserosal layer. No vascular
					or nerve invasion. Metastasis in 0/6 lymph
					nodes pT4aN0M0
7	ZNC47	Female	56	Colorectal	Moderately differentiated adenocarcinoma,
				cancer	invading subserosal fat tissue. No vascular,
					nerve, or lymphatic invasion. Metastasis in
					3/13 lymph nodes pT4aN2M0

Isolating T cells from patients' tumor IDs ZNC34, ZNC35, ZNC39, ZNC41, ZNC42, ZNC46, ZNC47, ZNL49, ZNL50, ZNL64, and priming with long peptide containing mutations or corresponding wild-type proteins performed similarly to steps (101)-(104) described in method 100, in which the sequence of mutant and wild-type peptides used for PBMCs stimulation in patients IDs ZNC34, ZNC35, ZNC39, ZNC41, ZNC42, ZNC46, ZNC47, ZNL49, ZNL50, ZNL64 are listed in Table 6 below.

TABLE 6
Sequence of mutant and wild-type peptides used for PBMCs
stimulation in patients IDs ZNC34, ZNC35, ZNC39, ZNC41,
ZNC42, ZNC46, ZNC47, ZNL49, ZNL50, and ZNL64

		SEQ		SEQ
No.	Mutant peptide	ID No.	Wild-type peptide	ID No.

1	TP53_p.V157F_MT	24	TP53_p.V157F_WT	91
2	TP53_p.R273H_MT	9	TP53_p.R273H_WT	76
3	EGFR_p.L858R_MT	65	EGFR_p.L858R_WT	132
4	TP53_p.R273C_MT	39	TP53_p.R273C_WT	106
5	KRAS_p.G12C_MT	5	KRAS_p.G12C_WT	72
6	KRAS_p.G12D_MT	4	KRAS_p.G12D_WT	71
7	PIK3CA_p.E545K_MT	13	PIK3CA_p.E545K_WT	80
8	RNF43_p.G659X_MT	7	RNF43_p.G659X_WT	74
9	TP53_p.Y220C_MT	21	TP53_p.Y220C_WT	88
10	KRAS_p.G12V_MT	2	KRAS_p.G12V_WT	69
11	TP53_p.R248Q_MT	10	TP53_p.R248Q_WT	77

Performing similarly to step 105 described in method 100, screening the stimulated PBMC in patients with IDs ZNC34, ZNC35, ZNC39, ZNC41, ZNC42, ZNC46, ZNC47, ZNL49, ZNL50, and ZNL64 based on response of T cells is measured by interferon-γ (IFN-γ) secretion to mutant peptides, which is more than twofold higher compared to its corresponding wild-type peptides. The results showed that stimulated PBMC in patients with IDs 35 and 49 had the fold change of spots for mutant peptides compared to the corresponding wild-type peptides (as shown in FIG. 4A), collecting T cells from patients IDs ZNC35 and ZNL49 from wells containing T cells stimulated with immunogenic neoantigens and the corresponding wild-type peptides (as shown in FIG. 4B), in which the immunogenic neoantigen in patient IDs ZNC35 and ZNL49 are respectively TP53_p.R273H and TP53_p.V157F.

Performing similarly to sub-steps (a1)-(a7) of step 106 described in method 100, identifying the clonotype-purified cell, in which the experimental data are illustrated in FIG. 5 to FIG. 10 include:

• • the results of cDNA length was measured using the Tapestation for the gene expression library (as shown in FIG. 5);
  • the results of cDNA length was measured using the Tapestation for the VDJ—T cell library (as shown in FIG. 6);
  • the results of single cell TCR and gene expression sequencing of the experimental scheme for neoantigen specific TCR from patient ID ZNC35 (as shown in FIG. 7); the quality control (QC) results for the gene expression library and the VDJ library following sequencing from patient ID ZNC35, in which metrics such as library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data (as shown in FIG. 8A-8B);
  • the results of single cell TCR and gene expression sequencing of the experimental scheme for neoantigen specific TCR from patient ID ZNL49 (as shown in FIG. 9); the quality control (QC) results for the gene expression library and the VDJ library following sequencing from patient ID ZNL49, in which metrics library size, read quality, and cell number distribution were evaluated to ensure the integrity and reliability of the sequencing data (as shown in FIG. 10A-10B).

Performing similarly to sub-steps (b1)-(b5) of step 107 described in method 100, ranking the clonotypes based on their IFNG expression and T cell activation score, and their ratio size to identify the TCR candidates for shared neoantigen;

• • in which result of quality control of selected clonotypes based on 200<nFeature_RNA <6000, and percent.mt<15% from patient IDs ZNC35 and ZNL49 (as shown in FIG. 11A-11B);
  • wherein the performing bioinformatics analysis from patient IDs ZNC35 and ZNL49 further comprises clustering the TCR clonotypes, in which the clustering comprises grouping the TCR clonotypes by CD8 or CD4 expression, gene function of differentially expressed genes, and the level of expression of each TCR, which performed through Uniform Manifold Approximation and Projection (or UMAP) to visualize patterns of clustering in high-dimensional data (as shown in FIG. 12-13);
  • the results average expression of 10 genes of top 20 genes in patient IDs ZNC35 and ZNL49 associated with T cell activation (as shown in FIG. 14-15), in which the 10 genes associated consisting of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1;
  • sub-step (b4), defining the clonotype as expanded in the Mutant group if the clonotype size ratio (Mutant/Wildtype) is ≥2; otherwise, it is considered non-expanded. The results showed the clusters as expanded in the mutant group compared to the wild type group in patient IDs ZNC35 and ZNL49 referred FIG. 16-17;
  • sub-step (b5), the result of ranking the clonotypes based on their fraction, expansion, their IFNG expression and T cell activation score of TP53_R.273H and TP53_V.157F in patient IDs ZNC35 and ZNL49 (as shown in FIG. 18-19). Referring a list of the isolated and identified TCR candidate for shared neoantigen TP53_R.273H in patient ID ZNC35 is listed in Table 7 below, and a list of the isolated and identified TCR candidate for shared neoantigen TP53_V.157F in patient ID ZNL49 is listed in Table 8 below.

TABLE 7
The list of the isolated and identified TCR candidate for shared
neoantigen TP53_R.273H in patient ID ZNC35

No.	TCR candidate for shared neoantigen TP53_R.273H	SEQ ID No.
1	TCR12.1_ZNC35348	135
2	TCR32_ZNC3548	136

TABLE 8
The list of the isolated and identified TCR candidate for shared
neoantigen TP53_R.273H in patient ID ZNC35

No.	TCR candidate for shared neoantigen TP53_R.273H	SEQ ID No.
1	TCR179_ZNL4901	137
2	TCR314_ZNL4901	138
3	TCR61_ZNL4901	139
4	TCR6_ZNL4901	140
5	TCR1034_ZNL4901	141
6	TCR1_ZNL4901	142

Performing similarly to step 108 described in method 100, validating the function of the TCR candidate for shared neoantigen through T cell activation bioassay using NEAT and using PBMCs to identify the neoantigen-reactive TCRs.

A first method: validating the function of the T cell receptors (TCRs) using jurkat (JKT) del beta/CD8 with nuclear factor of activated T-cells (NFAT) reporter gene comprising steps performed in the following specific order:

• • (d1) generating of JKT del beta/CD8/NFAT/TCR12.1 comprising:
    • establishing JKT del beta stably expressing CD8 to obtain a stable JKT/CD8 cells;
    • establishing JKT del beta/CD8 (JKT/CD8) expressing NFAT reporter gene (JKT/CD8/NFAT) to obtain a JKT/CD8 expressing NFAT;
    • establishing JKT/CD8/NFAT cells expressing an identified TCR candidate to obtain a JKT/CD8/NFAT cells expressing TCR12.1; and
    • validating the expression of the TCR on the JKT/CD8/NFAT cells by staining with a constant beta-mouse antibody;
  • (d2) measuring luminescence and comparing intensity between mutant and wild type peptide of the JKT/CD8/NFAT cells expressing TCR12.1 candidate to assess a TCR function, comprising:
    • pulsing K562 cells expressing HLA-1 molecules, selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, and HLA-C0702, with peptides corresponding to TP53_R.273H;
    • coculturing the peptide-pulsed K562 cells with the JKT/NFAT/TCR12.1 cells expressing the identified TCR candidate at a 2:1 effector-to-target (E:T) ratio in a 96-well plate; and
    • incubating the coculture at 37° C. with 5% CO₂ for 6 hours.

The TCR according to the first method may be considered to have antigenic specificity for mutated TP53 peptide if a Jurkat del beta cell expressing the NFAT-luciferase reporter and the TCR produces at least twice as much luminescent signal when cocultured with HLA molecule-positive target cells pulsed with a mutated TP53 peptide, compared to the luminescent signal observed in a negative control. The negative control is prepared by performing steps (d1)-(d2) similarly, with the difference being that:

• • Jurkat del beta cells expressing the NFAT-luciferase reporter and the TCR, cocultured with (a) wild-type peptide and applicable HLA molecule-positive target cells pulsed with the same concentration of a wild-type peptide, or (b) DMSO; or
  • untransduced Jurkat del beta cells (derived from Jurkat del beta cells that do not express TCR) cocultured with (a) wild-type peptide and applicable HLA molecule-positive target cells pulsed with the same concentration of a wild-type peptide, or (b) DMSO.

A second method: validating the function of identified T cell receptors (TCRs) using JKT del beta/CD8 cells to obtain a functional validation of TCRs, comprising:

• • (e1) generating of JKT del beta/CD8/TCR12.1 comprising:
    • establishing JKT del beta stably expressing CD8 to obtain a stable JKT/CD8 cells the establishment;
    • establishing JKT del beta/CD8 (JKT/CD8) cells expressing an identified TCR candidate to obtain a JKT/CD8 cells expressing TCR12.1; and
    • validating the expression of the TCR on the JKT/CD8 cells by staining with a constant beta-mouse antibody;
  • (e2) measuring cytotoxicity of the JKT/CD8 cells expressing TCR 12.1 candidate to assess a TCR function, comprising:
    • pulsing K562 cells expressing HLA-1 molecules, selected from the group consisting of HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, and HLA-C0702, with peptides corresponding to TP53_R.273H;
    • staining the peptide-pulsed K562 cells with carboxyfluorescein succinimidyl ester (CFSE);
    • coculturing the peptide-pulsed K562 cells with the JKT/CD8 cells expressing TCR12.1 candidate at a 5:1 effector-to-target (E:T) ratio in a 96-well plate;
    • incubating the coculture at 37° C. with 5% CO₂ for 6 hours;
    • collecting, washing, and staining the cells with a dead/live marker; and
  • (e3) measuring activation markers IL2 or CD69 on the JKT/CD8 cells expressing TCR candidate to validate a TCR functionality, comprising:
    • pulsing K562 cells expressing HLA-1 molecules with peptides corresponding to the mutated TP53 peptide;
    • coculturing the peptide-pulsed K562 cells with the JKT/CD8 cells expressing TCR12.1 candidate at a 5:1 effector-to-target (E:T) ratio in a 96-well plate;
    • incubating the coculture at 37° C. with 5% CO₂ for 24 hours;
    • collecting, washing, and staining the JKT/CD8 cells with antibodies specific to IL2 or CD69.

The TCR according to the second method may be considered to have antigenic specificity for mutated TP53 if a jurkat del beta cell expressing the TCR secretes at least twice as much CD69 when cocultured with HLA molecule-positive target cells pulsed with a mutated TP53 peptide, compared to the IFN-γ secretion observed in a negative control. The negative control is prepared by performing steps (e1)-(e3) similarly, with the difference being that:

• • a jurkat del beta cells expressing the TCR, coculture with (a′) wild type peptide, applicable HLA molecule positive target cells (HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-C0702) pulsed with the same concentration of an wild type peptides or (b′) DMSO; or
  • untransduced jurkat del beta cell (derived from a jurkat del beta cells, which do not express TCR) cocultured with (a′) wild type peptide, applicable HLA molecule positive target cells pulsed with the same concentration of an wild type peptides or (b′) DMSO, in which CD69 expression is measured by, for example, flow cytometry after co-culture with target cells pulsed mutated TP53.

The test results by the second method include:

• • the results showed of TCR-Transduced JKT/CD8 cells to K562 expressing monoallelic HLA-A1101 that the fluorescent signal of the positive control sample had a positive CD8+ ratio (17.21%) and a positive CD4+ ratio (1.35%) higher than the negative control by 1.62% and 0.20%, respectively;
  • the results showed of TCR-Transduced JKT/CD8 cells to K562 expressing monoallelic HLA-B5401 that the fluorescent signal of the positive control sample had a positive CD8+ ratio (3.21%) and a positive CD4+ ratio (0.43%) higher than the negative control by 2.06% and 0.18%, respectively;
  • the results showed of TCR-Transduced JKT/CD8 cells to K562 expressing monoallelic HLA-A0206 that the fluorescent signal of the positive control sample had a positive CD8+ ratio (3.66%) lower than the negative control by 4.87%, but a positive CD4+ ratio (0.51%) higher than the negative control by 0.23%;
  • refer to FIG. 20 illustrates the killing ability of TCR-transduced JKT/CD8 cells assessed at different effector-to-target (E:T) ratios respectively 0.5:1 and 2:1, in which each plot represents target cells (K562) expressing different HLA-I molecules, (HLA-A1101, HLA-A0206, HLA-B5401, and HLA-C0102);
  • refer to FIG. 21 illustrates the killing ability of TCR-transduced JKT/CD8 cells assessed at different target-to-effector (T:E) ratios (1:1 and 1:10), in which each plot represents target cells (K562) expressing different HLA-I molecules, including no HLA-1, HLA-C0102, and HLA-A1101; and
  • the results showed the killing ability of TCR-transduced JKT/CD8 cells to K562 expressing monoallelic HLA-A1101 higher than untransduced JKT/CD8 cells, the killing ability of TCR-transduced JKT/CD8 cells to K562 expressing monoallelic HLA-B5401 also higher than untransduced JKT/CD8 cells, and the killing ability of TCR-transduced JKT/CD8 cells to K562 expressing monoallelic HLA-A0206 also higher than untransduced JKT/CD8 cells.

Finally, a third method: validating the function of an identified T cell receptor (TCR) using peripheral blood mononuclear cells (PBMCs) to obtain a functional validation of TCRs, comprising:

• • (f1) generating of TCR12.1-PBMC with efficiency of 50% comprising:
    • establishing PBMCs expressing identified TCR candidate (PBMCs/TCR12.1);
    • transducing PBMCs obtained from healthy donors with the lentivirus supernatants; and
    • validating the expression of the TCR on the transduced PBMCs by staining the cells with a constant beta-mouse antibody;
  • (f2) measuring cytotoxic activity of the TCR-expressing PBMCs to evaluate a TCR function, the measurement comprising:
    • pulsing K562 cells expressing HLA-I molecules, selected from the group consisting of HLA-A1101, and HLA-C0102 with peptides corresponding to the mutated TP53 peptide;
    • staining the peptide-pulsed K562 cells with carboxyfluorescein succinimidyl ester (CFSE);
    • coculturing the peptide-pulsed K562 cells with the TCR-expressing PBMCs at a 5:1 effector-to-target (E:T) ratio in a 96-well plate;
    • incubating the coculture at 37° C. with 5% CO₂ for 6 hours;
    • collecting, washing, and staining the cells with a dead/live marker; and
  • (f3) measuring functional markers IFNg or CD107a on the TCR-expressing PBMCs to validate a TCR functionality, comprising:
    • pulsing K562 cells expressing HLA-I molecules with peptides corresponding to the mutated TP53 peptide;
    • coculturing the peptide-pulsed K562 cells with the TCR-expressing PBMCs at a 5:1 effector-to-target (E:T) ratio in a 96-well plate;
    • incubating the coculture at 37° C. with 5% CO₂ for 24 hours;
    • collecting, washing, and staining the TCR-expressing PBMCs with antibodies specific to IFNg or CD107a.

The TCR according to the third method may be considered to have antigenic specificity for mutated TP53 if a T cell expressing the TCR secretes at least twice as much IFN-γ when cocultured with HLA molecule-positive target cells pulsed with a mutated TP53 peptide, compared to the IFN-γ secretion observed in a negative control. The negative control is prepared by performing steps (f1)-(f3) similarly, with the difference being that:

• • T cells expressing the TCR, coculture with (aa) wild type peptide, applicable HLA molecule positive target cells (HLA-A1101, HLA-A0206, HLA-B5401, HLA-C0102, HLA-C0702) pulsed with the same concentration of an wild type peptides or (bb) DMSO; or
  • untransduced T cells (derived from PBMCs, which do not express TCR) cocultured with (aa) wild type peptide, applicable HLA molecule positive target cells pulsed with the same concentration of an wild type peptides or (bb) DMSO. The number of cells secreting IFNg may be measured by methods known in the art such as, for example, enzyme-linked immunospot (ELISpot) assay.

The efficiency of TCR transduction into PBMCs of healthy donor 1, in which the results showed of TCR-transduced PBMCs that the fluorescent signal of the positive control sample had a positive CD8+ ratio (31.35%) and a positive CD4+ ratio (30.94%) higher than the negative control by 0.05% and 0.10%, respectively (as shown in FIG. 22A-22B). Refer to FIG. 23 illustrates the killing ability of TCR-transduced T cells assessed at different effector-to-target (E:T) ratios respectively 0.5:1 and 2:1, in which each plot represents target cells (K562) expressing different HLA-I molecules (HLA-A1101, HLA-A0206, HLA-B5401, and HLA-C0102); and their capacity to secrete IFN-γ of the TCR-transduced T cells which cocultured with K562 expressing monoallelic HLA-A1101 or HLA-C0102 pulsed with DMSO, wild type peptides, mutant peptides (as shown in FIG. 24). Refer to FIG. 25 shows a representative ELISpot image for IFN-γ secretion, highlighting the immunogenic activity of the TCR-transduced T cells.

The TCR according to the third method may be considered to have antigenic specificity for mutated TP53 if T cells expressing the TCR kill target cells expressing HLA-A1101, or HLA-C0102 as measured by, for example, flow cytometry after co-culture with target cells pulsed mutated TP53. Refer to FIG. 26 illustrates the killing ability of TCR-transduced T cells assessed at different target-to-effector (T:E) ratios (1:1 and 1:10), in which each plot represents target cells (K562) expressing different HLA-I molecules, including HLA-I is HLA-A1101 or HLA-C0102 pulsed with DMSO, wild type peptides, mutant peptides.

The killing ability of TCR-transduced into T cells of healthy donor 2 assessed at different target-to-effector (T:E) ratios (as shown in FIG. 27A), and the CD69 expression of TCR-transduced T cells cocultured with target cells pulsed with peptides, highlighting their activation status (as shown in FIG. 27B).

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes” and/or “including,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, element components, and/or groups thereof.

While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.

The description of the present invention has been presented for purposes of illustration and description but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

The flow diagrams depicted herein are just one example. There may be many variations to this diagram or the steps (or operations) described therein without departing from the spirit of the invention. For instance, the steps may be performed in a differing order, or steps may be added, deleted, or modified. All of these variations are considered a part of the claimed invention.

While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.

The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated. The scope of the invention should therefore be construed in accordance with the appended claims and any equivalents thereof.