The screening, identification, design and clinical application of tumor-specific neoantigens for TCR-T cells

Abstract

Recent advances in neoantigen research have accelerated the development of tumor immunotherapies, including adoptive cell therapies (ACTs), cancer vaccines and antibody-based therapies, particularly for solid tumors. With the development of next-generation sequencing and bioinformatics technology, the rapid identification and prediction of tumor-specific antigens (TSAs) has become possible. Compared with tumor-associated antigens (TAAs), highly immunogenic TSAs provide new targets for personalized tumor immunotherapy and can be used as prospective indicators for predicting tumor patient survival, prognosis, and immune checkpoint blockade response. Here, the identification and characterization of neoantigens and the clinical application of neoantigen-based TCR-T immunotherapy strategies are summarized, and the current status, inherent challenges, and clinical translational potential of these strategies are discussed.

Keywords: ACT; Immunotherapy; Neoantigen; TCR-T; TSA.

Fig. 1

Characteristics of tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Sources of TSA and TAA. In contrast to normal tissues, TSA is only expressed in tumors. TAA is underexpressed in normal tissues and overexpressed in tumors. The advantages and disadvantages of each antigen type of TSA and TAA and its representation of related tumors are shown

Fig. 2

Computational workflow of neoantigen prediction. a The general route of neoantigen prediction. b The workflow of clinical sample collection and single-cell sequencing. c The neoantigen sources can develop at the genomic level through SNV mutation, INDEL mutation, fusion mutation, integrated viral ORF and splice variants (the display of prediction software for mutation calling), at the transcriptomic level through alternative splicing, polyadenylation (pA), RNA editing and allegedly noncoding regions, and at the proteomic level through dysregulated translation and PTMs. d HLA typing prediction and display by prediction software tools. e and f Mutant polypeptides are produced by proteasome-mediated decomposition of endogenous proteins, which are subsequently transported to the ER by antigen-processing associated transporters (TAP). They may be loaded into MHC-I and MHC-II for binding to specific peptides produced by mutated proteins that breakdown in the endosomal pathway. These peptide-MHC-II/MHC-I (pMHC) complexes are then transported to the cell surface, where they are recognized by T cells. g pMHC complex binding prediction and the display of prediction software tools. h The prediction of T-cell recognition of pMHC complexes and the display of prediction software tools. i T-cell validation of neoantigens. Coculture of patient TILs or PBMCs with autologous antigen-presenting cells (APCs) expressing candidate neoantigens (TMG or peptides) allows for the identification of neoantigen-reactive T cells based on functional data such as IFN-γ release or 4-1BB expression. On the one hand, it could be injected into patients for cell therapy. On the other hand, the related functions of neoantigen reactive T cells have been verified by different experiments

Fig. 3

Rapid identification of neoantigen-specific TCRs for personalized engineered TCR-T-cell therapy. Tumor (T) and normal (N) DNA are used to conduct WES and RNA-seq to identify cancer-specific nonsynonymous mutations. Candidate neoantigens are used to design tandem minigenes (TMGs) encoding mutant peptides and synthesize mutant peptide libraries (Step 1). TILs and PBMCs are isolated from single-cell suspensions taken from patient samples. TILs and PBMCs are analyzed using single-cell TCR-CITE-Seq, and a combined tag of gene and surface protein expression is used to predict candidate neoantigen-reactive T cells (Step 2). Candidate antigen-reactive T cells are cocultured with autologous antigen-presenting cells (APCs) expressing candidate neoantigens (TMG or peptides), and antigen-specific T cells are amplified (Step 3). Antigen-specific T cells are selected by flow cytometry, and neoantigen-reactive TCRs are identified and screened by scTCR-CITE-seq or deep sequencing. Then, the TCRα/β chain is reverse transcribed by single-cell multiplex nested RT-PCR, and its related plasmid is constructed (Step 4). T cells expressing candidate reactive TCRs are generated by cloning the selected TCR sequence into a retrovirus vector and transducing T cells. The recognition of neoantigens by T cells transduced by the TCRα/β chain is verified by different screening experiments (Step 5). PBMCs are obtained from patients, and the reactive T cells of the neoantigen-specific TCRα/β chain are amplified by the above methods (Step 6). Validated neoantigen reactive TCRα/β chains are selected to design final personalized TCR-engineered T (TCR-T) cell products that will be injected into patients for cell therapy (Step 7)

Fig. 4

Classification and characteristics of neoantigen-based therapy. a Immunotherapies targeting neoantigens mainly include ACTs (TCR-T, TILs, CAR-T, CAR-NK/NKT, CAR-γδT), bispecific antibodies, cancer vaccines and combination therapy regimens. b Advantages and disadvantages of neoantigen-based immunotherapy

Fig. 5

Combined antitumor strategies based on neoantigens. The diagnosis and routine treatment of tumor patients (Step 1). The formation of tumor cells initiates the immune function of T cells, and the tumor cells die and lyse, resulting in the release of neoantigens (Step 2). Neoantigens produced by tumors are released and captured by DCs. The DC transmits the collected neoantigens on the MHC-I and MHC-II molecules to the T cells (Step 3). Immunotherapies targeting neoantigens (neoantigen-based adoptive cell therapy) mainly include TCR-T cells, TILs, CAR-T cells, CAR-NK/NKT cells, CAR-γδ T cells and bispecific antibodies (Step 4). Adoptive back transport of ACT cells and chemotaxis into the tumor play an antitumor role (Step 5). Neoantigen-based DC vaccine therapy is also initiated (Step 6). Immune cells are primed and activated in the lymph node (Step 7). Effector cells develop into effector memory cells through lymphatic homing (Step 8). Effector memory ACT cells target and kill tumor cells (Step 9). After a series of treatments, clinical evaluation and efficacy monitoring are performed (Step 10). In brief, the “Cancer-Immunity Cycle” includes enhancing neoantigen release by chemotherapy, radiation therapy and oncolytic viruses, increasing the quantity and quality of tumor-reactive T cells through cancer vaccines and ACTs, and boosting the infiltration and cytotoxicity efficacy of immune cells via checkpoint inhibitors

Fig. 6

Challenges in the clinical application of neoantigen TCR-T-cell therapy. a Low neoantigen load results in a lack of suitable neoantigen targets. b At present, the accuracy of neoantigen prediction technology is limited. c Downregulation of MHC expression causes tumor cells to lose neoantigen targets. d The loss of pMHC molecules leads to the interruption and reduction of neoantigen presentation. e The expression of adhesion molecules and stroma-rich and abnormal blood vessels in tumor tissues is downregulated, which limits the effective penetration of T cells. f Immunosuppressive tumor microenvironments inhibit T-cell function. g The technical bottleneck of ACT leads to the production of neoantigen-specific T cells. h Tumor heterogeneity leads to the singleness of specific tumor therapeutic targets and the absence of universal neoantigen targets. i The neoantigen epitopes developed thus far are mainly for HLA-A2 targets. j Safety of TCR-T-cell therapy itself