Targeting cancer with precision: strategical insights into TCR-engineered T cell therapies

Abstract

T cell receptor-engineered T (TCR-T) cell therapies are at the forefront of cancer immunotherapy, offering a transformative approach that significantly enhances the ability of T cells to recognize and eliminate cancer cells. This innovative method involves genetically modifying TCRs to increase their affinity for tumor-specific antigens. While these enhancements improve the ability of T cells to recognize and bind to antigens on cancer cells, rigorous assessment of specificity remains crucial to ensure safety and targeted responses. This dual focus on affinity and specificity holds significant promise for the treatment of solid tumors, enabling precise and efficient cancer cell recognition. Despite rapid advancements in TCR engineering and notable progress in TCR screening technologies, as evidenced by the growing number of specific TCRs entering clinical trials, several technical and clinical challenges remain. These challenges primarily pertain to the specificity, affinity, and safety of engineered TCRs. Moreover, the accurate identification and selection of TCRs that are both effective and safe are essential for the success of TCR-T cell therapies in cancer treatment. This review provides a comprehensive examination of the theoretical foundations of TCR therapy, explores strategies for screening specific TCRs and antigens, and highlights the ongoing challenges in this evolving therapeutic landscape.

Keywords: T cell receptor-engineered T cell therapy; cancer immunotherapy; screening strategy.

Figure 1

Overview of T-cell immunity against tumors. A) Tumor antigens are recognized by DCs, which activate naïve T cells and stimulate the generation of antigen-specific cytotoxic CD8⁺ T cells and helper CD4⁺ T cells. CD8⁺ T cells release PFN and GZMB to induce cancer cell apoptosis. CD4⁺ T cells secrete cytokines such as IFN-γ and TNF-α, enhancing CD8⁺ T cell cytotoxicity and promoting macrophage activation and B cell-mediated humoral immunity. B) Tumors evade immune surveillance through multiple mechanisms. They upregulate inhibitory molecules like PD-L1, which engage co-inhibitory receptors such as CTLA-4 on CD8⁺ T cells, thereby suppressing their effector functions. Additionally, tumors downregulate MHC class I molecules, reducing the ability of CD8⁺ T cells to recognize tumor antigens via TCR, leading to impaired recognition and elimination of cancer cells. Tumor-secreted cytokines, including IL-6, TGF-β, and IL-10, further inhibit the activity of CD8⁺ T cells and NK cells. These cytokines, along with IL-35, also promote the proliferation of Tregs, which play a crucial role in suppressing anti-tumor immune responses, further enabling tumor immune evasion. C) Strategies include CAR T cells and TCR-engineered T cells to enhance the number and affinity of tumor-reactive T cells. Immune checkpoint inhibitors such as pembrolizumab and atezolizumab restore T cell activity by counteracting the immunosuppressive TME. Additional approaches focus on improving T cell infiltration, persistence, and combining therapies for synergistic effects against cancer. The images in the figures were created using BioRender (https://www.biorender.com/).

Figure 2

Mechanism of T cell activation through TCR signaling. The TCR complex recognizes antigenic peptides presented by MHC I molecules on APCs. CD8 co-receptor binds to MHC I, stabilizing the interaction. Upon antigen recognition, the ITAMs on CD3 are phosphorylated by the associated kinase LCK, leading to the recruitment and activation of ZAP70. This initiates downstream signaling cascades involving pathways such as PI3K-AKT-mTOR, NFAT, NF-κB, and MAPK, which ultimately result in T cell activation, proliferation, and effector functions. Co-stimulatory signals, provided by CD28 binding to CD86 and 4-1BBL binding to 4-1BB, are crucial for full T cell activation. These signals enhance the activation of downstream signaling pathways, promoting T cell survival, proliferation, and cytokine production. In contrast, co-inhibitory signals, mediated by CTLA-4 and PD-1 interacting with CD80 and PD-L1, respectively, dampen T cell activation. These inhibitory signals play a critical role in maintaining immune homeostasis and preventing overactivation, but in the context of cancer, they can contribute to immune evasion by tumors. Balancing co-stimulatory and co-inhibitory signals is essential for regulating T cell responses in both immunity and immunotherapy. The images in the figures were created using BioRender (https://www.biorender.com/).

Figure 3

Classification of tumor antigens. A) Differentiation antigens are restricted to specific cell lineages and are expressed during cell differentiation. These antigens, such as Melan-A in melanoma, are highly immunogenic and can serve as targets for immunotherapy. Melan-A is selectively expressed in melanocytes and melanoma cells, and immunotherapies targeting this antigen have been developed to elicit specific anti-tumor immune responses. B) Overexpressed antigens are present at elevated levels in tumor cells compared to normal tissues. Examples include MUC1 in breast cancer, PSA in prostate cancer, NY-ESO-1 in lung cancer, and CEA in colon cancer. These antigens are frequently utilized as biomarkers and therapeutic targets due to their high expression levels in tumors, allowing for more selective cancer treatments and monitoring of disease progression. C) TSAs are derived from tumor-specific mutations or viral infections and are exclusively expressed by cancer cells. Neoantigens, arising from somatic mutations, are key targets for personalized immunotherapies. Viral antigens, such as those associated with HPV in cervical cancer, HBV in hepatocellular carcinoma, and EBV in nasopharyngeal carcinoma, also represent critical targets for immune-based interventions. TSAs play a central role in driving specific immune responses against tumors, minimizing off-target effects on normal tissues. D) TCR-T can target either TSAs or TAAs. Targeting TSAs is associated with a higher likelihood of selectively killing cancer cells without affecting normal cells due to the tumor-specific nature of these antigens. However, TCR-T targeting TAAs, which are overexpressed in tumors but also present in normal tissues, can result in "on-target, off-tumor" toxicity, leading to adverse effects such as tissue damage, inflammation, and autoimmune disorders. The images in the figures were created using BioRender (https://www.biorender.com/).

Figure 4

Processing and presentation of different types of antigens. A) Endogenous proteins are ubiquitinated and degraded into peptides by the proteasome. Peptides are transported by the TAP1-TAP2 complex into the endoplasmic reticulum, where they are trimmed by ERAAP and loaded onto MHC I molecules. Calreticulin and calnexin assist in peptide loading and stabilization of MHC I. Once the peptide-MHC I complex is formed, it is transported to the cell surface to be recognized by CD8⁺ T cells, promoting cytotoxic T cell responses against tumor cells expressing these antigens. Co-stimulatory molecules such as CD80/CD86 and CD70 further enhance T cell activation. B) Exogenous antigens are taken up by APCs via endocytosis or phagocytosis. These antigens are processed in lysosomes and loaded onto MHC II molecules within the MIIC compartment, facilitated by HLA-DM, which removes CLIP from the MHC II complex. The peptide-MHC II complex is transported to the cell surface, where it is recognized by CD4⁺ T cells. This interaction triggers helper T cell activation and subsequent immune responses. C) Exogenous antigens can also enter the cytoplasm of APCs and be cross-presented on MHC I molecules, enabling CD8⁺ T cell activation. Additionally, endogenous antigens can be processed through autophagy, with autophagosomes delivering cytoplasmic contents, including endogenous antigens, to the MIIC for loading onto MHC II. Both cross-presentation and autophagy-mediated presentation allow for the activation of distinct subsets of T cells, broadening immune recognition of tumor antigens. The images in the figures were created using BioRender (https://www.biorender.com/).

Figure 5

Cell-based TCR screening strategies. A) Target cells are labeled with radioactive chromium (⁵¹Cr) and co-cultured with cytotoxic T cells. Upon recognition and lysis of target cells by T cells, chromium is released into the supernatant. The level of chromium release is measured to assess T cell cytotoxicity, providing a quantitative evaluation of the T cell-mediated killing of cancer cells. B) T cells from patients or donors are co-cultured with target cells expressing a library of antigens. The proliferation of T cells is monitored by the incorporation of BrdU into newly synthesized DNA during cell division. The level of BrdU incorporation, detected through fluorescence microscopy, reflects the strength of the TCR-pMHC interaction, providing insights into T cell responses against specific antigens. C) ELISpot is used to detect cytokine secretion from individual T cells in response to target cells. T cells are co-cultured with target cells, and cytokine release is captured on a membrane pre-coated with specific antibodies. After incubation and washing, spots representing single T cell cytokine release are visualized. Flow cytometry-based sorting can then be used to isolate specific subsets of T cells for further analysis. D) In T-Scan, T cells are cultured with target cells expressing an antigen library. Granzyme B release upon target cell recognition is measured, serving as an indicator of cytotoxic activity. Target cells are sorted based on granzyme B reporter expression, and next-generation sequencing is used to identify the antigen that triggered the T cell response, enabling the identification of novel antigens. The images in the figures were created using BioRender (https://www.biorender.com/).

Figure 6

TCR screening using display libraries and pMHC tetramer-based assays. A) TCR libraries displayed on yeast, phages, and mammalian cells enable the high-throughput screening of antigen specificity. Yeast display incorporates TCRs anchored to the cell wall via Aga2p, facilitating the screening against labeled pMHC complexes. Phage display vectors present TCRs on capsid proteins, allowing for rapid affinity maturation through successive biopanning rounds. CHO cells expressing TCR-pMHC constructs provide a mammalian context for functional analyses. The resulting interactions are analyzed via biochemical screening, flow cytometry, and other downstream assays to identify TCRs with desired antigen specificities. B) pMHC tetramer-based screening utilizes DNA barcode-labeled pMHC tetramers linked to fluorochromes, facilitating simultaneous analysis of multiple TCR-pMHC interactions through flow cytometry. The barcodes allow for precise identification and quantification of TCR engagement, while TCR sequencing and droplet digital PCR provide detailed insights into the TCR repertoire. Mass spectrometry complements these analyses by identifying specific peptide-MHC interactions, enhancing the understanding of TCR specificity and affinity. The images in the figures were created using BioRender (https://www.biorender.com/).