Synergizing adoptive cell therapy: Boosting Cancer Cell Targeting with Dual TCR/CAR Engineering


The T cell receptor (TCR) is a composite of multiple proteins with two main functions: antigen recognition (mediated by TCRα and TCRβ chains) and intracellular signaling (triggered by the CD3γ, CD3δ, CD3ε and CD3ζ chains) leading to cell activation. In contrast, a chimeric antigen receptor (CAR) is a synthetic receptor that comprises an extracellular antigen recognition domain and one or more intracellular signaling domains. CAR-based cell therapy targets cell surface tumor antigens with NK or T cells that are genetically modified to express a CAR, enabling them to target and kill cancer cells. In turn, intracellular antigens presented by the major histocompatibility complex (MHC) class I on the surface of tumor cells are recognized by TCRs. TCR adoptive cell therapy (ACT) is typically based on autologous CD8 T lymphocytes, but allogenic NK cells can be adapted to express a TCR, if they are also transfected with CD3 and CD8 genes.

Human papillomavirus type 16 (HPV16), is an oncogenic virus that causes cervical cancer and other anogenital malignancies as well as head and neck carcinomas. The HPV16 E6 and E7 genes encode oncoproteins that promote degradation of TP53 and inactivate RB, respectively, and their continuous expression is necessary for tumor progression, which makes them ideal targets for ACT.

However, the effectiveness of TCR-modified immune cells as a standalone therapy against solid tumors, like cervical cancer, has been limited. Eventually, the MHC of cancer cells does not present enough of the E6 and E7 oncoproteins to be effectively recognized and targeted by the modified immune cells. Moreover, solid tumors often create a microenvironment that is hostile to immune cells and suppress their activity, making them less effective at targeting and destroying cancer cells. While the strategy of targeting HPV16 E6 and E7 oncoproteins is appealing, the practical implementation faces significant limitations. There is a need for new approaches to improve ACT, potentially combining TCR-based cell therapy with other strategies.

One way to overcome these limitations is shown in a new study published in Journal Cancer Letters by Dr. Mansour Poorebrahim, Dr. Isaac Quiros-Fernandez (both contributing equally to the study) and Dr. Angel Cid-Arregui from the Targeted Tumor Vaccines and Applied Tumor Immunity group, German Cancer Research Center (DKFZ), Heidelberg, Germany together with Dr. Frederik Marmé from Heidelberg University and Dr. Stefan Burdach from Technical University of Munich. The researchers devised a strategy that combined a TCR specific to the HPV16 E7 oncoprotein (E7-TCR) with a CAR targeting TROP2, a cell surface antigen overexpressed in various types of cancer, including HPV-associated cancers. This was shown by a meta-analysis of the gene expression profile of cervical cancer samples, available in the Gene Expression Omnibus (GEO) public database, which was compared with the gene expression data of healthy tissues.

In their study, they constructed a gene coding a CAR against TROP2, which comprised the human CD8a leader signal peptide, a Myc tag, the scFv of an anti-TROP2 antibody, and the intracellular signaling domains of CD28 and 4-1BB. Then they used a lentiviral vector to transduce Jurkat J76 and NK-92 cell lines with the TROP2-CAR gene. In addition, they synthesized human CD3 and CD8 synthetic genes that were needed to generate NK-92 cells stably expressing all CD3 and CD8 subunits, which are required for proper surface expression and functionality of the E7-TCR in the NK-92 cells. Furthermore, primary CD8 T cells were electroporated with plasmids carrying the E7-TCR and T-CAR to test their activation capacity upon co-culture with HPV16+ and TROP2+ tumor cells. Various assays were conducted to measure cell-cell binding affinity, avidity and activation of the effector cells upon co-culture with tumor cells, and the cytotoxic capacity of the NK-92/CD3/CD8 cell line and the engineered primary CD8 T cells expressing the E7-TCR and T-CAR constructs.

The authors demonstrated that NK-92/CD3/CD8 cells expressing both E7-TCR and TROP2-CAR have significantly enhanced antigen-specific activation and cytotoxicity against HPV16+ tumor cells. The TROP2-CAR design focused on targeting a tumor-associated antigen, thereby reducing the risk of on-target off-tumor toxicity. The experiments with primary CD8 T cells isolated from healthy donors showed enhanced activation upon co-culture with HPV16+/TROP2+ tumor cells compared with controls, corroborating the efficacy of the dual TCR/CAR strategy.

The integration of TCR and CAR technologies in NK cells represents a promising advancement in cancer immunotherapy. This study showing enhancing the activation and cytotoxicity of engineered immune cells against HPV16+ tumors not only highlights the potential of this approach in treating cervical and other HPV-associated cancers but also sets a precedent for future research in the field of adoptive cell therapy. The dual receptor approach, targeting both a viral antigen and an overexpressed tumor antigen, could be a paradigm shift in the immunotherapy of solid tumors, mitigating some of the limitations of current immunotherapeutic strategies.

Synergizing adoptive cell therapy: Boosting Cancer Cell Targeting with Dual TCR/CAR Engineering - Medicine Innovates
The combination of a TCR recognizing a neoantigen and a CAR against a tumor-associated antigen such as TROP2 enhances the signaling strength and contributes to reduce on-target off-tumor toxicity.


Poorebrahim M, Quiros-Fernandez I, Marmé F, Burdach SE, Cid-Arregui A. A costimulatory chimeric antigen receptor targeting TROP2 enhances the cytotoxicity of NK cells expressing a T cell receptor reactive to human papillomavirus type 16 E7. Cancer Lett. 2023;566:216242. doi: 10.1016/j.canlet.2023.216242.

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