High specificity of engineered T cells with third generation CAR (CD28-4-1BB-CD3-ζ) based on biotin-bound monomeric streptavidin for potential tumor immunotherapy

Front Immunol. 2024 Sep 19:15:1448752. doi: 10.3389/fimmu.2024.1448752. eCollection 2024.

Abstract

Introduction: Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity.

Method: Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain.

Results: We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells.

Discussion: First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity.

Keywords: Chimeric Antigen Receptor; Treg; engineered cells; immunosuppression; streptavidin-based CAR.

MeSH terms

  • Animals
  • Biotin
  • CD28 Antigens* / immunology
  • CD3 Complex / immunology
  • Cell Line, Tumor
  • Humans
  • Immunotherapy, Adoptive* / methods
  • Neoplasms / immunology
  • Neoplasms / therapy
  • Receptors, Chimeric Antigen* / genetics
  • Receptors, Chimeric Antigen* / immunology
  • Receptors, Chimeric Antigen* / metabolism
  • Streptavidin* / chemistry
  • T-Lymphocytes* / immunology

Substances

  • Streptavidin
  • Receptors, Chimeric Antigen
  • CD28 Antigens
  • Biotin
  • CD3 Complex

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grants co-funded by ERDF (FEDER) Funds from the European Commission “A way of making Europe” from the Instituto de Salud Carlos III (ISCIII) PI18/00506, PI22/00473, and DTS23/00037. It was also financed by grants from the consortium ACT4COVID funded by Cellnex-Telecom and by Foundation La Caixa (SP23-00007). JG-V was supported by the Predoctoral Research Training Contract Program by the Health Research Institute Carlos III and FEDER Funds (FI19/00173). SG-M was supported by the Youth Employment Program co-financed by the Madrid community and FEDER Funds (PEJ-2020-AI/BMD-17954). JR-M was supported by the Predoctoral Research Training Contract Program by the Health Research Institute Carlos III and FEDER Funds (FI23/00086). The funders had no role in study design, data collection, or analysis; the decision to publish; or the preparation of the manuscript.