Allogeneic CAR-T Therapy Technologies: Has the Promise Been Met? Caroline Lonez and Eytan Breman Celyad Oncology SA, Belgium; ebreman@celyad.com Correspondence: clonez@celyad.com; Tel.: +32-(0)-10-39-41-00 Abstract: Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has become a real treatment option for patients with B-cell malignancies, while efforts are being made to extend this therapy to other malignancies and broader patient populations. However, several limitations remain, including those associated with the time-consuming and highly personalized manufacturing of autologous CAR-Ts. Technologies to establish "off-the-shelf" allogeneic CAR-Ts with low alloreactivity are currently being developed, with a strong focus on gene-editing technologies. Although these technologies have many advantages, they have also strong limitations, including double-strand breaks in the DNA with multiple associated safety risks as well as the lack of modulation. As an alternative, non-gene-editing technologies provide an interesting approach to support the development of allogeneic CAR-Ts in the future, with possibilities of fine-tuning gene expression and easy development. Here, we will review the different ways allogeneic CAR-Ts can be manufactured and discuss which technologies are currently used. The biggest hurdles for successful therapy of allogeneic CAR-Ts will be summarized, and finally, an overview of the current clinical evidence for allogeneic CAR-Ts in comparison to its autologous counterpart will be given. Keywords: allogeneic; chimeric antigen receptor; off-the-shelf; gene editing Citation: Lonez, C.; Breman, E. Allogeneic CAR-T Therapy Technologies: Has the Promise Been Met? Cells 2024, 13, 146. https://doi.org/10.3390/cells13020146 Academic Editor: Kazuhiko Ikeda Received: 29 November 2023; Revised: 9 January 2024; Accepted: 10 January 2024; Published: 12 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 1. Introduction Over the past decades, immunotherapy has become an important treatment option for patients with cancer. Among the most promising options, T-cells engineered to express chimeric antigen receptors (CARs) aim to strengthen the power of T-cells to recognize and eliminate tumor cells inAllogeneic CAR-T Therapy Technologies: Has the Promise Been Met? Caroline Lonez and Eytan Breman Celyad Oncology SA, Belgium; ebreman@celyad.com Correspondence: clonez@celyad.com; Tel.: +32-(0)-10-39-41-00 Abstract: Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has become a real treatment option for patients with B-cell malignancies, while efforts are being made to extend this therapy to other malignancies and broader patient populations. However, several limitations remain, including those associated with the time-consuming and highly personalized manufacturing of autologous CAR-Ts. Technologies to establish "off-the-shelf" allogeneic CAR-Ts with low alloreactivity are currently being developed, with a strong focus on gene-editing technologies. Although these technologies have many advantages, they have also strong limitations, including double-strand breaks in the DNA with multiple associated safety risks as well as the lack of modulation. As an alternative, non-gene-editing technologies provide an interesting approach to support the development of allogeneic CAR-Ts in the future, with possibilities of fine-tuning gene expression and easy development. Here, we will review the different ways allogeneic CAR-Ts can be manufactured and discuss which technologies are currently used. The biggest hurdles for successful therapy of allogeneic CAR-Ts will be summarized, and finally, an overview of the current clinical evidence for allogeneic CAR-Ts in comparison to its autologous counterpart will be given. Keywords: allogeneic; chimeric antigen receptor; off-the-shelf; gene editing Citation: Lonez, C.; Breman, E. Allogeneic CAR-T Therapy Technologies: Has the Promise Been Met? Cells 2024, 13, 146. https://doi.org/10.3390/cells13020146 Academic Editor: Kazuhiko Ikeda Received: 29 November 2023; Revised: 9 January 2024; Accepted: 10 January 2024; Published: 12 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 1. Introduction Over the past decades, immunotherapy has become an important treatment option for patients with cancer. Among the most promising options, T-cells engineered to express chimeric antigen receptors (CARs) aim to strengthen the power of T-cells to recognize and eliminate tumor cells in