New Optimized Academic Anti-BCMA CAR (CARTemis-1): How Does the Manufacturing Process Impact CAR-T Cell Features?
Authors: Sierro-Martínez Belén , Escamilla Gomez Virginia , Perez-Ortega Laura , Guijarro-Albaladejo Beatriz , Hernández-Díaz Paola , de la Rosa-Garrido María , Lara-Chica Maribel , Reguera Juan Luis , Sanoja Flores Luzalba , Arribas-Arribas Blanca , Montiel-Aguilera Miguel-Ángel , Carmona Gloria , Robles Maria-Jose , Caballero Teresa , Briones Javier , Einsele Hermann , Hudecek Michael , Pérez-Simón Jose A. , Garcia-Guerrero Estefania 
Published: December 2023

Introduction
CAR therapy targeting BCMA (B-cell maturation antigen) is under research as treatment for multiple myeloma (MM). However, given the lack of a plateau in most studies, it becomes imperative to pursue more effective alternatives. BCMA downmodulation, the presence of soluble BCMA or the immune dysfunction observed in MM are some limitations that may affect efficacy. Next-generation CAR-T cells are being designed exploring new approaches to overcome restrictions to CAR-T efficacy in MM. We present a novel optimized anti-BCMA CAR (CARTemis-1) and its translation to the clinic. We have specifically focused on the study of the dynamics of the immunophenotype of CARTemis-1 along the manufacturing process, analyzing how these variables might impact the quality of the final product and influence the release decision.
Materials and methods
CARTemis-1 was evaluated in preclinical in vitro and in vivo models. CARTemis-1 generation from three healthy donors was validated under GMP-conditions in CliniMACS Prodigy with IL-7/IL-15. Flow cytometry was performed to characterize the immunophenotype along the manufacturing process. Quality controls of the final CARTemis-1 product were performed according to the criteria of the Spanish Medicine Agency. Stability of the final cryopreserved CARTemis-1 cell product was also analyzed.
Results
CARTemis-1 is a second generation anti-BCMA CAR that co-expresses the truncated epidermal growth factor receptor (EGFRt) as a transduction marker and suicide gene. Two different versions of CARTemis-1 were synthesized with different spacer regions (short vs long) with the long version showing significantly greater anti-myeloma function in vitro compared to the short version (p&lt;0.001) (Figure 1A). During the preclinical validation, CARTemis-1 demonstrated potent anti-tumor capacity in vitro and in vivo, showing no susceptibility to the presence of soluble BCMA. Expansion with IL-2 or IL-7/IL-15 was compared (n=8), obtaining increased proliferation (mean 1.59x10 7 vs 3.07x10 7, respectively, p=0.002), increased stem cell memory and naïve population (mean 37.48% vs 43.95%, respectively, p=0.0052), and less PD1+LAG3+ levels (1.38% vs 0.55%, respectively, p=0.0338) with IL-7/IL-15. The generation of CARTemis-1 was validated under GMP-conditions closely monitoring the immunophenotype dynamics from leukapheresis to the final product. Through comprehensive evaluations, the optimal time-point for product release to achieve the best-fitness product was &gt;6 (specifically, day 8-10) with reduced levels of exhaustion markers (Figure 1B) maintaining hallmarks of T cell activation and anti-tumor potency. In vivo biodistribution of CARTemis-1 cells was studied in mice models without myeloma cells, finding preserved tissue structure without relevant morphological alterations in brain, cerebellum, intestine, lung, endometrium, ovary, testicle, spleen, and liver. We performed graft-versus-host disease (GVHD) in vivo mice models and found no increase in GVHD incidence following CARTemis-1 inoculation. Additionally, we assessed the cryopreserved product stability, and it met all specifications and retained its functionality for up to 12 months after cryopreservation. With this positive outcome and having obtained the approval of the Spanish Medicine Agency, we plan to proceed with a phase I/II clinical trial using CARTemis-1 for patients with multiple myeloma who have experienced relapse following allogeneic transplantation (EudraCT 2021-001955-15).
Conclusions
CARTemis-1 has been rationally designed to increase anti-tumor efficacy and overcomes sBCMA inhibition. To our knowledge, this is the first study analyzing the impact of the manufacturing process in the dynamics of the CAR-T immunophenotype highlighting the importance of the immunophenotypic characterization of CAR-T cells throughout the manufacturing process to define the optimal cell culture protocol and expansion time to increase CAR-T cell product fitness.