Initial Phenotypic Characterization of Anti-SEM4A CAR T Cells
Authors: Celichowski Piotr , Nhu Nguyen Quynh , Charvatova Sandra , Durech Michal , Kotulova Jana , Radova Eva , Venglar Ondrej , Jelinek Tomas , Hajek Roman , Simicek Michal 
Published: December 2024

Background: The utilization of Chimeric Antigen Receptor (CAR) T cells has revolutionized cancer immunotherapy. Despite the initial promising outcomes, continuous response is limited due to resistance mechanisms associated with tumor tissue and issues intrinsic to CAR T cells. Recent studies suggested Semaphorin 4A (SEM4A) as a potential immunotherapeutic target in myeloma (Anderson et al., 2022, Blood). However, the presence of SEM4A on activated T cells may challenge the production and efficacy of anti-SEM4A CAR T cells.
Aim: This study aims to develop and evaluate the efficacy of anti-SEM4A CAR T cells, with a focus on early phenotypic alterations in T cells upon the introduction of SEM4A CAR.
Methods: Anti-SEM4A CAR constructs were designed by fusing an in-house developed single-chain variable fragment specific for SEM4A to the 4-1BB and CD3ζ signaling domains. Human T cells isolated from healthy donors' buffy coats (n=5) were transduced with the anti-SEM4A CAR or CD19 CAR using a lentiviral vector. The transduced CAR T cells were expanded and characterized for CAR expression, cytotoxicity, cytokine production, and proliferation in vitro. Non-transduced and anti-CD19 CAR T cells were used as controls to monitor immunophenotypic changes in anti-SEM4A CAR T cells.
Results: Anti-SEM4A CAR T cells demonstrated specific cytotoxicity against SEM4A-positive myeloma cell lines, accompanied by significant levels of pro-inflammatory cytokines such as IFN-γ and IL-2 upon antigen recognition. Compared to non-transduced or anti-CD19 CAR T cells, anti-SEM4A CAR T cells exhibited slower proliferation and significant changes in certain T cell subsets.
Flow cytometry analysis showed SEM4A surface expression in non-activated T cells predominantly in CD8+ T cells (8.2%), with only 2.1% of CD4+ cells expressing SEM4A. After activation, SEM4A expression in CD8+ cells of both non-transduced and anti-CD19 CAR T cells increased to 19.6% (p&lt;0.001) by day 14, while SEM4A expression in CD4+ T cells peaked at 30.8% (p&lt;0.001) on day 6 before declining to 6% (p&lt;0.001) by day 14. SEM4A was undetectable (&lt;1%, p&lt;0.001) in all anti-SEM4A CAR T cells from day 3 post-activation. Consequently, CD8+ T cells were significantly less abundant in anti-SEM4A CAR T cells (11%) compared to non-transduced and anti-CD19 CAR T cells (38.5% and 39.5%, p&lt;0.001 and p&lt;0.001, respectively) on day 14. Conversely, CD4+ T cells represented 83.5% of SEM4A CAR T cells, while untransduced and CD19 CAR+ T cells consisted of 56.7% and 55.4% CD4+ cells, respectively (p&lt;0.001).
Immunoprofiling of T cell subsets revealed that, in addition to fratricide, expression of anti-SEM4A CAR facilitated the preferential development of specific T cell subsets. A significant shift towards a T cell effector memory phenotype (CD27- CD45RA-) was observed, represented by 38.7% CD4+ and 44.6% CD8+ cells in anti-SEM4A CAR T cells on day 14 post-activation. In non-transduced and anti-CD19 CAR T cells, effector memory T cells constituted 10.5% and 13.8% of CD4+ (p&lt;0.001 and p&lt;0.001), and 16.7% and 13.9% of CD8+ (p&lt;0.01 and p&lt;0.001) on day 14, respectively. Naïve T cells (CD27+, CD45RA+) were significantly diminished in anti-SEM4A CAR T cells (17.4% CD4+ and 18.1% CD8+ cells) compared to non-transduced and anti-CD19 CAR T cells (55.9% and 45.3% CD4+, p&lt;0.001 and p&lt;0.001, respectively) and (61.5% and 50.3% CD8+, p&lt;0.001 and p&lt;0.001, respectively) on day 14. Slight differences in other CD4+ and CD8+ T cell subsets (central memory, TEMRA) were observed between controls and SEM4A CAR T cells.
Conclusion: This study provides the initial phenotypic characterization of novel anti-SEM4A CAR T cells. Our results highlight the critical importance of SEM4A in T cell differentiation. Early fratricide of SEM4A+ CD8+ T cells and a lack of transient SEM4A expression in CD4+ cells result in the preferential development of effector memory T cells and reduction of naïve T cell subsets upon introduction of anti-SEM4A CAR. These findings underscore the need for further refinement of production protocols for anti-SEM4A CAR T cells, particularly enhancing strategies to promote central memory T cells to create a safer and more persistent therapeutic product.