Structure Guided Design of CCL27 CAR T-Cells Against CCR10 for Multiple Myeloma

• Published in: Blood 2024-11, Vol.144, p.2036-2036
• Main Authors: Chilakapati, Nikhil, Patino-Escobar, Bonell, deMontagnac, Jonathan, Johnson, Haley, Ramos, Emilio, Phojanakong, Paul, Salangsang, Fernando, Steri, Veronica, Karlon, William J, Wiita, Arun P
• Format: Article
• Language: English
• ISSN: 0006-4971
• DOI: 10.1182/blood-2024-206274

Background: Even in the BCMA CAR-T era, many multiple myeloma (MM) patients often relapse and need additional treatment options. Our group identified CCR10 as a therapeutic target for MM and showed it is correlated with significantly worse outcomes and relapse. Additionally, we developed anti-CCR10 CAR-Ts that utilized the natural ligand CCL27 as the binder (Ferguson et al., Nat Comm 2022). However, these proof-of-concept CAR-Ts only had moderate efficacy in in vitro experiments. Here, we further validated CCR10 as a therapeutic target for MM and utilized structure-guided and computational modeling approaches to improve the performance of these CCL27 CAR-Ts to the level of current BCMA CAR-Ts. Methods: CAR-Ts were generated by lentiviral transduction of CD3+ human T cells. In vitro and in vivo performance of CAR-Ts was assessed against cell lines engineered to express luciferase. Statistical analysis on cytotoxicity was performed by two-way ANOVA. We used the Seurat and Harmony packages in R for data analysis and integration of single-cell RNAseq data. AlphaFold3 was utilized for generating predicted structural models, visualized by ChimeraX. Murine studies were performed with 1e6 MM.1S intravenously implanted in NSG mice, with 3e6 CAR-Ts administered 8 days after tumor inoculation (n=3 mice/arm). Results: Flow cytometry of 16 primary MM samples, 10 previously published and 6 new cases, showed that all samples were positive for CCR10. We also confirmed the expression of CCR10 on 7 MM cell lines by flow cytometry. Analyzing single-cell RNAseq data from 9 MM patients and 7 healthy donors, we found CCR10 was highly expressed in myeloma patient plasma cells, with lower expression in healthy donor plasma cells. To improve on our proof-of-concept CAR-Ts, we used homologous chemokine-chemokine receptor structures, the limited literature on CCL27, and AlphaFold models to identify the importance of the N-terminus of CCL27 in its interaction with CCR10. This structure-guided approach suggested that there was scope to improve the binding of CCL27 by modifying N-terminal residues and that our N-terminal MYC tag may have prevented CCR10 binding. From this, we created a small mutational library of 10 unique CCL27 CAR-T constructs with modified N-terminal residues, while moving the MYC tag to the C-terminus. Screening this library for in vitro cytotoxicity against the MM cell lines MM.1S and AMO1, we identified two mutants that performed similarly to BCMA CAR-Ts in one o