Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.1922-1922
• Main Authors: O'Neal, Julie, Ritchey, Julie K., Gladney, Susan, Haas, Gabriel J., Abboud, Ramzi, Rettig, Michael P., Bruges, Cedric, Eissenberg, Linda, DiPersio, John F.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-183025

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P