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
Authors: O'Neal Julie , Ritchey Julie K. , Gladney Susan , Haas Gabriel J. , Abboud Ramzi , Rettig Michael P. , Bruges Cedric , Eissenberg Linda , DiPersio John F. 
Published: December 2023

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&lt;.001, 947 P&lt;.003, 977 P&lt;.0002, 980 P&lt;.008 BCMA P&lt;.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.