Bicistronic CAR T Cell Against CD229 and BCMA Effectively Controls Multiple Myeloma

INTRODUCTION. Multiple myeloma (MM) is an incurable disease despite great advances in its therapeutic landscape. Thus, there is great interest in developing new therapies, such as chimeric antigen receptor (CAR) T cell therapy. BCMA is the most predominant antigen used as a CAR T cell target in MM, with encouraging outcomes. Despite high response rates, many patients relapse (Munshi, NEJM 2021; Berdeja, Lancet 2021). MM cells with negative/low BCMA expression are implicated as a reservoir preceding relapse (Brudno, JCO 2018; Cohen, JCI 2019; Da Vià, Nat Med 2021). For this reason, new potential myeloma antigens are currently being tested. CD229 (Ly9) antigen is homogenously expressed on the surface of MM tumor cells and it is essential for MM cell survival. In addition, it could be expressed on myeloma precursor cells, which are believed to be responsible for relapses (Olson, Clin Immunol 2019). Moreover, the decrease or loss of antigen expression is another recently described mechanism for relapse. Thus, dual targeting of different antigens may be another possible strategy to avoid it. A BCMA-BBζ CAR T cell product (ARI2h; NCT04309981) has been developed at our Institution for relapsed/refractory MM patients with encouraging results (Fernández de Larrea, EHA 2022). Hence, we aimed to generate a novel CD229 CAR T cell product for MM, with the idea to develop a bicistronic CAR T cell against CD229 and BCMA to avoid relapses due to loss of BCMA expression.

METHODS AND RESULTS. We developed hybridoma clones against the extracellular domain of CD229 and selected two clones that specifically bound to the 1st (hLy9 10.169) and 2nd Ig-like domains (hLy9 1.84) of the extracellular domain. The sequences of the heavy (HV) and light variable (LV) regions of each antibody were used to generate a total of 4 single-chain variable fragments (scFv) domains. We designed 4 CD229 CARs with a CD8α hinge and transmembrane domain, a 4-1BB costimulatory and CD3ζ signaling domains. The 2 CARs targeting the 1st domain were discarded, as they were not expressed on the T cell surface. We compared our two remaining CD229 CAR T cell products (CD229-VHVL-BBζ and CD229-VLVH-BBζ) with ARI2h. We confirmed that both monospecific CD229 CAR T cells lyse, proliferate, and secrete cytokines in response to BCMA⁺ CD229⁺ MM cell lines (U266^wt, MM1S^wt and OPM2^wt). The in vivo activity was determined using NSG mice after engraftment with U266^wt cells. We found that the group that received CD229-VLVH-BBζ had better survival compared to CD229-VHVL-BBζ. However, ARI2h was still better than both anti-CD229 products. To recapitulate human MM, we modeled the established BCMA heterogeneous disease by mixing 15% MM1S BCMA CRISPR KO (MM1S^{BCMA KO}) into bulk MM1S^wt. Treatment with a low (1x10⁶) dose of CAR T cells showed that the CD229-VLVH-BBζ group had a survival advantage compared to ARI2h. MM1S cells recovered from bone marrow at the time of euthanasia were BCMA^-/CD229⁺ (ARI2h group) and BCMA⁺/CD229⁺ (CD229-VLVH-BBζ group).

To generate dual-target CAR constructs simultaneously targeting CD229 and ARI2h, we cloned CD229-VLVH-BBζ and ARI2h genes in a single vector and associated a 2A self-cleaving peptide. Two new CARs were obtained, CD229-VLVH-BBζ[2A]ARI2h and ARI2h[2A]CD229-VLVH-BBζ. We confirmed the expression of both CARs at the surface of the same T cells when using the bicistronic CARs. Both bicistronic CAR T cells could lyse, proliferate, and secrete cytokines in response to tumor cell lines that expressed one or both target antigens (BCMA⁺/CD229^- [K562^{BCMA KI}], BCMA^-/CD229⁺ [MM1S^{BCMA KO}], and BCMA⁺/CD229⁺ [U266^wt, MM1S^wt and OPM2^wt]). In vivo analysis revealed effective and comparable tumor control in NSG mice carrying MM1S^wt cells when treated with ARI2h, and both bispecific products (Fig 1A). Furthermore, the in vivo model established with BCMA heterogeneous disease (MM1S^wt 85% + MM1S^{BCMA KO} 15%) and with a low (1x10⁶) dose of CAR T cells showed that mice receiving CD229-VLVH-BBζ[2A]ARI2h had longer survival compared to ARI2h and ARI2h[2A]CD229-VLVH-BBζ (Fig 1B).

CONCLUSION. We have developed the first bispecific CD229/BCMA CAR T cell against MM, demonstrating that a single bicistronic vector encoding BCMA- and CD229-targeted CAR T cells effectively controls BCMA-expressing and non-expressing MM cells. This could be translated into complete mitigation of escape mediated by BCMA loss.

Pérez-Amill:Gyala Therapeutics S.L.: Current Employment. Moreno:Janssen: Honoraria, Other: Travel grants. Ortiz-Maldonado:Janssen: Honoraria; BMS: Honoraria; Novartis: Honoraria; Kite: Honoraria. Cibeira:Celgene: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akcea Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosinol Dachs:BMS-Celgene: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; GlaxoSmithKline: Honoraria. Bladé Creixenti:Janssen, Calegen/BMS,Amgen, Takeda, Oncopeptides: Honoraria. Juan:Gyala Therapeutics S.L.: Research Funding. Urbano-Ispizua:Miltenyi: Consultancy; Celgene: Consultancy; Gilead: Consultancy. Fernandez de Larrea:BeiGene: Consultancy, Honoraria; Pfizer: Honoraria; Sanofi: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.