Chimeric Antigen Receptor (CAR) T Cells Specific for CD229: A Potentially Curative Approach for Multiple Myeloma

Abstract

Background: Chimeric Antigen Receptor (CAR) T cells targeting B Cell Maturation Antigen (BCMA) have proven effective in Multiple Myeloma (MM). However, BCMA only shows substantial expression in a subgroup of patients and it is only expressed on terminally differentiated plasma cells. We have previously shown that the surface antigen CD229 is homogenously expressed on the tumor cells of all MM patients and that it is also expressed on MM precursor cells, which are thought to be responsible for relapses after chemotherapy. In addition, we have shown that CD229 is essential for the survival of MM cells potentially preventing the patient's tumor cells from losing its expression under the selective pressure of a CAR T cell immunotherapy.Methods and Results: Considering CD229 a target with curative potential, we aimed at generating CD229-specific CAR T cells as a novel adoptive immunotherapy for MM. We generated the first fully human single-chain variable fragment (scFv) domains for use in our CD229 CAR constructs using antibody phage display. We selected 23 unique CD229 binders and using a high-throughput surface plasmon resonance (SPR) assay, we determined that our antibodies had low nanomolar affinities to CD229 and that they did not bind to other SLAM receptors. When expressed as CARs using a CD8α hinge and transmembrane domain with a CD3ζ signaling and a 4-1BB co-stimulatory domain, 15 / 23 constructs showed high surface expression and CD229 binding. From these clones we then selected clone 2D3, which we confirmed to strongly and specifically bind to 293T cells transfected with a CD229 construct. Using a commercially available antibody we had previously observed that in addition to MM cells, CD229 is also expressed on T cells, B cells, and NK cells. However, our clone 2D3 only showed minor staining of some resting T cells and B cells, and no staining of NK cells and activated T cells. However, it showed strong binding to MM and Burkitt's lymphoma cell lines and even stronger staining of primary myeloma cells. We found that the differential binding may be related to a difference in the expression of CD229 isoforms in these cell types.Using lentiviral gene transfer we engineered primary human T cells expressing a CD229 CAR based on 2D3. We then compared our CD229 CAR to a well-characterized CD19 CAR regarding CAR T cell manufacturing and function. Importantly, we found that CD229 CAR T cells expanded almost identically to CD19 CAR T cells (Fig. 1A). In addition, we determined PD-1 expression during CAR T cell manufacturing and did not observe any signs of sustained spontaneous T cell activation in the absence of antigen, called tonic signaling (Fig. 1B). Analyzing the T cell phenotype during CAR T cell production we found that CD229 CAR T cell phenotypes mirrored those of CD19 CAR T cells, including preferential expansion of CD8+ CAR T cells.We next determined the cytotoxic activity of our CAR T cells using various target cells. CD229 CAR T cells showed strong and specific cytotoxic activity against K562 cells transduced with a CD229 expression construct. We also observed strong cytotoxic activity against CD229+ MM cell lines, such as U-266 (Fig. 1C). We did not observe any cytotoxic activity of our CD229 CAR T cells against normal activated T cells or NK cells, with only limited cytotoxicity observed against B cells and some resting T cells. Importantly, we also did not observe any cytotoxicity against purified CD34+ hematopoietic stem cells. Finally, we determined the in vivo efficacy of CD229 CAR T cells using immunocompromised NOD.Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice after engraftment with U-266 myeloma cells. We found that CD229 CAR T cells had completely eradicated MM cells expressing luciferase after only 18 days, while mice treated with control CD19 CAR T cells or PBS still showed strong bioluminescence signal (Fig. 1D).Conclusions: We have developed the first fully human antibodies against CD229, as well as CD229 CAR T cells and have demonstrated their strong cytotoxic activity and selectivity for MM cells in vitro and in vivo. We plan to initiate clinical trials evaluating the safety and efficacy of our CAR T cells in patients with relapsed/refractory MM and other types of lymphomas. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.