ALBERT

Description: Targeting CAR T resistance due to CD19 loss with CD79b-specific CAR T cells in B-cell malignancies Fuliang Chu, JingJing Cao, Jingwei Liu, Xiaoyun Cheng, Swathi Karri, Adithi Reddy, Shao Qing Kuang, Jinsheng Weng, Sattva S. Neelapu Department of Lymphoma and Myeloma, The University of Texas M. D. Anderson Cancer Center. Houston, TX 77030 USA Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in B-cell malignancies and two CD19-targteing CAR T-cell therapy products were recently approved by the US FDA for relapsed or refractory B-cell acute lymphoblastic leukemia and/or large B cell lymphoma. In pivotal trials, durable remissions lasting more than 1 year have been observed in ~40-50% of these patients. However, relapse or progression occurs in ~50-60% and a major cause of resistance appears to be due to CD19 antigen loss. Thus, there is an urgent need to develop CAR T-cell therapies against novel targets to further improve outcomes in these patients. CD79b is a pan B-cell linage marker and an important component of the B-cell receptor complex. CD79b is broadly expressed in normal B cells and B-cell malignancies and its expression is usually retained in CD19 negative tumors progressing after CD19-specific CAR T-cell therapy. Here, we report our results with a novel CD79b-specific CAR T cell product in in vitro and in vivo models. We generated five murine monoclonal antibodies against human CD79b by hybridoma technology and demonstrated that they bind specifically to recombinant human CD79b, have high affinity (Kd range of 1.44-18.1 nM), and stain multiple B-cell lymphoma cell lines comparable to the commercial CD79b fluorochrome-labeled antibodies. Next, we cloned the variable regions of the heavy and light chains of the CD79b antibodies, and developed lentiviral constructs for CD79b-specific CARs with CD3z and CD28 costimulatory domains. We demonstrated that the CD79b-CAR lentiviral constructs can be transduced into primary CD4+ and CD8+ T cells from healthy donors to 〉85% transduction efficiency. We observed that the CD79b-specific CAR T cells but not untransduced T cells had significant cytotoxic activity that was comparable to control CD19-specific CAR T cells against Daudi Burkitt lymphoma and Mino mantle cell lymphoma cell lines. More importantly, CD79b- but not CD19-specific CAR T cells lysed CD19-CD79b+ lymphoma cells. Significant CD107a/b degranulation was also observed in both CD4+ and CD8+ CD79b-specific CAR T cells when they were co-cultured with lymphoma cell lines. Multiplex cytokine analysis of co-culture supernatants revealed predominantly release of IFN-γ. The efficacy of CD79b-specific CAR T cells was also examined in vivo against Mino xenograft models in NSG mice. Luciferase-labeledMino mantle cell lymphoma cell line was injected IV into NSG mice at 2x106 tumor cells/mouse. After 18 days, mice were treated with untransduced primary T cells, CD19-specific CAR, or CD79b-specific CAR T cells via tail vein at 10x106 T cells/mouse. Bioluminescence imaging was used to assess tumor burden. Tumor growth was inhibited and survival was markedly improved in mice treated with CD19- and CD79b-specific CAR T cells but progressive tumor growth was observed in the control group treated with untransduced T cells. Additional studies using CD19-CD79b+ lymphoma cell line xenograft model are ongoing and will be presented at the meeting. In conclusion, these preclinical results indicate that CD79b is a novel target for CAR T-cell therapy and support its evaluation in patients with B-cell malignancies. Importantly, our results suggest that targeting CD79b could be a novel strategy to overcome resistance due to CD19 loss after CD19-specific CAR T-cell therapy. Disclosures No relevant conflicts of interest to declare.

Description: Chimeric antigen receptor (CAR)-modified T-cell therapy targeting CD19 induces high response rates in patients with relapsed or refractory B-cell lymphomas. However, about 60% of patients experience primary or secondary resistance after CD19-targeted CAR T-cell therapy and a major of cause of failure appears to be due to loss of CD19 expression on the tumor. Therefore, novel targets for adoptive T-cell therapeutic approaches are needed to further improve clinical outcome in these patients. T-cell leukemia/lymphoma antigen1 (TCL1) is an oncoprotein that is overexpressed in multiple B-cell malignancies including follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and chronic lymphocytic leukemia (CLL). Importantly, it has restricted expression in only a subset of B cells among normal tissues. We previously identified a TCL1-derived HLA-A2-binding epitope (TCL170-79 SLLPIMWQLY) that can be used to generate TCL1-specific CD8+ T cells from peripheral blood mononuclear cells of both HLA-A2+ normal donors and lymphoma patients. More importantly, we showed that the TCL1-specific CD8+ T cells lysed autologous primary lymphoma cells but not normal B cells (Weng et al. Blood 2012). To translate the above discovery into clinic, we cloned the T-cell receptor (TCR) alpha and beta chains from a TCL1-specific CD8+ T-cell clone and showed that this TCL1-TCR could be transduced into polyclonal donor T cells using a lentiviral system with a transduction efficiency of 〉40% as determined by TCL170-79 tetramer positive T cells. Furthermore, we demonstrated that the TCL1-TCR-transduced T cells recognized T2 cells pulsed with TCL170-79 peptide producing IFN- γ 〉8 ng/ml and IL-2 〉350 ng/ml but were not reactive to control HIV-Gag peptide (IFN- γ