(OA-10) MZB1-Directed HLA-Dependent CAR T Cells for Targeting B-Cell Malignancies

BCMA-directed CAR T cell therapies have improved outcomes for MM patients, and several other targeted CAR T cells are in development. However, one of the main hurdles is the limited availability of tumor-specific cell surface molecules. Unlike conventional CAR T cells, TCR-like CAR T cells can recognize intracellular tumor antigens presented on MHC molecules, mimicking T cell receptors (TCR), and significantly expanding the range of potential cancer targets. We identified MZB1 as a tumor-associated intracellular antigen in MM and predicted putative MZB1 peptides that can be presented in the context of HLA-A*02:01, the most frequent HLA allele worldwide. Importantly, we also evaluated the expression of MZB1 in additional hematological malignancies and observed high levels of MZB1 in Waldenström Macroglobulinemia (WM) and non-Hodgkin lymphoma cells.

Methods: Based on expression data, we generated a novel antibody recognizing MZB1 bound to HLA-A*02:01 on the cell surface. Using a human scFv antibody yeast surface display library, we identified scFv clones specific to the MZB1/HLA-A2 complex. We confirmed MZB1- and HLA-A2-specific binding by flow cytometry and histology. Next, we used the MZB1-A2 scFv to generate MZB1 CAR T cells. Human T cells from donor PBMCs were transduced to express MZB1 CAR.

Results:

We confirmed the specificity of MZB1 CAR T cells to MZB1/A2 complex and their inability to bind irrelevant pMHCs. In vitro killing assays showed MZB1 CAR T cells had significant cytotoxicity against MZB1⁺ and HLA-A2⁺ MM and WM cells, but not against HLA-A2^neg and/or MZB1^neg cells, in a dose-dependent manner. MZB1 CAR T cells secreted higher levels of IFN-γ, sFas ligand, and granzyme A/B compared to untransduced T cells when co-cultured with MZB1⁺ HLA-A2⁺ MM cells. Antitumor activity was evaluated against primary patient myeloma and Waldenström cells. MZB1 CAR T cells displayed significant activity against primary MZB1⁺ HLA-A2⁺ CD138⁺ from BM of ND and relapsed MM patients and MZB1⁺ CD19⁺ WM cells. In an in vivo disseminated orthotopic NSG MM mouse model, a single infusion of MZB1 CAR T cells significantly reduced tumor burden.

Importantly, we also observed an HLA cross-reactivity of our antibody as well as CAR T product, with MZB1 also being recognized by other HLA-A alleles, such as HLA-A*24:02 and HLA-A*23:01, which are more common in African Americans and Asian Americans, respectively, suggesting broad applicability of our antibody across patient groups.

Conclusions:

In conclusion, we have generated a MZB1-directed HLA-dependent CAR T cell therapy that exhibit significant activity and specificity against MZB1 and HLA-A2 positive MM and WM cells, both in vitro and in vivo. This study therefore provides the rationale to evaluate MZB1 TCR-like CAR T cell therapy as a therapeutic approach in MM and WM and provide the logic and framework for similar therapies to be developed against other intracellular antigens in MM and other cancers.