Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors
Authors: Mardiana Sherly , Shestova Olga , Ruella Marco , Gill Saar 
Published: February 2021

BACKGROUND
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19, CD22, or B cell maturation antigen (BCMA). Antigen density is an important factor modulating CAR T cell responses, and therefore antigen expression below a certain threshold may fail to induce optimal CAR-T cell response. This presents a significant problem as antigen expression varies highly in many cancers including lymphoma, leukemia, multiple myeloma, and solid tumors, both between and within patients. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two antigens will result in enhanced CAR T cell signaling, allowing them to attack low antigen density (LAD) tumors that would otherwise escape CAR T cells.
METHODS
Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 (FMC63), CD22 (CD22-12), or both, herein referred to as CAR19, CAR22, or CAR19/22, respectively. Tumor cells expressing variegated antigen densities were generated using lentivirus transduction or mRNA electroporation. For in vitro functional characterization, co-culture assay of T cells and tumor cells was performed, and CAR T cell anti-tumor potency was determined by assessing T cell functional parameters including T cell cytotoxicity in real-time using the Incucyte® systems for live-cell imaging and analysis. In addition, cytokine production and CFSE-based proliferation were also assessed using flow cytometry.
RESULTS
Using CAR19/22, we found LAD tumors are indeed more susceptible to bi-specific CAR T cells compared to either CAR19 or CAR22 T cells, as demonstrated by significantly higher amounts of cytokines produced by bi-specific CAR T cells such as IL-2 (p&lt;0.0001) and IFNg (p&lt;0.0001) compared to CAR19 or CAR22 T cells. Results from cytotoxicity assay revealed increased killing of LAD tumors by CAR19/22 T cells compared to CAR19 (p&lt;0.0001) or CAR22 (p=0.0002) T cells. Further, we found bi-specific CAR19/22 T cells have increased proliferation capacity (p&lt;0.0001) compared to either of the mono-specific CAR T cells. Using NanoString-based RNA analysis, we confirmed that graded antigen density on tumor cells modulates CAR T cell gene expression, with cytotoxicity-associated genes being more susceptible to modulation by antigen expression than cytokine-associated genes. Upcoming experiments aim to test and compare therapeutic efficacy of bi-specific and mono-specific CAR T cells in vivo against LAD tumors. Further characterization of the immune synapse using confocal microscopy as well as analyses of early and late events in downstream CAR signaling will further illuminate the mechanism by which bi-specific CARs can overcome the challenge of low tumor-associated antigen density.
CONCLUSIONS
Here we showed that antigen density on tumors modulates T cell transcriptional profiles. Our results demonstrated that bi-specific CAR19/22 T cells are superior than mono-specific CAR19 or CAR22 T cells against LAD tumors, as demonstrated by their enhanced cytokine-producing function, cytotoxic capacity and proliferation. Results from this study will provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing the risk of antigen-negative escape.

Disclosures
Ruella: Abclon, BMS, NanoString: Consultancy; Abclon: Consultancy, Research Funding; UPenn/Novartis: Patents & Royalties. Gill:Tmunity Therapeutics: Research Funding; Sensei: Consultancy; Aileron: Consultancy; Fate: Consultancy; Carisma Therapeutics: Patents & Royalties, Research Funding; Novartis: Research Funding.