A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

An APC-conjugated anti-6XHis antibody was used for BsAb detection by flow cytometry.

CAR). ET190L1-CAR-T cells or FMC63-CAR-T cells were incubated with targets cells for 16
hours at an E:T of 5:1. Cytotoxicity was measured by LDH release assay (n= 3 technical replicates). Error bars, SEM.

Generation of ET190L1 (human anti-CD19 antibody)
A collection of human single-chain variable fragment (scFv) antibody phage display libraries (diversity= 10x10 10 ) constructed at Eureka Therapeutics (E-ALPHA® Phage Display) was used for the selection of human antibodies specific to human CD19. Eureka's E-ALPHA® Phage Display library is comprised of both naïve and semi-synthetic human scFv antibodies. The scFv libraries were used in panning against recombinant human CD19 ECD-Fc fusion protein and human CD19 positive cells including Raji and 3T3 cells engineered to express CD19. Three rounds of panning were performed to enrich for scFv phage clones that specifically bound the extracellular region of human CD19. These panning campaigns identified 8 specific and unique (by sequence) phage clones that bound human CD19. Affinity maturation and clone optimization led to the selection of a lead clone (ET190L1) for further characterization and pre-clinical testing.

Antibodies Target
Clone Antibodies were purchased from Biolegend. All flow cytometry samples were run on a FACS Canto II or Fortessa X-20 (BD Biosciences) and the data was analyzed using FlowJo software (TreeStar).

T-cell activation and degranulation assays:
ET190L1-CAR-T cells and ET190L1-AbTCR-T cells were activated with target cells at an E:T ratio of 2:1 for 16 hours. Expression of activation markers (CD25 and CD69) and exhaustion markers (PD-1, LAG-3, and TIM-3) were determined by flow cytometry. For the degranulation assay, ET190L1-AbTCR-T cells (1x10 5 ) were pre-mixed with anti-CD107a antibody and 2M Monensin prior to activation with target cells. Target cells (1x10 5 ) were added at an E:T ratio of 1:1 for 4 hours. Unstimulated cells served as a negative control. Degranulation was determined by flow cytometry.

ET190L1-CAR-T cells or ET190L1-AbTCR-T cells were serum starved overnight in RPMI + 2%
FBS and then labeled with 1M CFSE (ThermoFisher Scientific) for 5 minutes at room temperature. The reaction was stopped by washing with PBS + 5% FBS. The labeled cells were re-suspended in RPMI +10% FBS and co-cultured with target cells at an E:T ratio of 2:1. To account for differences in transduction efficiency, donor-matched un-transduced T cells were used to normalize the percentage of receptor-positive cells. Cell division was monitored by flow cytometry.
ET190L1-CAR-T cells and ET190L1-AbTCR-T cells were co-cultured with target cells at an E:T ratio of 5:1 for 16 hours. To account for differences in transduction efficiency, donor-matched untransduced T cells were used to normalize the percentage of receptor-positive cells. The amount of LDH released into the supernatant was measured following the manufacturer's protocol. Cell lysis was calculated using the following formula: (Sample -Target Background -Effector Background ) / (Maximum Target lysis -Target Background ). Maximum target lysis was achieved by adding lysis solution (Promega) to the target cells.