Construction and Pre-Clinical Evaluation of a New Anti-CD19 Chimeric Antigen Receptor
Authors: Kramer Anne Marijn , Ghorashian Sara , Cheung Gordon Weng-Kit , Vetharoy Winston , Moulding Dale , Mekkaoui Leila , Onuoha Shimobi , Amrolia Persis J , Pule Martin A 
Published: October 2019

Abstract
Relapsed and refractory B-lineage acute lymphoblastic leukemia remain the leading cause of cancer related death in children and young adults. Clinical studies of adoptive cell immunotherapy, re-directing T cells against CD19 by endowing them with a chimeric antigen receptor (CAR), have shown considerable clinical responses. To date, 3 different binding domains (scFv) targeting CD19 have been used in CARs taken forward in clinical trials and we have constructed a new CD19-CAR, derived from a different anti-human CD19 antibody, clone CAT. Whether different binding affinities of the CD19 targeting domain, when significantly different, could affect CAR-mediated T cell functionality has not been evaluated in depth. We therefore investigated the impact of scFv affinity on CAR-mediated T cell function in vitro, as well as on anti-tumour efficacy in vivo.
We have generated 3 CD19-CARs only differing in their scFv, which were derived from 3 anti-human CD19 antibodies (Clones FMC63, 4G7 & CAT) respectively. All other structural variables of the CAR and the use of the 4-1BB endodomain were identical. The Kd values obtained by Biacore Surface Plasmon resonance (SPR) analysis ranged from 8.8 x 10-10 to 1.1 x 10-7. Differences in affinity were predominantly determined by the off-rates, leading to significantly quicker dissociation from its target in CAT scFv compared to FMC63 and 4G7. CAT-CAR transduced T-cells showed enhanced cytotoxic responses to the CD19+ cell line SUPT1-CD19 in 51Cr release assays (p<0.001) compared to 4G7 and FMC63. Moreover, CAT+ T-cells demonstrate an increased proliferative capacity following antigen specific stimulation and an increased capacity to produce IL-2 and TNFα (p<0.001).
A quick dissociation rate has been described to be of particular importance when targeting cells with low levels of antigen expression, as T cell functional avidity can be detrimentally affected when dissociation is prolonged (Thomas et al, Blood 2011). We therefore investigated cytotoxicity of CAR transduced T cells against a cell line engineered to express CD19 at very low levels. This demonstrated increased cytotoxicity by CAT+ T-cells as well as greater CD107a degranulation in response to low CD19 expressing targets compared to FMC63 or 4G7-transduced T cells. Similarly, CAT+ T-cells showed greater killing of NALM 6 cells at very low effector:target ratios, reflecting the ability of serial killing by CAT+ T-cells by virtue of their rapid dissociation from target cells. Live cell imaging studies by confocal microscopy analysis confirmed a higher number of serial engagements by CAT+ T-cells (p<0.001), as well as greater motility (p<0.001).
We are now studying the relative potency in a xenogeneic model of ALL, using a CAR-T cell dose that is purposefully lowered to a suboptimal range to study kinetic differences and tumor clearance. Preliminary data suggests that, transferred after exposure to leukemia, CAT+ T cells have a less exhausted phenotype and higher effector:target ratios 2 weeks after infusion. Further experiments, in which recipient mice are re-challenged with the same tumor, will assess differences in the ability of adoptively-transferred CAR T cells to form memory.
In conclusion, we have developed a novel CD19-CAR which confers enhanced cytotoxicity and proliferative responses compared to existing CD19-CARs. Our work indicates that the scFv binding kinetics impacts the functional avidity of CAR-transduced T cells, providing important implications for the design of future CARs, especially when tumour cells expressing low levels of antigen are targeted.

Disclosures
Onuoha: Autolus Ltd: Employment, Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.