Construction and Pre-Clinical Evaluation of a New Anti-CD19 Chimeric Antigen Receptor

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.

T Cell Exhaustion and CAR-T Immunotherapy in Hematological Malignancies

“…Relapse after CAR-T cell therapy in patients with relapsed/refractory ALL can be roughly divided into CD19-positive recurrence and CD19-negative recurrence [ 57 , 58 ]. The positive recurrence is manifested as poor proliferation and killing function of CAR-T cells, which may be due to the application of cytotoxic drugs before CAR-T cell infusion, the presence of persistent leukemic blasts in bone marrow and blood, and the defective scFv (single-chain variable fragment) binding kinetics and co-stimulatory molecules [ 59 ]. Therefore, the reversal of cancer-related T cell exhaustion is the key point to enhance the therapeutic effects of CAR-T immunotherapy.…”

Acute lymphoblastic leukemia relapse after CD19-targeted chimeric antigen receptor T cell therapy

“…Conversely, high leukemia burden could lead to CART19 rapid exhaustion; however, the importance of a lack of stimulation in this setting remains unknown, as more favorable outcomes have been observed in patients with lower leukemia burdens. In addition, proliferation could be influenced not only by the quantities of signaling as determined by leukemia burden, but also by the qualities of CAR-CD19 interaction, as indicated by binding kinetics [47], influenced by the scFv sequence. Optimizing the scFv sequence has been reported in the 2016 American Society of Hematology meeting to increase CART19 proliferation [47].…”

“…In addition, proliferation could be influenced not only by the quantities of signaling as determined by leukemia burden, but also by the qualities of CAR-CD19 interaction, as indicated by binding kinetics [47], influenced by the scFv sequence. Optimizing the scFv sequence has been reported in the 2016 American Society of Hematology meeting to increase CART19 proliferation [47]. Lymphodepletion is another factor.…”