Activity and biophysical properties related to clinical evaluation of a first-in-class EHL ROR1xCD3 T Cell Engager.
Authors: Granger David , Bhatt Shivam , Henne Patricia , Baccaro Annalisa , Fernando Dinusha , Gore Jasmine , Gbajumo Ephraim , O'Donovan Kieran , Chester Kerry , Nathwani Amit C. 
Published: June 2022

e14505 Background: NVG-111, a T cell engager (TCE) targeting ROR1 and CD3, is in clinical development for the treatment of Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Leukemia (MCL). This 55KDa molecule consisting of humanised tandem scFvs is administered into patients as a continuous intravenous infusion. The intrinsically short half-life of NVG-111 has its advantages, such as rapid management of CRS by cessation of infusion. Early safety and efficacy clinical data supports the development of an extended half-life (EHL) ROR1xCD3 TCE for ease of administration and convenience for patients, building on the favorable activity and biophysical attributes of NVG-111. A next generation therapy possessing such an EHL has potential to improve patient experience by offering a different drug administration paradigm. Methods: Several formats were designed and evaluated, incorporating different EHL technology solutions by increasing the size and enabling FcRn-mediated recycling. EHL TCEs and non-EHL TCEs were cloned in-format with Fc, HSA, or an HSA binding moiety into a suitable expression vector. The resultant clones were expressed using Expi293 cells and the potency of TCEs were evaluated by co-culture assays using MCL derived JeKo-1 target cells and T cells from healthy donor PBMCs. Cytotoxic responses and T cell activation as determined by CD69 expression were measured by flow cytometry. T cell activation was also assessed by measuring CD3 signalling in a reporter gene assay. Biophysical properties were evaluated by determining the post-purification expression titer, the aggregation profile using analytical SEC, and the impact of stability challenge in formulation buffer and serum on target cell binding and activity. Results: Reformatting NVG-111 into an IgG-like heterodimeric bispecific scFv-Fc resulted in loss of potency, which may be due to a change in geometry of binding and/or poorer target engagement. Activity and expression titer were also deleteriously affected by directly fusing NVG-111 to human serum albumin (HSA), or to an HSA binding moiety. Fusing NVG-111 directly to Fc resulted in maintained potency in cytotoxicity and T cell activation assays, the expression titer of parental NVG-111, and exhibited very low levels of aggregation. Stability studies performed in formulation buffer and in human serum showed that NVG-111-Fc was at least as stable as parent. Furthermore, other studies with this molecular format indicate that the half-life in mice is comparable to a heterodimeric bispecific scFv-Fc. Conclusions: NVG-111 has been successfully engineered into an EHL format that increased the molecular size and has the potential to engage in FcRn-mediated recycling. This format maintains functional activity, is stable, and expresses well with a favorable aggregation profile. Further molecular refinements are being actively evaluated in readiness for IND enabling studies.