NVG-222: A First-in-Class Autoregulating Half-Life Extended ROR1xCD3 T Cell Engager Heralding a New Class of Safer Drugs
Authors: Granger David , Muczynski Vincent , Henne Patricia , Baccaro Annalisa , Fernando Dinusha , Shah Mittal , Devlin John , O'Donovan Kieran , Nathwani Amit C. 
Published: December 2023

Background
T cell engagers (TCEs) are realizing their promise in both liquid and solid tumours, with several approvals in the last two years and many more in clinical development. However, the mechanism of action (MOA) that drives efficacy and the impressive clinical response rates comes at a cost. Activation and inflammatory signals induced by TCEs can push T cells into overdrive, resulting in grade 3+ treatment related adverse events (TRAEs). While step up dosing and pre-medication can help in limiting the severity of toxicity, there is a clear clinical need for safer TCEs with a lower frequency of high grade TRAEs to improve tolerability and widen the therapeutic index, which may ultimately result in better efficacy and responses. Our autoregulation (AR) platform technology, applicable across different biologic modalities and multiple indications, enables the deactivation of a drug in response to biological cues emanating from the MOA but only when levels associated with a high rate of toxicity are reached. AR's threshold-based, response-mediated negative feedback loop has the potential to improve safety without affecting potency and is based on similar physiological regulatory pathways that maintain homeostasis.
Objective
Establish proof-of-concept with AR in the context of NVG-222, our next generation half-life extended (HLE) ROR1-targeting TCE.
Methods
AR peptides sensitive to the proteolytic activity of immune cell specific protease granzyme B were carefully selected to maintain integrity at the low concentrations of granzyme B associated with TCE potency, whilst undergoing proteolysis at the high concentrations reported in patients with grade 3+ TRAEs. These peptides were embedded in the linker between both targeting arms of ROR1xCD3 TCEs formatted as tandem scFvs. Cytotoxic response and cytokine release potential of TCEs with and without AR were tested in a T cell coculture assay using the ROR1 + Mantle Cell Lymphoma cell line JeKo-1. Safety and efficacy was evaluated in vivo using a model of severe cytokine-induced toxicity in NOD-Scid gamma (NSG) mice engrafted with a ROR1 + solid tumour of MDA-MB-231 cells and reconstituted with human T cells. PK was assessed following a single intravenous injection in Tg32 mice.
Results
Selected AR peptides were exposed to a range of proteases present in circulation or in the tumour microenvironment and showed a specific and threshold-dependent sensitivity to granzyme B. A first prototype of an AR TCE was engineered from NVG-111, a ROR1xCD3 TCE in clinical development (NCT04763083), and showed uncompromised cytotoxic potency in vitro with an EC 50 in the pM range. This indicates that insertion of a granzyme B sensitive AR peptide does not lead to premature inactivation of the drug. Further evaluation in vivo showed that exposure of tumour-bearing mice to high levels of parental NVG-111 induced severe symptoms of cytokine-induced toxicity, including rapid weight loss, that led to the ethical euthanizing of mice. In contrast, no symptoms of toxicity were observed in the cohort exposed to high levels of AR NVG-111, with all mice surviving to the end of the study. Both parental and AR NVG-111 induced the same level of tumour growth inhibition. The cohort of mice treated with AR NVG-111 had a low or undetectable rate of metastasis.
AR technology was then applied to our next generation HLE TCE NVG-222, which builds on the clinical success of NVG-111 by using the same structure at its core, but with a longer half-life that provides more convenient dosing. The cytotoxic potency of AR NVG-222 was shown to be largely equivalent to that of AR NVG-111 (EC 50=38pM vs 13pM respectively), suggesting that similar clinical efficacy should be observed with equivalent dosing. Furthermore, a PK study in Tg32 mice, known to correlate to human PK, showed a t 1/2 of ~12 days and further allometric scaling suggests a potential t 1/2 of ~14 days in clinic.
Conclusion
Our AR platform technology has the potential to improve the therapeutic index of immunotherapies leading to improved safety and efficacy outcomes. This technology has been applied to our next generation ROR1-targeting TCE NVG-222, which is in IND-enabling studies with the aim of beginning clinical development for hematological and solid malignancies in 2024.