e14506 Background: Bispecific antibodies provide an opportunity to treat a diverse range of disorders because of their ability to simultaneously attenuate several different pathways precisely and effectively, thus overcoming many of the limitations of monoclonal antibodies. The adaptability of bispecifics has facilitated the generation of compelling preclinical data covering a range of malignant and non-malignant disorders. Proof-of-concept early phase clinical trials with bispecific antibodies, including bispecific T-cell engagers (TCE) have been hindered by manufacturing challenges, including a heavy burden in terms of time, quality and costs associated with generating a stable producer cell line. We took a more agile approach, using transient transfection to manufacture a ROR1×CD3 bispecific TCE drug product (DP [NVG-111]) for a first in human, Phase I/II clinical trial. Methods: A HEK293T cell line was expanded into 10-layer cell factories. The cells were transfected with a ROR1xCD3 scFv bispecific antibody (NVG-111) plasmid and incubated. Post incubation, the supernatant containing NVG-111 was clarified by filtration and subjected to solvent/detergent viral inactivation. This was followed by a concentration/buffer exchange step and subsequent DNA reduction using an endonuclease. NVG-111 was captured using a bind/elute Protein A column and the eluate polished on a multimodal anion exchange column in flow through mode to remove residual impurities. The eluate was formulated and concentrated to the target protein concentration, viral and sterile filtered and QC tested. The bulk drug substance was 0.2 µm filtered into Din 2R vials and the DP stored at ≤-65°C pending release. Results: The transient transfection approach resulted in the rapid tech transfer and GMP manufacture of clinical NVG-111 DP. Two batches of DP were manufactured producing 1,971 vials of NVG-111, sufficient to support the Phase I/II clinical trial in patients with advanced Chronic Lymphocytic Leukaemia and Mantle Cell Lymphoma (Clinical Trial.gov NCT04763083). Both batches met the approved specifications for identity, potency, purity/impurities, strength and safety. The exercise took less than 7 months from contract signature to DP in the freezer and cost ̃50% of the more traditional producer cell line approach. Conclusions: We established a novel GMP process in 7 months, using transient transfection to manufacture NVG-111 for Phase I/II trials. The process costed less than the more conventional manufacturing approach of using a producer cell line. The strategy offers a rapid, and very efficient way of reaching a first in human study without a trade-off between time, quality and cost.
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.
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