Background IL-12 is a potent cytokine linked to activation of innate and adaptive immune system for anti-tumour immunity.1 The clinical development of IL-12 therapy has been constrained with severe toxicity reported from systemic administration of IL-12. Toxicity is attributed to poor pharmacokinetics of IL-12, necessitating frequent dosing, and nonspecific distribution. Recently, there has been a renewed interest in IL-12 therapy with various strategies to improve half-life and reduce toxicity, with lead candidates in preclinical and early clinical stages of development.2 Here, we describe novel bispecific BCA356 with an affinity matured humanized anti-CAIX antibody and attenuated IL-12 subunits fused to each of the heavy chains at the C-terminus by a linker in a knob-in-hole format.
Methods In silico mutational screening was performed on the p40 and p35 subunits of IL-12 to identify optimally attenuated IL-12 variants (IL-12vs). CAIX as a tumour-targeting antigen was selected based on high expression of CAIX across several solid tumour types. Expression of cellular and soluble CAIX across tumour types was evaluated by immunohistochemistry (IHC) on tissues and ELISA on tumour-matched plasma. BCA356 was identified after IL-12vs were screened in a HEK-Blue™ IL-12 assay (figure 1) followed by phosphorylated STAT4 expression on CD8+ T cells and IFNγ release by PHA-stimulated PBMCs/IL-2 primed NK-cells. In in vitro 2 and 3D co-culture assays with CAIX-overexpressing cell line and PBMCs, cytotoxicity and cytokine/chemokine release were assessed across days. Efficacy and safety of BCA356 were evaluated in PBMC-based humanized mice models and human IL-12 and IL-12 receptor gene knock-in transgenic mice.
Results IHC studies confirmed high CAIX expression in clear cell renal carcinoma3 and multiple other tumour types, a significant number of samples, expressed high and moderate CAIX expression (figure 2). BCA356 significantly attenuated IFNγ release by stimulated PBMCs, activated NK-cell and pSTAT4 expression in CD8 T cells as compared to IL-12(wt). In co-culture assays, BCA356 showed cytotoxicity of cancer cells comparable to IL12(wt) (figure 3) without significant cytokine release unlike IL12(wt) (figure 4). Finally, efficacy studies in PBMC-based humanized mice models and transgenic mice models confirm that BCA356 is efficacious and safe in CAIX-overexpressing tumour bearing mice.
Conclusions BCA356 specifically targets CAIX-expressing tumour cells and similar to wild type IL-12 cytokine, has the potential to reduce tumour proliferation with optimal activation of pro-inflammatory cytokines. Through these in vitro and in vivo studies, we demonstrate that BCA356 by its CAIX-targeted IL-12v delivery approach is both efficacious and safe.
Acknowledgements The authors would like to acknowledge the help extended by Biocon Limited in running this study. We would also acknowledge the assistance provided by personnel at Syngene vivarium facility for supporting the animal studies at their dedicated vivarium.
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Holder PG, Lim SA, Huang CS, Sharma P, Dagdas YS, Bulutoglu B, et al. Engineering interferons and interleukins for cancer immunotherapy. Adv Drug Deliv Rev 2022;182:114112
Pastorekova S, Gillies RJ. The role of carbonic anhydrase IX in cancer development: links to hypoxia, acidosis, and beyond. Cancer Metastasis Rev 2019;38:65–77
Ethics Approval Mice were maintained as per the regulations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India and Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. All animal experiments were approved by institutional ethical committee and performed under approved protocols. All animals were maintained at dedicated Syngene vivarium
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