ABSTRACT
We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 anti-body in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively-spliced EDB domain of fibronectin), retained the activity of the parental cyto-kine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent anti-tumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with check-point blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anti-cancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.
NOVELTY AND IMPACT STATEMENT In this study, we describe the generation of a novel fusion protein consisting of murine inter-leukin-12 fused to the L19 antibody (specific to the EDB domain of fibronectin). L19-mIL12 revealed favourable tumor to organ ratios 24h after intravenous administration and it was able to cure 60% of CT26 tumor-bearing mice. From a clinical perspective, the rapid clearance from circulation should ease the administration to patients as infusions could be stopped upon onset of side-effects.
Footnotes
Posted on non-comercial preprint servers: The data that support the findings of this study are openly available in bioRxiv at https://doi.org/10.1101/684100 (DOI: 10.1101/684100).
Financial Suppport: D.N. receives financial support of the ETH Zürich, the Swiss National Science Foundation (Grant Nr. 310030_182003/1), the ERC Advanced Grant “ZAUBERKUGEL” and the Federal Commission for Technology and Innovation (KTI, Grant Nr. 12803.1 VOUCHLS).
Conflict of interest disclosure: D.N. is co-founder and shareholder of Philogen, a biotech company that owns the L19 antibody. S.C., B.G., S.W., M.M. and A.V. are employees of Philochem AG. The authors have no additional financial interests.
Abbreviations used: ADCC: Antibody-dependent cellular cytotoxicity; APCs: Antigen presenting cells; CHO cells: Chinese hamster ovary cells; CTLA-4: cytotoxic T-lymphocytes-associated protein 4; ELISA: Sandwich enzyme-linked immunosorbent assay; Foxp3: Forkhead box P3 HE: haematoxylin and eosin; HPV: Human papillomavirus; IL12: interleukin 12; IFN-γ: interferon- γ; IP-10: interferon-γ-induced protein LLC: Lewis Lung carcinoma; MCC: Merkel Cell Carcinoma; MIG: Monokine induced by γ interferon MTD: Maximal Tolerated Dose NK cells: Natural killer cells; NKG2D: Natural Killer Group 2D; PD-1: Programmed cell death-1; PD-L1: Programmed cell death-ligand 1; Tregs: regulatory T cells; PAMPs: as pathogen associated molecular patterns; SDS-PAGE: Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis; TH1: Type 1 T helper cell; Tumor dLN: Tumor draining lymphnodes; VCAM-1: Vascular cell adhesion protein 1
-added list of abbreviations -added "data availability" statement -adapted statement regarding ethical approval of animal studies -Added DOI of preprint version of the manuscript -Cell lines are now listed with RRID identifier -Added that all experiment are performed with mycoplasma-free cells