Abstract 5757: Preclinical development and testing of a therapeutic antibody against LRG1

Abstract

Introduction: The purpose of this study was to generate a therapeutic function-blocking antibody against leucine-rich alpha-2-glycoprotein 1 (LRG1), a novel regulator of pathological angiogenesis that is up-regulated in many cancers, and demonstrate efficacy in proof of principle experiments in mouse models. Experimental Procedures: Mice were immunized with recombinant human LRG1 and hybridomas generated. More than 100 monoclonal antibodies (mAbs) were first screened by surface plasmon resonance (SPR) to measure affinity for target, and then in a co-culture angiogenesis assay to evaluate functional activity. The most promising candidates were then tested in vivo in the mouse model of laser-induced choroidal neovascularization, and from this study a lead was humanized and deimmunized. The resulting antibody is a hinge-stabilized IgG4 that is now undergoing manufacturing and pre-clinical safety and toxicology testing. Results: Of the original set of mouse mAbs, an initial screen to eliminate low affinity (KD greater than 1nM) binders resulted in 25 progressing to functional testing in angiogenesis assays. The seven most effective blocking mAbs were then tested in vivo in the mouse model of laser-induced choroidal neovascularization. Intraocular injections of mAb yielded two that exhibited equivalent inhibition of neovascularization to a therapeutic VEGF-blocker, and of these, the higher affinity mAb (15C4) was selected for humanization. Epitope mapping identified an amino-acid sequence within human LRG1, that in the form of a synthetic peptide was able to compete the binding of 15C4 to LRG1. Multiple species sequence alignment revealed the epitope to be partially conserved with the corresponding mouse sequence, and SPR analysis showed that 15C4 binding to recombinant mouse LRG1 was several orders of magnitude lower than to human LRG1. Nevertheless, administration of 15C4 to tumor-bearing mice significantly inhibited tumor growth in a range of subcutaneous and genetic models. Furthermore, due to the vessel normalizing effects of 15C4, combinations of mAb with both immunotherapy and cytotoxics were significantly more effective at restricting tumor growth than either monotherapy. Conclusions: We have generated and characterized a novel vessel-normalizing, fully humanized function-blocking mAb against LRG1 that shows efficacy in mouse tumor models. GMP manufacture is on-going, and early stage clinical trials are planned in 2019. Financial Support: The work was supported by the Medical Research Council, the Rosetrees Trust and UCL Business. Citation Format: Stephen Moss, David Kallenberg, Vineeta Tripathi, Sterenn Davis, Jestin George, Marie O'Connor, Laura Dowsett, John Greenwood. Preclinical development and testing of a therapeutic antibody against LRG1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5757.

Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis

“…5D). To rule out any direct effect of LRG1 on tumor cell extravasation, we used an LRG1-neutralizing antibody 15C4 ( 35 , 36 ). Mice were preconditioned with a single injection of either 15C4 or control-IgG (immunoglobulin G) before intravenous injection of melanoma cells (fig.…”

“…To assess the therapeutic potential of blocking LRG1 during metastatic progression, we administered an LRG1-neutralizing antibody 15C4 ( 35 , 36 ) as postsurgical adjuvant therapy in two spontaneously metastasizing mouse models, the LLC and the MMTV-PyMT model (Fig. 6A).…”

“…Lastly, to assess the therapeutic potential of the LRG1-neutralizing antibody 15C4 28 in clinically relevant settings, we adopted two therapeutic strategies -short-term perioperative and longterm postsurgical adjuvant therapy (Fig. 4f).…”