Pathological angiogenesis contributes to morbidity in a number of diseases including cancer, diabetic retinopathy and the neovascular form of age-related macular degeneration, leading to significant efforts to develop effective anti-angiogenic therapeutics for these conditions. The field is dominated by inhibitors of vascular endothelial growth factor (VEGF), yet angiogenesis can also be driven and modified by other factors. We have previously demonstrated that leucine-rich alpha-2-glycoprotein 1 (LRG1) contributes to abnormal vessel growth by activating a TGFß switch. Here we report the development and characterisation of a function-blocking fully humanised IgG4 and its Fab fragment, that bind to LRG1 with high affinity and specificity and inhibit vascular leakage in the mouse model of laser-induced choroidal neovascularisation. In summary, we have developed a therapeutic antibody that targets a VEGF-independent signalling axis, which may be effective in a number of conditions either as monotherapy or in combination with other vascular targeted therapies.

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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.

There is great individual variation in response to general anesthetics (GAs) leading to difficulties in optimal dosing and sometimes even accidental awareness during general anesthesia (AAGA). AAGA is a rare, but potentially devastating, complication affecting between 0.1% and 2% of patients undergoing surgery. The development of novel personalized screening techniques to accurately predict a patient’s response to GAs and the risk of AAGA remains an unmet clinical need. In the present study, we demonstrate the principle of using a fluorescent reporter of the membrane dipole potential, di-8-ANEPPs, as a novel method to monitor anesthetic activity using a well-described inducer/noninducer pair. The membrane dipole potential has previously been suggested to contribute a novel mechanism of anesthetic action. We show that the fluorescence ratio of di-8-ANEPPs changed in response to physiological concentrations of the anesthetic, 1-chloro-1,2,2-trifluorocyclobutane (F3), but not the structurally similar noninducer, 1,2-dichlorohexafluorocyclobutane (F6), to artificial membranes and in vitro retinal cell systems. Modulation of the membrane dipole provides an explanation to overcome the limitations associated with the alternative membrane-mediated mechanisms of GA action. Furthermore, by combining this technique with noninvasive retinal imaging technologies, we propose that this technique could provide a novel and noninvasive technique to monitor GA susceptibility and identify patients at risk of AAGA.

Immunocore’s ImmTAC™ (Immune Mobilising Monoclonal TCR Against Cancer) platform combines affinity-enhanced T-cell receptor (TCR)-based targeting with an anti-CD3 scFv effector function to activate a cytotoxic T-cell response against cancer cells. A key part to this process is the identification of tumour epitope specific TCRs from tumor antigen-reactive T-cells. Here, we describe an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes, coupled with rapid identification of TCR chains from individual clones using single cell sequencing. The process involves first strand cDNA generation and universal amplification using SmartSeq2 chemistry, followed by targeted sequencing of the TCR alpha and beta chains using next-generation sequencing (NGS). We have also leveraged the 10x Genomics VDJ/5’ counting platform to label and pool multiple experimental clones for repertoire sequencing within a single run. Together with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), we can reliably assign each T-cell clone to its sample of origin paired with transcriptomic information of epitope specific T-cell populations, linking TCR sequences to their functional phenotype. Citation Format: Karolina Lech, Lucia Correia, Max Beckmann, Maria Busz, Sean Collison, Sterenn Davis, Paraskevi Mallini, Sarah Scaife, Joseph Dukes, Bent K. Jakobsen, Luke Williams, Michelle Teng. Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B024.