We aimed to develop a quantitative antibody-based near infrared fluorescence (NIRF) approach for the imaging of oxidized LDL in atherosclerosis. LO1, a well- characterized monoclonal autoantibody that reacts with malondialdehyde-conjugated LDL, was labeled with a NIRF dye to yield LO1-750. LO1-750 specifically identified necrotic core in ex vivo human coronary lesions. Injection of LO1-750 into high fat (HF) fed atherosclerotic Ldlr−/− mice led to specific focal localization within the aortic arch and its branches, as detected by fluorescence molecular tomography (FMT) combined with micro-computed tomography (CT). Ex vivo confocal microscopy confirmed LO1-750 subendothelial localization of LO1-750 at sites of atherosclerosis, in the vicinity of macrophages. When compared with a NIRF reporter of MMP activity (MMPSense-645-FAST), both probes produced statistically significant increases in NIRF signal in the Ldlr−/− model in relation to duration of HF diet. Upon withdrawing the HF diet, the reduction in oxLDL accumulation, as demonstrated with LO1-750, was less marked than the effect seen on MMP activity. In the rabbit, in vivo injected LO1-750 localization was successfully imaged ex vivo in aortic lesions with a customised intra-arterial NIRF detection catheter. A partially humanized chimeric LO1-Fab-Cys localized similarly to the parent antibody in murine atheroma showing promise for future translation.

Aberrant WNT signaling is associated with the formation and growth of numerous human cancer types. The low-density lipoprotein receptor-related protein 6 (LRP6) is the least redundant component of the WNT receptor complex with two independent WNT ligand-binding sites. Using domain antibody (dAb) technology, a bispecific antibody (GSK3178022) to LRP6 was identified that is capable of blocking stimulation in the presence of a range of WNT and R-spondin (RSPO) ligands in vitro. GSK3178022 was also efficacious in reducing WNT target gene expression in vivo, in both cancer cell line and patient-derived xenograft models, and delays tumor growth in a patient-derived RSPO fusion model of colorectal cancer.Implications: This article demonstrates the inhibition of a key oncogenic receptor, intractable to mAb inhibition due to multiple independent ligand interaction sites, using an innovative dAb approach.

HighlightsER and ERR transcription levels were unaffected in Marisa cornuarietis exposed to 17β-estradiol or 4-tert-Octylphenol.The mollusc ER protein interacts with the phytoestrogen genistein in transfected HEK-293 cells.The mollusc ERR protein interacts weakly with bisphenol-A in transfected HEK-293 cells.The mollusc ER protein binds to the vertebrate consensus estrogen response element (ERE) sequence.

Background We aimed to study oxidised low density lipoprotein (oxLDL) as a near infrared fluorescence (NIRF) molecular imaging target, using a monoclonal anti-oxLDL autoantibody (LO1) in mouse and rabbit atherosclerosis models. Methods LO1 and an IgG3k control antibody were labelled with a NIRF dye to give LO1–750 and IgG3–750. Targeting to atherosclerosis in mice was compared to a reporter of matrix-metalloproteinase activity (MMPSense FAST) by Fluorescence Molecular Tomography (FMT) combined with micro-CT. Uptake by rabbit aorta was detected with a custom-built intra-arterial NIRF catheter, using IVUS for morphological assessment. We further developed LO1 into a molecularly expressed humanised Fab construct with a cysteine-tag (LO1-Fab-Cys). Results Injection of LO1–750 into high fat (HF) fed Ldlr -/- atherosclerotic mice led to specific focal localization within a region of interest enclosing the aortic arch and its branches. Ex vivo studies confirmed LO1–750 localisation to subendothelium and partially to macrophages within atherosclerotic lesions. Both LO1–750 and the MMP reporter quantified significant increases in arterial targeting related to genotype, age, and duration of HF diet. Following HF diet withdrawal, MMPSense FAST targeting was affected more dramatically, exhibiting a lower intermediate signal than that of LO1–750. In the rabbit, LO1–750 localization was identified in aortic lesions with the intra-arterial catheter. The LO1-Fab-Cys construct localised to atherosclerosis in the mouse model in a similar fashion to the parent antibody. Conclusion We demonstrated the utility of LO1 and derivative molecularly-expressed constructs, either alone or in combination with other reporters as multi-modality optical imaging agents for the evaluation of atherosclerosis.

7549 Background: Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is a type I transmembrane protein is highly expressed on an array of haematological and solid tumours. NVG-111 is a humanised, tandem scFv ROR1xCD3 bispecific antibody previously shown to elicit potent killing of tumour cells in vitro and in vivo by engaging a membrane-proximal epitope in the Wnt5a-binding Frizzled domain of ROR1 and redirecting T cell activity. The in vitro potency and pharmacodynamic responses to NVG-111 were assessed to support progression to a first-in-human study. Methods: The potency of NVG-111 in vitro was determined by evaluating the concentration response for cytotoxicity, T cell activation, and cytokine release in co-cultured Jeko-1 and unstimulated human T cells. Comparative data were generated for the marketed CD19xCD3 bispecific antibody, blinatumomab. Potency data for NVG-111 were used together with allometric scaling from murine PK studies to inform planned clinical doses. Results: NVG-111 demonstrated T cell-dependent cytotoxicity, T cell activation and levels of cytokine release similar in potency to blinatumomab. Cytotoxic responses of both NVG-111 and blinatumomab were more potent than T cell activation and cytokine release. Dose response curves for NVG-111 showed a decrease in activity beyond the concentration of maximal response (ie “hook effect”). We hypothesise this is due to receptor saturation, inhibiting synapse formation. NVG-111 has progressed to a Phase 1/2 first-in-human study in patients with debulked, relapsed/refractory chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), the drug given as add-on to ≥2nd line therapy with a Bruton’s tyrosine kinase inhibitor, or venetoclax. Phase 1 includes escalating doses of 0.3 to 360 µg/day via continuous infusion over 3 cycles (each 21 days on, 7 days off) to establish safety, PK, pharmacodynamics (PD) and recommended phase 2 dose (RP2D). Predicted exposure at 0.3 µg/day is ̃EC20 for cytotoxicity in vitro and below the lowest EC10 for cytokine release. PD biomarkers in the study include systemic cytokines. Phase 2 will study efficacy and safety of the RP2D in CLL and MCL, with primary endpoint complete response rate; other efficacy endpoints include minimal residual disease and progression free survival. Conclusions: NVG-111 shows potent T-cell mediated lymphoma cell cytotoxicity in vitro at concentrations well below those associated with extensive cytokine release. NVG-111 is in an ongoing Phase 1/2 study and may present a novel option for adoptive immunotherapy in patients with non-Hodgkin lymphoma and potentially other cancers. Clinical trial information: 2020-000820-20. [Table: see text]