Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.

Abstract. Rabbit antithrombin III (AT), purified by heparin-agarose, was labeled with iodine-131 by either the glucose oxidase-lactoperoxidase or iodine monochloride techniques. When intravenously injected, the disappearance of the '3'I-AT from plasma was characterized by rapid initial decreases, and three-exponential equations were required for best fit of the plasma disappearance curves. This rapid '3'I-AT removal was not caused by denaturation, as shown by comparison with results obtained when '3'I-AT was biologically screened (injected into a first rabbit, and then transferred 16 h later in whole plasma to a second for kinetic evaluation) before injection. Thus, the same rapid initial loss ofplasma '3'I-AT was observed with screened preparations, and the plasma fractional catabolic rates of 0.716±0.048 and 0.673±0.051 day-' for unscreened and screened '3'I-AT were not significantly different. These results support the hypothesis that a vascular-endothelial AT compartment is present in rabbit. The fractions of the total-body AT in the plasma, the vascular-endothelial and the extravascular compartments were 0.337±0.031, 0.178+0.056, and 0.485±0.069, respectively.Two three-compartment kinetic models are discussed. The first pictures AT as distributing independently between plasma and two other compartments, and the second sees AT as first passing to the vascular-endothelial compartment, and then directly into the extravascular

Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer’s disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia.

Methylthioadenosine phosphorylase, (MTAP) is a key enzyme in the adenine and methionine salvage pathways. MTAP is encoded on human chromosome 9p21 in close proximity to the p16INK4a and p14ARF tumor suppressor genes and is frequently co-deleted with p16INK4a in many cancers. Deletion of MTAP has been reported to create a reliance of MTAP-/tumors on de novo purine synthesis to maintain adequate pools of AMP, leading to increased sensitivity to purine synthesis inhibitors, such as L-alanosine. The 'Achilles heel' created by the loss of MTAP in cancer cells provides a unique therapeutic opportunity whereby MTAP-/tumors could be selectively targeted with purine synthesis inhibitors and normal tissues could be preferentially rescued with MTAP substrates, such as MTA. We demonstrate that, in contrast to published literature, MTAP-/cells are not more sensitive to inhibition of de novo purine synthesis than MTAP +/+ cells. Although MTA can preferentially rescue MTAP +/+ cells from purine-synthesis inhibitor toxicity in vitro, MTA protects cells of both genotypes from L-alanosine equivalently in vivo. Our data demonstrate that in vivo, adenine salvaged from plasma and adjacent tissues is sufficient to protect MTAP-/tumors from the effects of purine synthesis inhibitors. These results suggest targeting MTAP-/tumors with de novo purine synthesis inhibitors is unlikely to provide significant benefit over other therapeutic strategies and may explain, at least in part, the lack of efficacy of Lalanosine in clinical trials.

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