Ryan John Hansen

Properties of a general PK/PD model of antibody-ligand interactions for therapeutic antibodies that bind to soluble endogenous targets

Antibodies that target endogenous soluble ligands are an important class of biotherapeutic agents. While much focus has been placed on characterization of antibody pharmacokinetics, less emphasis has been given to characterization of antibody effects on their soluble targets. We describe here the properties of a generalized mechanism-based PK/PD model used to characterize the in vivo interaction of an antibody and an endogenous soluble ligand. The assumptions and properties of the model are explored, and situations are described when deviations from the basic assumptions may be necessary. This model is most useful for in vivo situations where both antibody and ligand levels are available following drug administration. For a given antibody exposure, the extent and duration of suppression of free ligand is impacted by the apparent affinity of the interaction, as well as by the rate of ligand turnover. The applicability of the general equilibrium model of in vivo antibody-ligand interaction is demonstrated with an anti-Aß antibody.

Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody.

Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a significant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused significant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action. PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen to accumulate.

In Vivo and In Vitro Characterization of Basal Insulin Peglispro: A Novel Insulin Analog

The aim of this research was to characterize the in vivo and in vitro properties of basal insulin peglispro (BIL), a new basal insulin, wherein insulin lispro was derivatized through the covalent and site-specific attachment of a 20-kDa polyethylene-glycol (PEG; specifically, methoxy-terminated) moiety to lysine B28. Addition of the PEG moiety increased the hydrodynamic size of the insulin lispro molecule. Studies show there is a prolonged duration of action and a reduction in clearance. Given the different physical properties of BIL, it was also important to assess the metabolic and mitogenic activity of the molecule. Streptozotocin (STZ)-treated diabetic rats were used to study the pharmacokinetic and pharmacodynamic characteristics of BIL. Binding affinity and functional characterization of BIL were compared with those of several therapeutic insulins, insulin AspB10, and insulin-like growth factor 1 (IGF-1). BIL exhibited a markedly longer time to maximum concentration after subcutaneous injection, a greater area under the concentration-time curve, and a longer duration of action in the STZ-treated diabetic rat than insulin lispro. BIL exhibited reduced binding affinity and functional potency as compared with insulin lispro and demonstrated greater selectivity for the human insulin receptor (hIR) as compared with the human insulin-like growth factor 1 receptor. Furthermore, BIL showed a more rapid rate of dephosphorylation following maximal hIR stimulation, and reduced mitogenic potential in an IGF-1 receptor–dominant cellular model. PEGylation of insulin lispro with a 20-kDa PEG moiety at lysine B28 alters the absorption, clearance, distribution, and activity profile receptor, but does not alter its selectivity and full agonist receptor properties.

Qualification of a free ligand assay in the presence of anti-ligand antibody Fab fragments.

The aim of this work was to develop and characterize an ELISA to measure free ligand concentrations in rat serum in the presence of a Fab to the same ligand. A variety of experiments were conducted to understand optimal assay conditions and to verify that only free ligand was detected. The parameters explored included sample incubation time on plate, the initial concentrations of Fab and ligand, and the pre-incubation time required for the Fab-ligand complex concentrations to reach equilibrium. We found the optimal experimental conditions to include a 10-minute on-plate incubation of ligand-containing samples, with a 24-hour pre-incubation time for test samples of Fab and ligand to reach equilibrium. An alternative approach, involving removal of Fab-ligand complexes from the solution prior to measuring concentrations of the ligand, was also used to verify that the assay only measured free ligand. Rats were dosed subcutaneously with Fab and the assay was used to demonstrate dose-dependent suppression of endogenous free ligand levels in vivo.

Issues, challenges, and opportunities in model‐based drug development for monoclonal antibodies

Over the last two decades, there has been a simultaneous explosion in the levels of activity and capability in both monoclonal antibody (mAb) drug development and in the use of quantitative pharmacologic models to facilitate drug development. Both of these topics are currently areas of great interest to academia, the pharmaceutical and biotechnology industries, and to regulatory authorities. In this article, we summarize convergence of these two areas and discuss some of the current and historical applications of the use of mathematical-model-based techniques to facilitate the discovery and development of mAb therapeutics. We also consider some of the current issues and limitations in model-based antibody discovery/development and highlight areas of further opportunity.

Addition of 20-kDa PEG to insulin lispro alters absorption and decreases clearance in animals

ABSTRACTPurposeDetermine the pharmacokinetics of insulin peglispro (BIL) in 5/6-nephrectomized rats and study the absorption in lymph duct cannulated (LDC) sheep.MethodsBIL is insulin lispro modified with 20-kDa linear PEG at lysine B28 increasing the hydrodynamic size to 4-fold larger than insulin lispro. Pharmacokinetics of BIL and insulin lispro after IV administration were compared in 5/6-nephrectomized and sham rats. BIL was administered IV or SC into the interdigital space of the hind leg, and peripheral lymph and/or serum samples were collected from both LDC and non-LDC sheep to determine pharmacokinetics and absorption route of BIL.ResultsThe clearance of BIL was similar in 5/6-nephrectomized and sham rats, while the clearance of insulin lispro was 3.3-fold slower in 5/6-nephrectomized rats than in the sham rats. In non-LDC sheep, the terminal half-life after SC was about twice as long vs IV suggesting flip-flop pharmacokinetics. In LDC sheep, bioavailability decreased to <2%; most of the dose was absorbed via the lymphatic system, with 88% ± 19% of the dose collected in the lymph after SC administration.ConclusionThis work demonstrates that increasing the hydrodynamic size of insulin lispro through PEGylation can impact both absorption and clearance to prolong drug action.

NOVEL MECHANISM FOR THE SUSTAINED DURABILITY OF PROPROTEIN CONVERTASE SUBTILISIN-KEXIN TYPE 9 MONOCLONAL ANTIBODY LY3015014

Monoclonal antibodies (Mabs) that neutralize proprotein convertase subtilisin-kexin type 9 (PCSK9) cause remarkable LDL cholesterol (LDL-C) lowering in human trials. It was reported that the efficacy is determined by the epitope of the PCSK9 Mab. Here we describe a novel mechanism by which the

Quantitative characterization of the mechanism of action and impact of a ‘proteolysis-permitting’ anti-PCSK9 antibody

ABSTRACT A recent report described a novel mechanism of action for an anti-proprotein convertase subtilisin-kexin type 9 (PCSK9) monoclonal antibody (LY3015014, or LY), wherein the antibody has improved potency and duration of action due to the PCSK9 epitope for LY binding. Unlike other antibodies, proteolysis of PCSK9 can occur when LY is bound to PCSK9. We hypothesized that this allowance of PCSK9 cleavage potentially improves LY efficiency through two pathways, namely lack of accumulation of intact PCSK9 and reduced clearance of LY. A quantitative modeling approach is necessary to further understand this novel mechanism of action. We developed a mechanism-based model to characterize the relationship between antibody pharmacokinetics, PCSK9 and LDL cholesterol levels in animals, and used the model to better understand the underlying drivers for the improved efficiency of LY. Simulations suggested that the allowance of cleavage of PCSK9 resulting in a lack of accumulation of intact PCSK9 is the major driver of the improved potency and durability of LY. The modeling reveals that this novel ‘proteolysis-permitting’ mechanism of LY is a means by which an efficient antibody can be developed with a total antibody dosing rate that is lower than the target production rate. We expect this engineering approach may be applicable to other targets and that the mathematical models presented herein will be useful in evaluating similar approaches.

Insulin-XTEN® Exhibits a Size-Dependent Alteration in Tissue Action in Rats

To optimize the action of exogenously administered insulin, we employed XTEN® technology to create insulins with variably sized XTEN amino acid polymers. Recombinant fusions of XTEN polymers linked to insulin lispro with an A21G mutation were prepared in various amino acid lengths. Insulin-XTEN molecules demonstrated 15-fold lower potency in binding and receptor phosphorylation than insulin lispro but did not differ from each other. These insulin-XTEN molecules were equally effective in lowering blood glucose at a 100nmol/kg dose in diabetic Sprague-Dawley rats. Furthermore, the larger insulin-XTEN molecules had a longer duration of glucose lowering. Insulin-XTENs were compared to insulin lispro in rat euglycemic clamp studies, using insulin doses that would elicit steady plasma insulin concentrations and equivalent increases in glucose infusion rate. Insulin-mediated suppression of endogenous glucose production was not significantly different among any of the administered insulins. However, plasma free fatty acids and soleus muscle glucose uptake were significantly decreased in an XTEN size-dependent manner when compared to insulin lispro. Additional studies demonstrated equal hepatic pAkt accumulation in rats treated with insulin lispro or any of the insulin-XTENs, but revealed a significant XTEN size-dependent reduction in skeletal muscle pAkt in rats administered insulin-XTENs compared to insulin lispro. These data suggest a possible XTEN size-dependent regulation of insulin action and that the differing sizes of the XTEN polymer may convey preferential tissue action. In conclusion, XTEN technology may permit “tuning” of the glucodynamic effects of the insulin, leading to an enhanced time extension and improved hepatic and peripheral pharmacodynamic action that could more closely mimic the action of endogenously secreted insulin into the portal circulation. Disclosure M.E. Christe: Employee; Self; Eli Lilly and Company. D. Konkol: None. J. Friedrich: None. J. Jacobs: None. E. Hawkins: Employee; Self; Eli Lilly and Company. J. Moyers: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. C. Zhang: Employee; Self; Eli Lilly and Company. S.D. Kahl: Employee; Self; Eli Lilly and Company. H.E. Baker: None. A.L. Cox: None. R.J. Hansen: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. A. Sperry: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. M. Michael: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. V. Schellenberger: None. D. Baldwin: None. J.M. Beals: Employee; Self; Eli Lilly and Company. A. Korytko: None.