Nancy Levin

Chemical generation of bispecific antibodies

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

Development of a Novel Long-Acting Antidiabetic FGF21 Mimetic by Targeted Conjugation to a Scaffold Antibody

Fibroblast growth factor (FGF)21 improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF21 mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF21 through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF21. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic β-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF21 protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF21 while preserving full therapeutic functionality.

FGF21 does not require interscapular brown adipose tissue and improves liver metabolic profile in animal models of obesity and insulin-resistance

FGF21 is a key metabolic regulator modulating physiological processes and its pharmacological administration improves metabolic profile in preclinical species and humans. We used native-FGF21 and a long-acting FGF21 (PF-05231023), to determine the contribution of liver and brown adipose tissue (BAT) towards metabolic improvements in Zucker rats and DIO mice (DIOs). FGF21 improved glucose tolerance and liver insulin sensitivity in Zuckers without affecting BW and improved liver function by decreased lipogenesis, increased fatty acid oxidation and improved insulin signaling. Through detailed lipidomic analyses of liver metabolites in DIOs, we demonstrate that FGF21 favorably alters liver metabolism. We observed a dose-dependent increase of [18F]-FDG-glucose uptake in interscapular BAT (iBAT) of DIOs upon FGF21 administration. Upon excision of iBAT (X-BAT) and administration of FGF21 to mice housed at 80 °F or 72 °F, the favorable effects of FGF21 on BW and glucose excursion were fully retained in both sham and X-BAT animals. Taken together, we demonstrate the liver as an organ that integrates the actions of FGF21 and provide metabolic benefits of FGF21 in Zucker rats and DIOs. Finally, our data demonstrates iBAT does not play a role in mediating favorable metabolic effects of FGF21 administration in DIOs housed at 80 °F or 72 °F.

Combined use of immunoassay and two-dimensional liquid chromatography mass spectrometry for the detection and identification of metabolites from biotherapeutic pharmacokinetic samples

Peptides and monoclonal antibodies have both emerged as important therapeutic modalities, but each has challenges which limit their use. Non-recombinant chemical conjugation of peptides onto antibodies has the potential to minimize or eliminate altogether many of these limitations. Once such approach, pioneered by CovX has created the possibility for rapid stoichiometric fusion of pharmacophores to a single antibody platform. These molecules, called CovX-Bodies, maintain both the pharmacologic properties of a given peptide and the pharmacokinetic properties of a monoclonal antibody. The result is a new class of molecules wherein each component contributes desirable traits. In this paper, we demonstrate the use of immunoassay and two-dimensional liquid chromatography mass spectrometry (2DLC/MS) in combination to investigate the antibody conjugates of Glucagon-like peptide-1 (GLP-1) and analogs for intact protein metabolite identification directly from mouse serum. The information gained from combining these approaches has helped guide and expedite the optimization of our drug product development efforts.

Evolution of potent and stable placental-growth-factor-1-targeting CovX-bodies from phage display peptide discovery.

Novel phage-derived peptides are the first reported molecules specifically targeting human placental growth factor 1 (PlGF-1). Phage data enabled peptide modifications that decreased IC(50) values in PlGF-1/VEGFR-1 competition ELISA from 100 to 1 μM. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold 1 (CVX-2000), generating bivalent CovX-Bodies with 2 nM K(D) against PlGF-1. In vitro and in vivo peptide cleavage mapping studies enabled the identification of proteolytic hotspots that were subsequently chemically modified. These changes decreased IC(50) to 0.4 nM and increased compound stability from 5% remaining at 6 h after injection to 35% remaining at 24 h with a β phase half-life of 75 h in mice. In cynomolgus monkey, a 78 h β half-life was observed for lead compound 2. The pharmacological properties of 2 are currently being explored.

Predictive Markers of Efficacy for an Angiopoietin-2 Targeting Therapeutic in Xenograft Models

The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials.

Abstract 4113: Discovery and optimization of potent SDF1α antagonist peptides.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The SDF1α / CXCR4 axis contributes to cancer metastasis, proliferation, angiogenesis and resistance to standard of care drugs. Here we describe novel phage display derived peptide SDF1α antagonists. Phage displayed peptides were optimized through a multistep process that resulted in a 400-fold improvement in activity, from 6.4 uM IC50 inhibition of SDF1α -driven Calcium flux to 16 nM IC50. First, peptides were affinity matured via panning of focused phage libraries. Peptides exhibiting enhanced potency were bioconjugated to the CovX antibody scaffold IgG (CVX-2000), generating bivalent CovX-Bodies with 5-fold improved potency in SDF1α driven Calcium flux assays. N and C-terminal extension of the peptide and introduction of unnatural amino acids produced an additional 11-fold improvement in activity to 16 nM. Constructs with optimal pharmacokinetic stability were identified, and demonstrated single agent tumor inhibition in a Ramos B cell lymphoma xenograft model. Citation Format: Julia A. Coronella, Yanwen Fu, Kimberly Johnson, Jingping Zhong, Lioudmila Campbell, Gang Chen, Dorian Willhite, Joselyn Del Rosario, Abhijit Bhat, Gary Woodnutt, Nancy Levin. Discovery and optimization of potent SDF1α antagonist peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2013-4113

Abstract 1386: Predictive markers of efficacy for an angiopoietin-2 targeting therapeutic in xenograft models

The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and in this era of personalized medicine biomarkers that can predict effect are sorely needed. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. The resulting therapeutic has shown promising activity in a phase 1 clinical trial, with several phase 2 clinical programs in progress/planning. In order to optimize the use of this compound in the clinic a set of predictive biomarkers are desired. Here we describe the construction of a predictive model based on the efficacy of CVX-060 in 13 conventional xenograft models, focusing on renal and ovarian cancer. Pretreatment size tumors from each of the models were profiled for the levels of ∼25 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for ∼12 genes implicated in tumor growth and/or vascularization. The markers chosen for evaluation were accumulated from angiogenesis literature and a differential gene expression analysis of CVX-060 treatment-induced changes. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested via prospective analysis in 7 additional models The results of this work reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-49% TGI, median = 38%). Notably, we define a subset of 5-7 proteins, the levels of which are predictive of TGI by Ang2 blockade. The majority of these proteins are proximal to the target of CVX-060 and the direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. The SNPs and somatic mutations examined here did not correlate with TGI. Our study describes a systematic approach to the problem of identifying predictive markers of anti-angiogenic affect, and demonstrates the significant ground work necessary to reveal treatment-sensitive predictive markers which can be quantitatively defined. This effort has revealed a set of predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1386. doi:1538-7445.AM2012-1386

Abstract 2849: Optimization and pharmacokinetic characterization of potent CXCR4 antagonist CovX-Bodies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Here we describe a series of CXCR4 antagonist CovX-Bodies, produced by covalently attaching CXCR4 antagonist peptides to a humanized IgG1 molecule with the goal of producing molecules with the potency and specificity of peptides and the pharmacokinetic parameters of an antibody. Extensive SAR was performed on peptides derived from T140, a highly charged 14 amino acid cyclic peptide containing 4 Arginine residues. Both the free peptides and the associated CovX-bodies were potent antagonists of CXCR4 in vitro, and SAR studies were consistent with a model of both allosteric and orthosteric inhibition. However, in vivo pharmacokinetic (PK) studies revealed an unexpected and CXCR4-independent disappearance of the CovX-Bodies from circulation, such that approximately 10% of the injected CovX-Body remained in circulation 1 hour after i.v. injection into mice. Based on the known nonspecific cell-penetrating properties of Arginine-rich peptides, we hypothesized that Arginine replacement and charge neutralization would improve the pharmacokinetics of the CovX-Bodies. We identified substitutions that could replace up to 3 of the 4 Arg residues while retaining potent CXCR4 binding and antagonism, but Arg2 was obligate. However, these CovX-Bodies were only modestly improved in PK, indicating that that the anomalous in vivo behavior was not solely a function of charge or the net number of Arg residues. Only the replacement of Arg2 significantly improved the pharmacokinetics of the CovX-Bodies, but also resulted in a loss of CXCR4 antagonism. Extensive SAR failed to identify a suitable replacement residue for Arg2, nor did alternative structural forms such as 5 member, bicyclic, or reversed ring molecules have significantly improved pharmacokinetics. Based on these data, we conclude that the specific property of the peptide that allows potent inhibition of CXCR4, of which Arg2 is an integral component, also mediates binding to moieties other than CXCR4, leading to the depletion of these molecules from circulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2849. doi:1538-7445.AM2012-2849