Joselyn Del Rosario

Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis, Tie2-Expressing Monocyte Infiltration, and Tumor Growth

Purpose: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1s function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold. Experimental Design: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC50s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration. Results: Bivalent Ang2 CovX-Bodies selectively block the Ang2–Tie2 interaction (IC50 < 1 nmol/L) with dramatically improved pharmacokinetics (T½ > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%–63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2+CD11b+ cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01). Conclusion: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic–pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents. Clin Cancer Res; 17(5); 1001–11. ©2011 AACR.

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.

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.

Development of a long acting human growth hormone analog suitable for once a week dosing

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

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.

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 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

Abstract 2327: Anti-angiogenic activity of a CovX bi-functional antibody conjugate targeting both activin-receptor like kinase type 1 (ALK-1) and vascular endothelial growth factor (VEGF)

Angiogenesis is essential for tumor growth and metastasis, and controlling tumor-associated angiogenesis is a proven therapeutic intervention to limit tumor progression. Several pathways have been involved in tumor-associated angiogenesis and the predominant target of current interventions is the vascular endothelial growth factor (VEGF) pathway. Recently, Activin-receptor like kinase type 1 (ALK-1) has been proven to be a novel and potent target of tumor angiogenesis. Our results indicate that inhibiting VEGF and ALK1 have different effects on tumor vasculature, suggesting that targeting both pathways could have a more potent effect than targeting either pathway alone. We have created a bi-functional antibody conjugate targeting both ALK-1 and VEGF by conjugating VEGF-trap peptides to an anti-ALK1 monoclonal antibody. We demonstrate that our bi-functional molecule binds both ALK-1 and VEGF with high affinity in vitro, blocks both ALK-1 and VEGF pathways in HUVEC primary human endothelial cells and has good pharmacokinetics properties in mice. Finally, in a MDA-MB-231 xenograft model in SCID mice, the ALK-1/VEGF bi-functional antibody conjugate slowed tumor growth and prolonged survival with greater efficacy than anti-VEGF therapy alone. 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 2327. doi:1538-7445.AM2012-2327

Abstract LB-396: FGFR4-antagonistic CovX-Body™ inhibits tumor growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL FGFR4 is the receptor for FGF19. Published data has shown FGF19 transgenic animals develop hepatocellular carcinoma (HCC), suggesting that the FGFR4:FGF19 axis may play a role in HCC. FGFR4 upregulation has been shown to be a negative prognostic indicator in multiple tumor types. CVX-63, an FGFR4-specific CovX-Body , was produced by fusing a chemically modified peptide to the specific Fab binding site of a specially designed antibody. CVX-63 was shown to inhibit FGF19 binding to FGFR4 in a competition ELISA with an IC50 of 10 nM, and had no cross reactivity with FGFR1–3. CVX-63 has a beta half life of 55 hrs in the mouse. The anti-tumor activity of CVX-63 was demonstrated in a staged Colo-205 xenograft model, with CVX-63 administered i.v. weekly. Maximal activity of 30% tumor growth inhibition was seen at dosing 10 mg/kg. Immunohistochemistry analysis of treated tumors shows a >90% reduction in phospho-ERK and 50% downregulation of FGFR4 on tumor cells. Ki67, a marker of proliferation was reduced, but active caspase 3 was not found in treated tumors. A patient-derived HCC xenograft was identified which expresses both FGFR4 and FGF19. When tested in this HCC model, CVX-63 did not show any effect inhibiting tumor growth. These data demonstrate that CVX-63 effectively inhibits FGFR4 activity in xenografts. FGFR4 inhibition has a modest effect on human tumor progression and FGFR4 does not seem to be a critical driver of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-396. doi:10.1158/1538-7445.AM2011-LB-396