Jacqueline F. Doody

Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation

Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human single-domain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules.

Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

Discovery of Dual VEGFR-2 and Tubulin Inhibitors with in Vivo Efficacy.

In an effort to develop potent, orally bioavailable compounds for the treatment of neoplastic diseases, we developed a class of dual VEGFR-2 kinase and tubulin inhibitors. Targeting the VEGFR receptor kinase and tubulin structure allows for inhibition of both tumor cells and tumor vasculature. Previously, a combination of two compounds, a VEGF receptor tyrosine kinase inhibitor and tubulin agent, was demonstrated to produce an enhanced antitumor response in animal studies. We have reaffirmed their results, with the added benefit that both activities are found in one compound.

Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class of VEGFR-2 inhibitors

We have discovered novel inhibitors of VEGFR-2 kinase with low nanomolar potency in both enzymatic and cell-based assays. Active series are heteroaryl-ketone compounds containing a central aromatic ring with either an indazolyl or indolyl keto group in the ortho orientation to the benzylic amine group (Fig. 1). The best compounds were demonstrated to be inactive against a small select panel of tyrosine and serine/threonine kinases with the exception of VEGFR-1 kinase, a close family member. In addition, the lead candidate 8 displayed acceptable exposure levels when administered orally to mice.

Abstract 3539: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian and cervical cancer. TAM proliferation, differentiation and survival is dependent on Colony Stimulating Factor - 1 Receptor (CSF-1R) activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. 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 3539. doi:1538-7445.AM2012-3539

Abstract A235: Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models.

In cancer, increased infiltration of macrophages within and surrounding the tumor mass correlates with increased tumor invasiveness and growth. In addition, presence of tumor-associated macrophages (TAMs) has been shown to correlate with poor prognosis, particularly in breast, prostate, ovarian, and cervical cancer. TAM proliferation, differentiation, and survival is dependent on CSF-1R activation, a type III integral membrane tyrosine kinase receptor selectively expressed on cells of the mononuclear phagocyte lineage. Given that TAMs enhance tumor growth and that activation of the CSF-1R pathway is required for TAM function, an antibody against mouse CSF-1R was generated for proof-of-principle studies. CS7, a monoclonal anti-mouse CSF-1R antibody inhibited both CSF-1 and IL-34 binding to mouse CSF-1R, leading to inactivation of the receptor and downstream signaling molecules. In addition, CS7 prevented monocyte proliferation and macrophage differentiation with IC50s of 0.1 nM and 0.75 nM, respectively. In murine models of breast cancer using CSF-1-secreting MDA-MB-231, Hcc1954, 4T1 or EMT6 tumor cells, CS7 treatment led to a marked reduction in TAMs and an associated decrease in tumor growth. In contrast, breast tumor xenografts with CSF-1 non-secreting breast tumor cell lines JimT1 and MCF-7 had limited or no decrease in tumor volume following CS7 treatment. In prostate models, DU145 tumor cell line xenografts (CSF-1-secreting) but not PC3 (CSF-1 non-secreting) tumor growth was inhibited by CS7, recaptitulating the results seen with breast xenografts. Thus, in breast and prostate preclinical models, CSF-1 secretion by tumor cells is a prerequisite for sensitivity to anti-CSF-1R treatment. Taken together, targeting CSF-1R with a monoclonal antibody inhibits CSF-1R signaling via CSF-1 and IL-34, prevents monocytic to macrophage differentiation, and reduces tumor volume in preclinical models, validating CSF-1R as a target for therapeutic application in cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A235. Citation Format: Yanxia Li, Sneha Mathew, Lan Wu, Ying Wang, Kris Persaud, Douglas Burtrum, Paul Balderes, David Surguladze, John Haurum, Dale Ludwig, Jacqueline Doody. Anti-CSF-1R antibodies reduce tumor-associated macrophages and inhibit tumor growth in preclinical models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A235.