John Lippincott

Targeting Activin Receptor-Like Kinase 1 Inhibits Angiogenesis and Tumorigenesis through a Mechanism of Action Complementary to Anti-VEGF Therapies

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.

Antitumor activity of an anti-CD98 antibody.

CD98 is expressed on several tissue types and specifically upregulated on fast‐cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98‐specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell‐line derived xenograft models and was as efficacious as standard of care carboplatin in patient‐derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase‐3 and ‐7‐mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.

Discovery of a novel class of targeted kinase inhibitors that blocks protein kinase C signaling and ameliorates retinal vascular leakage in a diabetic rat model

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

Monoclonal antibody binding-site diversity assessment with a cell-based clustering assay

The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.

Abstract 1765: Inhibition of Notch signaling by a Notch1 monoclonal antibody induces robust anti-tumor efficacy in T-cell acute lymphoblastic leukemia and breast cancer

Notch signaling is deregulated in T-cell acute lymphoblastic leukemia (T-ALL) and advanced solid tumors, making it an attractive target for oncology drug development. In this present report, we describe a novel mouse monoclonal antibody, Notch1 mAb, that specifically binds to the negative regulatory region (NRR) of human Notch1 receptor. Using a T-ALL cell line, HPB-ALL, that harbors mutations in Notch1 heterodimerization and PEST domains, we demonstrated that Notch1 mAb blocked Notch signaling by reduction of Notch1 intracellular domain (NICD) and down-regulation of Notch target genes, Hes-1 and cMyc. Notch1 mAb caused cell growth inhibition of HPB-ALL and several other T-ALL cell lines, via induction of cell cycle arrest and apoptosis. Notch1 mAb treatment resulted in robust NICD reduction and marked antitumor efficacy in HBP-ALL xenograft model. Additional mechanism-of-action studies revealed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the anti-tumor activity of Notch1 mAb may be mediated by its direct effects on tumor cell growth or survival. Furthermore, this antibody led to a significant reduction in leukemia progenitor cells (LPCs) baring NOTCH1 mutation in bioluminescent humanized T-ALL LPC mouse models. In addition to its inhibitory effect on mutant Notch1, Notch1 mAb also displayed robust neutralization activity on wild type Notch1 and caused tumor growth inhibition in breast cancer models MDA-MB-231 and MX-1 by targeting the wild type receptor in these tumor types. Interestingly, using a gamma secretase inhibitor PF-03084014, we showed that Notch1 mAb and PF-03084014 elicited similar degree of biology responses in HPB-ALL cells. Our results indicate that antibodies that bind to NRR can act as potent inhibitors of Notch1 signaling and provide opportunities for development of novel cancer therapeutics for T-ALL and solid tumors. 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 1765. doi:10.1158/1538-7445.AM2011-1765