Daniel T. Dransfield

Selective Inhibition of Matrix Metalloproteinase-14 Blocks Tumor Growth, Invasion, and Angiogenesis

Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.

Blockade of MMP14 Activity in Murine Breast Carcinomas: Implications for Macrophages, Vessels, and Radiotherapy

BACKGROUND Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor β (TGFβ) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFβ and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION MMP14 blockade decreased immunosuppressive TGFβ, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.

A Human Monoclonal Antibody against Insulin-Like Growth Factor-II Blocks the Growth of Human Hepatocellular Carcinoma Cell Lines In vitro and In vivo

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR–directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1–104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (KD value of 49 and 10 pmol/L, respectively) compared with IGF-I (∼10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor–binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 Å. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I–induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A. Mol Cancer Ther; 9(6); 1809–19. ©2010 AACR.

Identification and characterization of a human monoclonal antagonistic antibody AL‐57 that preferentially binds the high‐affinity form of lymphocyte function‐associated antigen‐1

LFA‐1 (αLβ2) mediates cell‐cell and cell‐extracellular matrix adhesions essential for immune and inflammatory responses. One critical mechanism regulating LFA‐1 activity is the conformational change of the ligand‐binding αL I domain from low‐affinity (LA), closed form, to the high‐affinity (HA), open form. Most known integrin antagonists bind both forms. Antagonists specific for the HA αL I domain have not been described. Here, we report the identification and characterization of a human antibody AL‐57, which binds to the αL I domain in a HA but not LA conformation. AL‐57 was discovered by selection from a human Fab‐displaying library using a locked‐open HA I domain as target. AL‐57 Fab‐phage bound HA I domain‐expressing K562 cells (HA cells) in a Mg2+‐dependent manner. AL‐57 IgG also bound HA cells and PBMCs, activated by Mg2+/EGTA, PMA, or DTT. The binding profile of AL‐57 IgG on PBMCs was the same as that of ICAM‐1, the main ligand of LFA‐1. In contrast, an anti‐αL murine mAb MHM24 did not distinguish between the HA and LA forms. Moreover, AL‐57 IgG blocked ICAM‐1 binding to HA cells with a potency greater than MHM24. It also inhibited ICAM‐1 binding to PBMCs, blocked adhesion of HA cells to keratinocytes, and inhibited PHA‐induced lymphocyte proliferation with potencies comparable with MHM24. These results indicate that specifically targeting the HA I domain is sufficient to inhibit LFA‐1‐mediated, adhesive functions. AL‐57 represents a therapeutic candidate for treatment of inflammatory and autoimmune diseases.

Selective Inhibition of Membrane Type 1 Matrix Metalloproteinase Abrogates Progression of Experimental Inflammatory Arthritis: Synergy With Tumor Necrosis Factor Blockade.

In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1‐MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen‐induced arthritis (CIA).

Selective inhibition of membrane type 1 matrix metalloproteinase abrogates progression of inflammatory arthritis: Synergy with TNF blockade

In rheumatoid arthritis (RA), destruction of articular cartilage by the inflamed synovium is considered to be driven by increased activities of proteolytic enzymes, including matrix metalloproteinases (MMPs). The purpose of this study was to investigate the therapeutic potential of selective inhibition of membrane type 1 MMP (MT1‐MMP) and its combination with tumor necrosis factor (TNF) blockage in mice with collagen‐induced arthritis (CIA).

Abstract A278: ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models.

Fibroblast growth factors (FGF) and their receptors (FGFR) play important roles in cell proliferation, cell differentiation, cell migration, cell survival, protein synthesis, and angiogenesis. The FGFR family consists of four genes encoding tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, and FGFR4). Dysregulation of FGFR signaling has been implicated in a number of developmental syndromes as well as cancers, e.g., squamous non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, liver, breast, ovarian, endometrial, and bladder carcinomas, fueling significant interest in FGFRs as targets for therapeutic intervention. ArQule, Inc. has discovered a novel, ATP competitive class of FGFR inhibitors from which ARQ 087 emerged as a candidate for advancement into clinical development. ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR activity against FGFR1, FGFR2, mutant FGFR2 (N549H), FGFR3, and FGFR4 kinases, all exhibiting IC 50 values in the low nanomolar range in biochemical assays. Ki versus FGFR1 and FGFR2 were 2.8nM and 0.68nM, respectively. ARQ 087 inhibited ectopically expressed FGFR1, 2, and 3 in COS-1 cells as well as, to a varying extent, the proliferation of BaF3 cells expressing the FGFR family of receptors (FGFR2≫FGFR1/FGFR3≫FGFR4). Cell proliferation studies suggested a correlation of FGFR2 mRNA amplification in gastric and other cancers with associated sensitivity to treatment with ARQ 087. Along these lines, ARQ 087 demonstrated potent inhibition of FGFR2 phosphorylation in NCI-H716, Kato III, SNU-16 and MFM223 cells; all demonstrated to be driven by FGFR2. The inhibition of cell growth was associated with an ARQ 087-induced G1 cell cycle arrest and subsequent induction in apoptosis that appears to be related to the levels of FGFR2 protein. Cell lines driven by FGFR2 activating mutations did not undergo apoptosis but did accumulate in G1 following ARQ 087 treatment. In vivo, ARQ 087 induced regressions in FGFR2-driven xenograft models (SNU-16, NCI-H716 and BaF3/FGFR2) and inhibited tumor progression in a model harboring an FGFR2-activating mutation (AN3CA). In addition, concentration-dependent inhibition of phosphorylation of FGFR2 and the downstream FGFR pathway signals (FRS2α, MEK, ERK, and AKT) was evident in response to ARQ 087 treatment in both in vitro and in vivo pharmacodynamic assays. In summary, ARQ 087 is an orally bioavailable kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven models, possessing good drug-like properties. A clinical development plan including a patient selection strategy is defined and the drug is currently in Phase I clinical studies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A278. Citation Format: Daniel Dransfield, Jennifer Lee, Carol Waghorne, Cathy Bull, Ronald E. Savage, Xiaolan Zhao, Shipeng Yuan, Edward Chang, Enkeleda Nakuci, Sudharshan Eathiraj, Susan Cornell-Kennon, Xiubin Gu, Syed Ali, Chang-Rung Chen. ARQ 087, a multi-tyrosine kinase inhibitor with potent in vitro and in vivo activity in FGFR2 driven 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 A278.

Crystallization and preliminary X-ray analysis of the complexes between a Fab and two forms of human insulin-like growth factor II

Elevated expression of insulin-like growth factor II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon and liver cancer. As IGF-II can deliver a mitogenic signal through both the type 1 insulin-like growth factor receptor (IGF-IR) and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is an attractive therapeutic option. One method of doing this would be to find antibodies that bind tightly and specifically to the peptide, which could be used as protein therapeutics to lower the peptide levels in vivo and/or to block the peptide from binding to the IGF-IR or IR-A. To address this, Fabs were selected from a phage-display library using a biotinylated precursor form of the growth factor known as IGF-IIE as a target. Fabs were isolated that were specific for the E-domain C-terminal extension and for mature IGF-II. Four Fabs selected from the library were produced, complexed with IGF-II and set up in crystallization trials. One of the Fab-IGF-II complexes (M64-F02-IGF-II) crystallized readily, yielding crystals that diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 106.9, c = 110.7 A. There was one molecule of the complete complex in the asymmetric unit. The same Fab was also crystallized with a longer form of the growth factor, IGF-IIE. This complex crystallized in space group P2(1)2(1)2(1), with unit-cell parameters a = 50.7, b = 107, c = 111.5 A, and also diffracted X-rays to 2.2 A resolution.

Production of a mouse monoclonal IgM antibody that targets the carbohydrate Thomsen-nouveau cancer antigen resulting in in vivo and in vitro tumor killing

The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = d-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.

Abstract 3640: Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site

Tumor-associated carbohydrate antigens (TACAs) historically have been challenging targets for antibody therapeutics. Sialyl-Tn (STn) is a cancer specific antigen that is expressed on the surface of carcinomas including ovarian, colon, prostate, and pancreatic tumors but is rarely present in normal tissue. STn expression has been linked to innate immune suppression, a chemoresistant phenotype, metastasis, and poor prognosis. Previous attempts to target this antigen in the clinic with synthetic glycan vaccines proved safe but lacked efficacy. We have developed humanized bispecific antibodies targeting STn and CD3 for T-cell recruitment and activation at the tumor site. These bispecific antibodies were selected for optimal tumor targeting using our glycan microarray that enriches for candidates whose binding is protein-independent and glycan specific. STn-selective binding was demonstrated. Current lead candidates exhibited low nanomolar EC50 binding in flow cytometric assays against both STn expressing tumor cells and T cells. Quantification of T-cell activation and T-cell induced tumor killing in vitro provides a basis for the further clinical development of these bispecific antibody candidates. Citation Format: David A. Eavarone, Jillian Prendergast, Patricia E. Rao, Jenna Stein, Jeff Behrens, Daniel T. Dransfield. Novel humanized anti-Sialyl-Tn, anti-CD3 bispecific antibodies demonstrate tumor and T-cell specificity for immune activation at the tumor site [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3640. doi:10.1158/1538-7445.AM2017-3640