Abstract C56: Characterization of hz515H7, a novel humanized anti-CXCR4 antibody: In vitro efficacy on CXCR4-associated signaling pathways and in vivo antitumor activity.
Authors: Klinguer-Hamour Christine , Berger Sven , Boute Nicolas , Broussas Matthieu , Beau-Larvor Charlotte , Akla Barbara , Robert Alain , Haeuw Jean-Francois , Goetsch Liliane , Bailly Christian , Corvaia Nathalie 
Published: June 2012

Chemokines are small, secreted peptides that control the migration of leukocytes along a chemical gradient of ligand, especially during immune and inflammatory reactions. They are divided into two major subfamilies, CC and CXC, based on the position of their NH2-terminal cysteine residues, and bind to G protein coupled receptors, whose two major sub families are designated CCR and CXCR. More than 50 human chemokines and 18 chemokine receptors have been discovered so far. The chemokine receptor CXCR4 and its ligand Stromal cell-derived factor-1 (SDF-1) play a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors: colon, breast, prostate, lung, ovary, pancreas. CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 overexpression correlated with poor prognosis in many types of cancer. A novel humanized monoclonal antibody hz515H7 was raised against the human CXCR4. It displayed efficacious antagonist properties for all major pathways associated with SDF-1-induced CXCR4 signaling in vitro. Its antitumor activity was investigated in vivo using several human tumor models. Materials and Methods: CHOK1 and NIH3T3 cells were stably transfected with human CXCR4. [125I]SDF1 and [35S]GTP S binding assays were performed on cell membranes containing CXCR4, using SPA-WGA beads. Calcium mobilization was monitored using Fluo-4NW dye. BRET assays were developed upon genetic fusion of CXCR4 to Rluc and β-arrestin-2 to YFP and transient co-expression in HEK293 cells. Xenograft model: 10.106 Ramos cells (B-cell lymphoma) were implanted s.c. into the right flank region of each SCID mouse and allowed to grow to the designated size before administration of antibodies. The mice were followed twice a week for the observation of xenograft growth. Tumor volume was calculated using the formula: π/6 × length × width × height.

Results: Hz515H7 Mab was found to strongly inhibit SDF-1 binding, Gα protein activation and -arrestin-2 recruitment. It also inhibited calcium release, and constrained by itself CXCR4 homodimers conformation. Hz515H7 reduced SDF-1-induced cell migration in vitro. Moreover we also demonstrated that hz515H7 Mab was able to significantly inhibit growth of xenograft tumors in mice.

Altogether, the data demonstrate that Mab hz515H7 behaves as a potent and efficacious antagonist of all major CXCR4-controlled signaling pathways and suggest that targeting CXCR4 is a promising way for the treatment of tumors.

285-286

C57 Targeting stromal platelet-derived growth factor receptorα (PDGFRα) inhibits lung cancer growth independent of tumor cell PDGFRα expression.

Colleen Burns1, David Gerber2, Puja Gupta2, Michael T. Dellinger2, Jason E. Toombs2, Michael Peyton2, Inga Dunignan1, Jennifer Malaby1, Timothy Bailey1, Rolf A. Brekken2, Nick Loizos1.

1ImClone Systems, New York, NY; 2University of Texas Southwestern Medical Center, Dallas, TX.

Platelet-derived growth factor receptor alpha (PDGFRα) is a type III receptor tyrosine kinase that is normally expressed on cells of mesenchymal origin (e.g. fibroblasts and smooth muscle cells) as well as on a variety of tumor types. In lung cancer, PDGFRα is expressed frequently by tumor associated stromal cells and, in a subset of tumors, by cancer cells themselves. The anti-human PDGFRα mAb, IMC-3G3, blocks the binding of PDGF-AA, -BB & - CC to PDGFRα, and was previously shown to inhibit tumor growth of glioblastoma, prostate, and sarcoma xenografts in mice. In our analysis, IMC-3G3 administered to mice 2× per week at 40 mg/kg significantly inhibited the growth of human H1703 non-small cell lung cancer (NSCLC) tumors with a T/C value of 35%. The PDGFRα-positive H1703 lung cancer line contains both PDGFRα and PDGF-CC gene amplifications and shows a co-dependency on this axis for proliferation. Co-dependence of this kind is a rare event in human lung cancer cell lines, suggesting that only a limited number of lung tumor patients might benefit from PDGFRα inhibition. However, this experimental system does not account for the potential therapeutic effects of stromal PDGFRα inhibition. In tumor stroma, the PDGF-PDGFRα axis functions in fibroblast activation, modulation of tumor interstitial pressure, and production and secretion of vascular endothelial growth factor (VEGF). To target stromal PDGFRα, an anti-mouse PDGFRα mAb (1E10) was generated and shown to inhibit PDGF-AA from binding to murine PDGFRα with an IC50 of 8.51 × 10−9M. MAb 1E10 inhibited PDGF-AA-induced phosphorylation of mouse PDGFRα but not human PDGFRα in cell-based assays; thus demonstrating the species specificity of 1E10. Tumor xenograft studies were performed using the PDGFRα-negative NSCLC cell lines Calu-6, H1993, and A549. MAb 1E10 treatment attenuated the tumor growth of A549 and Calu-6 xenografts in nude mice with T/C values of 67% and 51%, respectfully. In Calu-6 xenografts, the combination of 1E10 plus an anti-VEGF antibody (S12) significantly inhibited tumor growth compared to control. Microvessel density was significantly decreased by both 1E10 and S12 given alone or in combination. MAb 1E10 enhanced the anti-tumor activity of cisplatin-gemcitabine chemotherapy in A549 xenografts. However, MAb 1E10 monotherapy or when combined with cisplatin-gemcitabline chemotherapy showed no anti-tumor effect on H1993 xenograft growth. PDGF-AA and VEGF-A levels were determined in cell lines resistant and sensitive to 1E10 in vivo treatment. Elevated cancer cell expression of PDGF-AA and low expression of VEGF were associated with response to stromal PDGFRα targeting. Specifically, sensitive cell lines had relatively low VEGF-A expression (VEGF-A/PDGF-AA ratio 1.96 in Calu-6 cells and 10.87 in A549 cells), while the resistant cell line H1993 had elevated VEGF-A (VEGF-A/PDGF-AA ratio 65.86) when grown in cell culture. This difference may suggest that the 1E10-sensitive tumors have a relatively greater dependence on PDGF-AA-induced production of stromal VEGF. Therefore, inhibition of stromal PDGFRα represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression.

286

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C56.