Supplemental figure legends 1-8, Supplementary tables 1-5, and Supplementary methods

Purpose Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models. Methods Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing. Results Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P < 0.0001) versus control. In a PDX lung model, 10 mg/kg cetrelimab (every 5 days for six cycles) increased frequency of peripheral T cells and reduced (P < 0.05) mean tumor volume versus control. Activity was consistent with that of established PD-1 inhibitors. Cetrelimab dosing was well tolerated in cynos and mean drug exposure increase was dose-dependent. Conclusion Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation.

Abstract The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

Abstract Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p<0.004). In a separate study, similar tumor regressions were noted in the FGFR-driven GEMM at 1 week of erdafitinib with or without anti-PD-1 treatment, while no such response to these treatments was noted in a KRAS-driven lung cancer GEMM. Immune profiling of tumor specimens revealed high baseline expression of programmed death ligand-1 (PD-L1) expression by IHC and flow cytometry. Following combination treatment, subsequent immunohistochemistry (IHC) analyses showed a significant decrease in Ki67 and PD-L1 positive tumor cells, accompanied by an increase in cluster of differentiation 3 (CD3) positive tumor-infiltrating cells in combination group as compared to control. T cell function is not inhibited by erdafitinib, as measured in vitro by mixed lymphocyte reaction and cytomegalovirus recall assays. Additional changes observed in lung tumors across treatment groups in immune cell infiltration, functionality, and T-cell clonality will be discussed. These data suggest that combination treatment of erdafitinib and PD-1 blockade drives improved survival in FGFR2-driven model of lung cancer by simultaneous inhibition of FGFR pathway in tumor cells and enhancement of anti-tumor immunity. Thus, data here provide rationale for the combined clinical testing of erdafitinib and PD-1 blockade in patients with FGFR-altered lung cancers. Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

Abstract Hormone resistance is a major challenge in treating breast cancer patients. Although anti-estrogens, such as tamoxifen, are initially successful controlling disease in estrogen receptor alpha positive (ERα+) tumors, patients with metastatic disease and as many as 40 % of patients receiving adjuvant tamoxifen acquire resistance to treatment, relapse, and die. The receptor tyrosine kinase, RET, its co-receptor, GFRα1, and their ligand, GDNF, are proteins that are emerging as potential contributors to hormone resistance in breast cancer. RET and GFRα1 were recently shown to be overexpressed in a subset of ERα+ breast cancer tumors, and GFRα1 expression was associated with lymphovascular invasion. In vitro, silencing RET with siRNA increased MCF7 cells' sensitivity to tamoxifen and, in the absence of estrogens, treatment of MCF7 cells with GDNF induced upregulation of ERα target genes. Additionally, GDNF stimulation of MCF7 cells enhanced proliferation, survival, and cell scattering. Together, clinical and preclinical data suggest that this pathway plays an important role in driving breast cancer invasion, metastasis, and hormone resistance. However, the role of GFRα1 has not been examined in tumor cell invasion and metastasis. In this study we tested the functional consequences of silencing GFRα1 in ERα + MCF7 cells on tumor cell migration, invasion, and RET signaling. We showed that targeted reduction of GFRα1 significantly inhibited GDNF-induced migration in both a transwell (p=<0.001) and scratch wound assay (p=<0.001 at 24 hours). In addition, GDNF-induced increases in viability were significantly decreased when MCF7 cells were treated with neutralizing antibodies against GFRα1 (p=<0.001). Silencing GFRα1 in GDNF-treated MCF7 cells led to reduced invasion through collagen I, although this difference was not significant. Lastly, siRNA targeted reduction of GFRα1 in MCF7 cells inhibited GDNF-induced phosphorylation of RET. Together the data demonstrate that GFRα1 is important in migration and cell viability as well as RET signaling in breast cancer cells. Citation Format: Catherine A. Ferrante, Nikki DeAngelis, Raluca Verona. GFRα1 is required for GDNF-induced viability, migration, and signaling through RET in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4048. doi:10.1158/1538-7445.AM2014-4048

Abstract Lung cancer is a very heterogeneous type of cancer. Among various subtypes, non-small-cell lung cancer (NSCLC) comprises about 80 % of all cases, and the KRAS mutant subtype, has a generally poor prognosis and response to chemotherapy or targeted therapeutics. There is an urgent unmet medical need for development of effective treatments. Anticancer drugs are often selected based on studies performed on adherent cultures of tumor cells where the cells grow in two dimensional monolayers. However, tumors grow in 3 dimensions and are not composed exclusively of malignant cells, but of a mixture of cancer cells and stromal cells embedded in an extracellular matrix. These interactions involve a complex mechanical and biochemical interplay that is missed when cells are cultured in classical monolayer-based models. NSCLC 3-D heterotypic models that preserve epithelial and stroma cell interactions in a well-defined 3-D microenvironment should more accurately identify drug candidates for lung cancer treatment. We developed a 3-D heterotypic in vitro lung tumor model that recapitulates the human tumor microenvironment and is useful for selection of novel anticancer drugs. This in vitro 3-D KRAS mutant NSCLC model includes H358 epithelial cancer cells, stromal cell types such as lung cancer-associated fibroblasts (CAFs), and microvascular endothelial cells (HMVECs) cultured on a relevant cellular substrate component such as collagen type I. Each cell type was fluorescently labeled and the cultures grew to form what we named a “tumoroid” structure. The tumoroid is a palpable 2 mm3, spheric, dense structure that resembles the architecture of a human lung tumor and has features such as a capillary-like vasculature structure and areas of epithelial/stromal interaction as confirmed by microscopy and H&E staining. This model was used to test agents impacting known pathways in the tumor and stromal microenvironment to determine their effect on tumoroid viability and apoptosis. Test compounds included a DR5 agonistic monoclonal antibody, CNTO 95 (pan αv integrin monoclonal antibody), Cetuximab (EGFR monoclonal antibody) and Bevacizumab (VEGF monoclonal antibody). Cetuximab, CNTO 95, and DR5 significantly inhibited H358 tumoroid viability, and Bevacizumab disrupted the tumoroid capillary-like structures. Conversely, Cetuximab and CNTO 95 did not inhibit H358 cell viability when grown in 2-D cultures and Bevacizumab did not have an impact on H358 cells grown on plastic. However, previous publications have shown that Cetuximab and Bevacizumab inhibit H358 tumor growth in animal models. The results suggest that the in vitro tumoroid model is more predictive of the activity seen in animal models than 2-D cell culture techniques. The tumoroid model may therefore be useful for anticipating the effectiveness of new drugs in vivo and could reduce the need for evaluating numerous compounds in animal models. Citation Format: Luciana F. Macedo, Elizabeth Kaiser, Bradley Heidrich, Barbara Bushey, Catherine Ferrante, Deborah Marshall. A 3-dimensional tumoroid model made up of lung cancer cells, fibroblasts, and endothelial cells is predictive of drug activity in animal models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2014-2035

The human genome contains a large number of repetitive elements that have been suspected to play a role in human diseases. Recent literature has shown that aberrant activation of one of these repetitive elements, a long terminal repeat (LTR), leads to lineage inappropriate expression of the CSF-1 receptor (CSF-1R) transcript in Hodgkin's lymphoma (HL) clinical samples and cell lines. CSF1-R, also known as Feline McDonough Sarcoma (FMS), is the receptor for CSF-1 and IL-34, and under normal circumstances it plays an important role in monocyte survival, proliferation, and differentiation as well as osteoclast generation. Aberrant expression of CSF-1R has been shown previously to support proliferation and survival in HL cells. To confirm the role of CSF-1R in HL, we determined that 17 of 17 HL clinical samples and 7 of 7 HL cell lines were positive for the aberrant CSF-1R transcript by TaqMan and traditional PCR, while non-HL cell lines were negative for the aberrant CSF-1R transcript. The HL lines were further characterized for surface expression of CSF-1R by flow cytometry and CSF-1 secretion by ELISA. L-1236 cells expressed the highest levels of CSF-1R and CSF-1. Treatment of the HL cell lines with CSF-1R-Fc, resulted in reduced viability in four of the five lines, consistent with the hypothesis that HL cell lines are sensitive to CSF-1 as a growth factor. Additionally, it was found that treating the L-1236 HL cell line with the CSF-1R small molecule inhibitor JNJ-40646527 had a significant effect on viability of the HL cell line L-1236, with an IC50 of 264 nM at 72 hrs. JNJ-40646527 did not have a significant effect on the viability of several other HL cell lines. These results indicate that aberrant LTR driven CSF-1R expression may play a role in the pathogenesis of Hodgkin's lymphoma (HL) and could be a potential target for Hodgkin's lymphoma therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B269. Citation Format: Liam Campion, Nikki Deangelis, Catherine Ferrante, Raluca Verona, Shobha Seetharam, Carl Manthey, Linda Snyder. Effects of blocking aberrantly expressed CSF-1R in Hodgkin lymphoma. [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 B269.

Abstract CCL2 (C-C chemokine ligand 2; also known as MCP-1) is a pleiotropic chemokine overexpressed by many types of tumors. CCL2 is believed to promote tumor growth by increasing macrophage infiltration, angiogenesis, tumor proliferation/survival, and metastasis. The purpose of these studies was to assess potential mechanisms of action associated with the efficacy of neutralizing CCL2 in human pancreatic xenograft models. Cell lines included BxPC-3, PANC-1 and AsPC-1 human pancreatic carcinoma, all of which are p53mut/krasmut, but each differentially expresses CCL2 and its receptor, CCR2. The three tumor cell lines were implanted subcutaneously in immunocompromised mice. When tumors reached ∼50-100 mm3, a cocktail of neutralizing antibodies to human CCL2, mouse MCP-1 and mouse MCP-5 (termed CCL2 blockade) was administered i.p. at 10 mg/kg each, twice a week for the study duration, either alone or in combination with gemcitabine i.p. at 120 mg/kg q3dx4. CCL2 blockade alone significantly inhibited primary tumor growth for BxPC-3 (66-78% tumor growth inhibition (TGI); P<0.001) or prolonged survival for PANC-1 (by seven days; P<0.004). CCL2 in combination with gemcitabine significantly inhibited AsPC-1 tumor growth compared to either therapy alone (55% TGI; P<0.034). By IHC analysis, macrophage infiltration in AsPC-1 tumors was reduced significantly by CCL2 blockade (P=0.049). In vitro studies were conducted to further understand the mechanism of action. Recombinant huCCL2 had no effect on pancreatic tumor cell proliferation in vitro, suggesting that the in vivo anti-tumor effect by CCL2 blockade may be through CCL2 neutralization within the host stroma. Co-culture studies to mimic the tumor-stroma interaction were conducted, using pancreatic tumor cells and normal human lung fibroblasts (NHLF). Secretion of CCL2, as well as IL-5, IL-6, IL-6R, IL-8 and GRO, was significantly enhanced 2-10-fold during co-culture of BxPC3 with NHLF (P<0.001), while only CCL2 was enhanced 2-fold during PANC-1/NHLF co-culture (P<0.001). Furthermore, cell-cell contact was not required for cytokine induction for both BxPC-3 and PANC-1 with NHLF. Tumor cell conditioned medium (CM) stimulated NHLF to produce cytokines, not vice versa, suggesting that a factor(s) in tumor CM is responsible for cytokine induction. Neutralizing antibodies to either CCL2 or IL-6 did not abolish induction of other cytokines in the BxPC-3/NHLF model. The inducers of chemokines/cytokines as well as in vitro migration and in vivo gene profiling studies are underway to better understand the impact of CCL2 blockade on pancreatic tumor growth. These results demonstrate the significant effect of CCL2 blockade on pancreatic tumor growth in vivo, and suggest that the tumor cell/fibroblast interaction may be an important source of CCL2 at the tumor site. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 555.

Tumor histopathology is an important method to understand the mechanism of action of anti‐neoplastic therapies and immunotherapeutics in animal models. An experimental metastasis model was established with the MDA‐MB‐231 human breast adenocarcinoma cell line in SCID beige mice. Compared to subcutaneous and orthotopic models, this model allows evaluation of multiple lesions at earlier time points. Mice were injected i.v. with MDA‐MB‐231 cells and sacrificed either 14 or 21 days post tumor cell injection. Lungs were processed for histology and stained with H&E. Multiple focal lesions were observed and these lesions had a random distribution in the lung and were variable in size. The lesions increased in size and number over time. These observations were confirmed by a rank order analysis on disease severity, and morphometry on size of the lesions. Morphometry also revealed a significant increase in proliferating cells within lung lesions at Day 21 compared to Day 14. Numerous infiltrating macrophages could also be identified within the lesions. No evidence of angiogenesis within the lesions was observed in this time course. It is hypothesized that the lesions studied have not yet crossed the angiogenic switch and therefore no new blood vessel formation was observed. Lesions can be further characterized by additional histologic methods.

Abstract CCL2, (CC-chemokine ligand 2 or monocyte chemoattractant protein-1 (MCP-1)), is overexpressed in many human tumors and is believed to exert pro-tumor effects by recruiting monocytes to the tumor, where these cells become tumor associated macrophages (TAMs). TAMs secrete growth factors that stimulate angiogenesis and tumor growth, as well as proteases to promote tumor invasion and metastasis. CCL2 expression levels in primary breast tumors have been correlated with macrophage infiltration and blood vessel density, which in turn is correlated with disease stage and prognosis. These correlations indicate that CCL2 is a key player in tumor macrophage infiltration and/or tumor growth/invasion, and suggest that neutralizing CCL2 could be an effective form of therapy for breast cancer patients.The objective of these studies was to investigate whether CCL2 blockade could inhibit tumor growth in mice bearing human breast tumors. The human breast tumor cell lines MDA-MB-231 (ER-, PR-, Her2-) and MDA-MB-361 (ER+, PR+, Her2+) were implanted orthotopically in immunocompromised mice, and in both models the primary tumors metastasized to lungs and brain. Neutralizing antibodies to human CCL2 (CNTO 888) and to the mouse orthologs, MCP-1 and MCP-5, were administered therapeutically, either as a cocktail (termed CCL2 blockade) or individually to study the relative roles of host vs tumor derived CCL2 in promoting tumor growth.In both tumor models, CCL2 blockade significantly inhibited the growth of established primary tumors in the mammary fat pad. In addition, CCL2 blockade inhibited metastasis to distant sites. As measured by Taqman, visual inspection and immunohistochemistry, mice with MDA-MB-361 tumors treated with CCL2 blockade showed significantly reduced metastasis to lungs and brain, while mice bearing MDA-MB-231 tumors showed significantly reduced metastasis to lungs.To define the relative roles of human tumor-derived CCL2 vs mouse host-derived MCP-1/MCP-5, in vivo monotherapy tumor studies were conducted using the individual neutralizing antibodies. These studies included the mammary fat pad model and a tail vein metastasis model. In both cases, only the treatment with the anti-mouse MCP-1 antibody significantly inhibited primary tumor growth and distant metastasis, indistinguishable from the effect of CCL2 blockade treatment. In the tail vein metastasis model, the antibody treatment resulted in significantly fewer detectable lesions with these lesions showing a significant reduction in both tumor size and growth fraction, suggesting antibody treatment inhibits tumor seeding and growth. Mechanistic studies are in progress to further understand the basis of the anti-tumor effect mediated by the antibody treatment. These results demonstrate that host-derived MCP-1, produced from the tumor microenvironment, plays the critical role in tumor growth and metastasis in these models of human breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6095.

Abstract CCL2, (CC-chemokine ligand 2 or monocyte chemoattractant protein-1 (MCP-1)), is overexpressed in many human tumors. CCL2 is believed to exert direct effects on tumor cells (via increased proliferation/survival and migration) and the tumor stroma (via recruitment of tumor associated macrophages and promotion of angiogenesis at the tumor site). CCL2 expression levels in primary breast tumors have been correlated with macrophage infiltration and blood vessel density, which in turn correlate with disease stage and worse prognosis. These correlations suggest that CCL2 could be a key player in breast tumor growth/metastasis. To study the effect of neutralizing CCL2 in vivo, the human breast tumor cell lines MDA-MB-231 and MAXF857, both ER−, PR−, Her2lo, were implanted orthotopically in the mammary fat pad in immunocompromised mice. When tumors reached 70–100 mm3, a cocktail of neutralizing antibodies to human CCL2 (CNTO 888) and to the mouse orthologs, MCP-1 and MCP-5 (collectively termed CCL2 blockade) was administered i.p twice a week at 10 mg/Kg each for the study duration. In both models, CCL2 blockade significantly inhibited the growth of primary tumors (P<0.01). In addition, CCL2 blockade significantly reduced the number of visible lung metastases in the MDA-MB-231 model (P<0.008), suggesting that CCL2 promotes metastasis as well as primary tumor growth. To further study the role of CCL2 in tumor growth and metastasis, in vitro studies were conducted to mimic the in vivo interactions between tumor and stroma. MDA-MB-231 cells migrated toward CCL2 in a dose-dependent manner (P<0.05), which was significantly inhibited by CNTO 888 (P<0.01). When MDA-MB-231 cells were incubated with normal human lung fibroblasts (NHLF) to model the tumor-lung interaction, the tumor cells induced a 3-fold increase in CCL2 expression by NHLF (P<0.001), through both direct and indirect co-culture. These results suggest that tumor-stroma crosstalk results in increased expression of CCL2 by lung fibroblasts, which could help drive the migration/growth of tumors in the lung. Taken together with the in vivo data, the results indicate that CCL2 plays a role in the migration, seeding and/or growth of tumor cells in distant sites. To determine if neutralizing CCL2 could increase efficacy if combined with other agents, CCL2 blockade was dosed as described above, either alone or in combination with bevacizumab (i.p. once a week 10 mg/Kg) or docetaxel (i.p q7dx3 30 mg/Kg) in the MDA-MB-231 orthotopic model (tumors were 70–100 mm3 at treatment initiation). The combination therapies showed significantly greater inhibition of primary tumor growth (P<0.018) and increased survival (P<0.028) as compared to treatment with CCL2 blockade, bevacizumab or docetaxel alone. In addition, only treatments that included CCL2 blockade significantly reduced visible lung metastases (P<0.008). These results indicate that neutralizing CCL2 in combination with a taxane or an anti-angiogenesis agent may lead to increased efficacy in the treatment of patients with breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B230.

CNTO 95 is a fully human monoclonal antibody that recognizes αv integrins. Previous studies have shown that CNTO 95 exhibits both anti-tumor and anti-angiogenic activities (Trikha M et al., Int J Cancer 110:326–335, 2004). In this study we investigated the biological activities of CNTO 95 on breast tumor cells both in vitro and in vivo. In vitro treatment with CNTO 95 decreased the viability of breast tumor cells adhering to vitronectin. CNTO 95 inhibited tumor cell adhesion, migration, and invasion in vitro. CNTO 95 treatment also induced tyrosine dephosphorylation of focal adhesion kinase (FAK), and the docking protein paxillin that recruits both structural and signaling molecules to focal adhesions (Turner CE, Int J Biochem Cell Biol 30:955–959, 1998; O’Neil GM et al., Trends Cell Biol 10:111–119, 2000). These results suggest that CNTO 95 inhibits breast tumor cell growth, migration and invasion by interruption of αv integrin mediated focal adhesions and cell motility signals. In vivo studies of CNTO 95 were conducted in an orthotopic breast tumor xenograft model. Treatment with CNTO 95 resulted in significant inhibition of both tumor growth and spontaneous metastasis of MDA-MB-231 cells to the lungs. CNTO 95 also inhibited lung metastasis in a separate experimental (tail vein injection) model of metastasis. The results presented here demonstrate the anti-tumor and anti-metastatic activities of CNTO 95 in breast cancer models and provide insight into the cellular and molecular mechanisms mediating its inhibitory effects on metastasis.