Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.
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.
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.
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