Abstract A107: Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues
Authors: zhang Ningyan , Gonzalez Anneliese , Deng Hui , Fan Xuejun , Vallecillo Luis Baez , zhang songlin , Brezski Randall , Roberson Emily , Jorden Robert , Strohl William , An Zhiqiang 
Published: May 2015

Growing evidence indicates that the tumor microenvironment has suppressive immune system and immune evasion is emerging as one of the cancer hallmarks. Immunoglobulin G (IgG) is an important component of human adaptive immunity in removing pathological cells including tumor cells. Recently, we reported that the cancer therapeutic antibody trastuzumab, a humanized IgG1, can be cleaved at the hinge region by matrix metalloproteinases (MMPs) in vitro, and the hinge cleaved trastuzumab (scIgG-T) showed significant loss of immune effector functions such as antibody dependent cellular cytotoxicity (ADCC) and anti-tumor efficacy in vivo (Fan et al., Breast Cancer Research 2012). The objectives of this study are 1) to investigate if the Fc hinge cleavage of IgG occurs in tumor tissues of breast cancer patients; 2) to understand the relationship among IgG Fc hinge cleavage, MMP expression, and anticancer immunity.

Methods: Surgically removed fresh tumor tissues (n=23, 11 with upfront surgery and 12 with neoadjuvant therapies) from breast cancer patients with informed consent (protocol # HSC-MS-10-0580) were snap frozen in liquid nitrogen and stored in a -80oC freezer until analysis. Normal breast tissues (n=20) were obtained from an outside vendor (Cureline Inc., CA) as snap frozen tissues. Peripheral blood mononuclear cells (PBMCs) were freshly prepared from patient blood samples collected before surgery and were preserved in liquid nitrogen storage tank until analysis. Hinge cleaved IgGs were quantified by ELISA and immuno-histochemistry (IHC) detection with a panel of anti-hinge specific antibodies developed at Johnson & Johnson. MMP expression was profiled using a reverse phase protein array method (Ray Biotech Inc., CA) and populations of immune effector cells in patient PBMCs were determined by multi-color flow cytometry.

Results: Levels of single hinge cleaved IgGs (scIgGs) were significantly higher in breast cancer tumor tissue than in normal breast tissue. The elevated Fc hinge cleavage of IgGs in breast tumor tissues was positively correlated with high levels of MMPs and expression of MMP-9 showed linear correlation (R=0.78) with the level of scIgGs in the tumor tissues. Notably, Fc hinge cleavage of IgGs in residual tumor tissues from patients treated with neoadjuvant therapies was similar to the levels in the normal breast tissues, while tumor tissues from patients with up-front surgery had significantly higher Fc hinge cleavage than both normal and neoadjuvant treated tumor tissues. In comparison with PBMCs from patients with the up-front surgery, PBMCs from patients with neoadjuvant therapies had an increased percentage of NK cells (CD56+) and immune cells with Fc gamma receptor IIa (CD32a) expression. In addition, percentages of immune effector cells with CD56+/CD16+ (Fc gamma receptor RIII), CD56+/CD64+ (Fc gamma receptor RI), and CD14+/CD16+ were also higher in PBMCs from patients treated with neoadjuvant therapies than those from patients with up-front surgery. Taken together, our results suggest that the reduced scIgG levels in neoadjuvant treated tumor tissue correlated with the better profiles of immune effector cells, while the increased Fc hinge cleavage in breast tumor tissue before therapeutic intervention is linked with immune suppression in the tumor microenvironment.

Citation Format: Ningyan zhang, Anneliese Gonzalez, Hui Deng, Xuejun Fan, Luis Baez Vallecillo, songlin zhang, Randall Brezski, Emily Roberson, Robert Jorden, William Strohl, Zhiqiang An. Elevated hinge cleavage of immunoglobulin G correlates with immune suppression and increased matrix metalloproteinases in breast tumor tissues. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A107.