Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo

Abstract

BackgroundCarbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated.MethodsThe role of human anti-CAIX mAbs on CAIX+ RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX+ RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining.ResultsOur studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ−/− mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in inhibition of CAIX+ tumor growth.ConclusionsOur findings demonstrate that these novel human anti-CAIX mAbs have therapeutic potential in the unmet medical need of targeted killing of HIF-driven CAIX+RCC. The orthotopic tumor xenografted humanized mouse provides an improved model to evaluate the in vivo anti-tumor capabilities of fully human mAbs for RCC therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users.

Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

“…Another important proof-of-principle experiment involved the injection of human NK cells into the blood of mice one day before their euthanasia. Our previous experience with this orthotopic ccRCC model in NSG mice showed that human NK cells last only for a few days in the mouse blood [31], and for this reason the injection of these NK cells was not performed until the end of the experiment. The results demonstrated the specific recruitment of human NK cells into the tumor of mice treated with anti-PD-L1 IgG1-secreting anti-CAIX CAR T cells.…”

Humanized Mice as Unique Tools for Human-Specific Studies

“…These inventions cascaded into a series of immunodeficient mice and their variants (BRG, NOG, NRG) (Ali et al 2012; Grover et al 2017; Ishikawa et al 2005; Katano et al 2014; Koboziev et al 2015; Shultz et al 2005) being innovated which enabled in-depth analysis in research areas, such as human hematopoiesis (Rongvaux et al 2011; Yong et al 2016), innate and adaptive immunity (Brehm et al 2010; Pearson et al 2008), autoimmunity (Gunawan et al 2017; Viehmann Milam et al 2014), infectious disease (Keng et al 2015; Lüdtke et al 2015; Wege et al 2012), cancer biology (Chang et al 2015; Her et al 2017; Morton et al 2016), and GvHD (King et al 2008; Kirkiles-Smith et al 2009; Zhao et al 2015), in-turn, facilitating the development of therapeutic agents and novel vaccines. An overview of genotypic and physiological characteristics of each model is outlined in Tables 1 and 2.…”

Humanized Mouse Xenograft Models: Narrowing the Tumor–Microenvironment Gap

“…Chang et al humanized an orthotopic renal cell carcinoma (RCC) model mouse with PBMCs to examine the effectiveness of an antibody targeting the carbonic anhydrase IX protein expressed in RCC showing that this antibody primed T cells and inhibited cancer growth (30). Finally, while examining a novel immunotherapy against melanoma, Hu et al generated HMs from CD34+ HSPCs engineered to express an HLA class I-restricted melanoma antigen (MART-1)-specific TCR gene (31).…”