The online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users.
De-Kuan Chang and Raymond J. Moniz contributed equally to this work.
The authors declare that they have no competing interests.
WAM, DKC, and RJM. contributed to the design of the experiments and drafted the manuscript. DKC. carried out experiments and analyzed data. RJM performed the cytotoxicity, animal study, and FACS staining and analyzed data. ZX performed the cytotoxicity, transwell, and migration studies. JS participated in animal study. SS conceived the study and participated histology staining. QZ conceived the study and helped draft the manuscript. All authors read and approved the final manuscript.
National Foundation for Cancer Research.
Carbonic 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.
The 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.
Our 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.
Our 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.
Additional file 1: Figure S1. The antibody-mediated killing activities on CAIX expressing RCC4 tumor cells. (a) Anti-CAIX antibodies (G37 and G119) were tested for ADCC activity with human PBMCs in a dose dependent manner. CAIX+ RCC4 cells were incubated with human PBMCs at an indicated ratio and 5 μg/ml of antibodies, and cytotoxicity measured by LDH release in the culture supernatant. Data represent triplicate wells of one experiment. (b) CDC activity was determined by culture of rabbit serum with RCC4 cells, and cytotoxicity measured as above after 6 h. All data points represent the mean value ± S.D. *, **, and *** represent p value of Student t-test < 0.05, 0.01, and 0.005, respectively.12943_2015_384_MOESM1_ESM.pdf
Additional file 2: Figure S2. Selection of human PBMC with high ADCC activity. Human PBMC, isolated from twenty-one healthy donors were cultured with CAIX+ SKRC-59 cells (25:1 PBMC: RCC) in the presence of the indicated concentration of anti-CAIX G37. ADCC activity was measured as described in Materials and Methods, with donor 7 (D7) was chosen as the source of human PBMC utilized in the in vivo mouse model construction. Data represent the mean of triplicate measurements, ± S.D.12943_2015_384_MOESM2_ESM.docx
Additional file 3: Figure S3. The antibody-mediated killing activities on CAIX expressing tumor cells through NSG blood or serum. (a) Human anti-CAIX mAbs (G37 or G119) and mouse antihuman CAIX antibody, MAB2188, were tested for ADCC activity against CAIX+ SKRC-59 cells. NSG mouse PBMCs were isolated from untreated NSG mice, and total PBMC counted as effector cells (E) in co-culture with the respective target CAIX+ SKRC-59 cells (T) for six hours. The concentration of antibodies was 5 μg/ml. (b) CDC activity was determined by culture of NSG mouse serum with CAIX+ SKRC-59 cells, and cytotoxicity measured as above after 6 h. Culture supernatant was examined for LDH as a measure of cytotoxicity. Data represent the mean of three independent experimental values, ± S.D.. * represents p value of Student t-test < 0.05.12943_2015_384_MOESM3_ESM.pdf
Additional file 4: Figure S4. IFN-γ and CD8+ T cells staining on orthotopic RCC tissues. Representative immunohistochemical staining for IFN-γ (upper two lanes) and CD8 (lower lane) in the tumor sections at day 32 was shown by the indicated treatment group. Anti-IFN-γ and anti-CD8 antibodies were detected by DAB (shown as brown particles) and indicated by arrows. Bars represent 50 μm.12943_2015_384_MOESM4_ESM.docx
Additional file 5: Figure S5. IHC staining for HRP-labeled secondary anti-rabbit antibody and mouse on mouse (M.O.M.) kit. Representative immunohistochemical staining for HRP-labeled anti-rabbit antibody (upper) and M.O.M. kit (lower) in the tumor sections at day 32 was shown by the G37 treatment group. Sections were further detected by DAB. Bars represent 100 and 50 μm in 40X and 20X, respectively.12943_2015_384_MOESM5_ESM.docx
Additional file 6: Table S1. The kinetic constants of all anti-CAIX antibodies were obtained by global analysis using a 1:1 langmuir binding model and the T100 evaluation software. Percentage of carbonic anhydrase inhibition by CAIX antibodies was estimated from an electrometric assay. Internalization measured by both flow cytometry and ImageStream. + denotes degree of internalization as measured by regular flow cytometry.12943_2015_384_MOESM6_ESM.pdf
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- Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo
Raymond J. Moniz
Wayne A. Marasco
- BioMed Central
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