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

Background: 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. Methods: 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. Results: 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. Conclusions: 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. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0384-3) contains supplementary material, which is available to authorized users

A vault nanoparticle vaccine induces protective mucosal immunity.

BackgroundGeneration of robust cell-mediated immune responses at mucosal surfaces while reducing overall inflammation is a primary goal for vaccination. Here we report the use of a recombinant nanoparticle as a vaccine delivery platform against mucosal infections requiring T cell-mediated immunity for eradication.Methodology/principal findingsWe encapsulated an immunogenic protein, the major outer membrane protein (MOMP) of Chlamydia muridarum, within hollow, vault nanocapsules (MOMP-vaults) that were engineered to bind IgG for enhanced immunity. Intranasal immunization (i.n) with MOMP-vaults induced anti-chlamydial immunity plus significantly attenuated bacterial burden following challenge infection. Vault immunization induced anti-chlamydial immune responses and inflammasome formation but did not activate toll-like receptors. Moreover, MOMP-vault immunization enhanced microbial eradication without the inflammation usually associated with adjuvants.Conclusions/significanceVault nanoparticles containing immunogenic proteins delivered to the respiratory tract by the i.n. route can act as "smart adjuvants" for inducing protective immunity at distant mucosal surfaces while avoiding destructive inflammation

Immunization with MOMP-vaults reduces local bacterial burden following genital infection.

<p>The bacterial burden of chlamydiae following a challenge infection was determined from vaginal swabs to be statistically reduced in the MOMP-vault immunized group and the positive control group immunized i.n. with live <i>C. muridarum</i> (Live CM) compared to the control GL-vault immunized group (two-way RM ANOVA, *p<0.005). Dunn's post-hoc test showed no difference between the Live CM and the MOMP-vault immunized groups.</p

Design of recombinant vault nanoparticles containing immunogenic proteins.

<p>(a) ELISA assay configured using vaults with the z peptide (cp-MVP-z) and without (cp-MVP) reacted with either mouse IgG or IgA. Data points represent duplicates, SD = 0.004–0.034 nm. (b) Schematic diagram of constructs used to prepare baculovirus recombinant vaults containing MOMP indicating their approximate locations within a vault. (c) Western blot of high speed pellet extracts of MOMP-vaults (5 µg/lane). Molecular weight markers (lane 1). The gel was probed with a monoclonal antibody against the VD1 region of MOMP (MoPn-40)(lane 2) or mouse 1023C monoclonal antibody against MVP (lane 3). The size of MOMP fused to mINT is shown in the box and is approximately 58 kDa. (d) Negative stain EM of cp-MVP and cp-MVP-z/MOMP-mINT recombinant vaults. Bar, 100 nm.</p

Dendritic cells are efficient at incorporating vaults.

<p>(a) BMDC (1×10⁶) were incubated with media, GL-vaults (500 µg) or FITC-dextran (250 µg) at 37°C for the indicated times. Cells were stained for a marker on mouse BMDC (CD11c-PE) and analyzed using a flow cytometer. (b) BMDC (red) incubated as above for 30 min were viewed on a fluorescent microscope (Carl Zeiss, LSM5 Pascal). The fluorescent particles (GL-vaults or FITC-dextran) appear green and mouse BMDC appear red due to staining with CD11c-PE. Results shown are representative of three experiments.</p

MOMP-vault immunization enhances the number and induces the redistribution of Th1 cells in the ILN.

<p>(a) Representative histogram showing CD3+CD4+ cells and IFNγ vs. IL-4 intracellular cytokine staining of ILN cells. The number of (b) Th1 or (c) Th2 cells in the ILNs of individual mice on days 7 and 15 after challenge infection were compared by Students' t-test (*p<0.02). Representative experiment with 4–5 mice per group where at least 50,000 cells were analyzed per mouse. The level of (d) IL-1α, (e) IFNγ, and (f) CXCL10 was measured in triplicate in OD tissue homogenates at two time-points after challenge infection in mice immunized with MOMP-vaults or given a lung infection with <i>C. muridarum</i> using Luminex assays (Milipore Corp). Cytokine levels were compared by Students' t-test (*p<0.05). One representative experiment of 3–5 mice per group.</p