PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin.

BACKGROUND: The success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is associated with poor outcome, thus emerging as an important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft models, Podxl expression promotes tumor growth and metastasis. Although a promising target for immunotherapy, the expression of Podxl on normal vascular endothelia and kidney podocytes could hamper efforts to therapeutically target this molecule. Since pathways regulating post-translational modifications are frequently perturbed in cancer cells, we sought to produce novel anti-Podxl antibodies (Abs) that selectively recognize tumor-restricted glycoepitopes on the extracellular mucin domain of Podxl. METHODS: Splenic B cells were isolated from rabbits immunized with a Podxl-expressing human tumor cell line. Abs from these B cells were screened for potent reactivity to Podxl+ neoplastic cell lines but not Podxl+ primary endothelial cells. Transcripts encoding heavy and light chain variable regions from promising B cells were cloned and expressed as recombinant proteins. Tumor specificity was assessed using primary normal tissue and an ovarian cancer tissue microarray (TMA). Mapping of the tumor-restricted epitope was performed using enzyme-treated human tumor cell lines and a glycan array. RESULTS: One mAb (PODO447) showed strong reactivity with a variety of Podxl+ tumor cell lines but not with normal primary human tissue including Podxl+ kidney podocytes and most vascular endothelia. Screening of an ovarian carcinoma TMA (219 cases) revealed PODO447 reactivity with the majority of tumors, including 65% of the high-grade serous histotype. Subsequent biochemical analyses determined that PODO447 reacts with a highly unusual terminal N-acetylgalactosamine beta-1 (GalNAcß1) motif predominantly found on the Podxl protein core. Finally, Ab-drug conjugates showed specific efficacy in killing tumor cells in vitro. CONCLUSIONS: We have generated a novel and exquisitely tumor-restricted mAb, PODO447, that recognizes a glycoepitope on Podxl expressed at high levels by a variety of tumors including the majority of life-threatening high-grade serous ovarian tumors. Thus, tumor-restricted PODO447 exhibits the appropriate specificity for further development as a targeted immunotherapy.

Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells.

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell-mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

A pan-cancer analysis of alternative splicing events reveals novel tumor-associated splice variants of matriptase.

High-throughput transcriptome sequencing allows identification of cancer-related changes that occur at the stages of transcription, pre-messenger RNA (mRNA), and splicing. In the current study, we devised a pipeline to predict novel alternative splicing (AS) variants from high-throughput transcriptome sequencing data and applied it to large sets of tumor transcriptomes from The Cancer Genome Atlas (TCGA). We identified two novel tumor-associated splice variants of matriptase, a known cancer-associated gene, in the transcriptome data from epithelial-derived tumors but not normal tissue. Most notably, these variants were found in 69% of lung squamous cell carcinoma (LUSC) samples studied. We confirmed the expression of matriptase AS transcripts using quantitative reverse transcription PCR (qRT-PCR) in an orthogonal panel of tumor tissues and cell lines. Furthermore, flow cytometric analysis confirmed surface expression of matriptase splice variants in chinese hamster ovary (CHO) cells transiently transfected with cDNA encoding the novel transcripts. Our findings further implicate matriptase in contributing to oncogenic processes and suggest potential novel therapeutic uses for matriptase splice variants.

A unique role of the cholera toxin A1-DD adjuvant for long-term plasma and memory B cell development.

Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG ∼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.

T cell-independent IgA class switch recombination is restricted to the GALT and occurs prior to manifest germinal center formation.

Recently, we reported that CD40(-/-) mice, exhibiting exclusively T cell-independent IgA class switch recombination (CSR), demonstrated near normal levels of IgA plasma cells in the gut lamina propria (LP), despite the complete lack of germinal centers (GCs). In this study, we have extended our analysis focusing on how to reconcile these findings using flow cytometry and molecular markers for IgA CSR. In agreement with our previous results with small intestinal LP, the colon LP was found to host IgA CSR only when lymphoid follicles were present. Thus, no IgA CSR was observed in the nonorganized colon LP. By contrast, the Peyer's patch (PP) was the dominant IgA CSR site in both CD40(-/-) and wild type (WT) mice, and they both hosted similar levels of mRNA expression for B cell activating factor of the TNF family, a proliferation inducing ligand, and inducible NO synthase, potential switch-factors for IgA. Unexpectedly, we found that PP B cells undergoing IgA CSR were GL7-intermediate. These cells had not undergone somatic hypermutations (SHMs), whereas GL7-high cells in WT PP, which exhibited GCs, were heavily mutated. Moreover, IgA plasma cells in the LP of CD40(-/-) mice demonstrated few mutations in their Ig V regions, whereas WT LP B cells from different sites showed extensive SHMs, which were also clonally related. Therefore, IgA CSR can occur in PP at a stage preceding manifest GC (GL7-intermediate), whereas SHM require GC formations (GL7-high). These findings reconcile that IgA CSR can occur in PP in the absence of GC with the fact that CD40(-/-) mice host near normal levels of IgA plasma cells in the LP.

Gut IgA class switch recombination in the absence of CD40 does not occur in the lamina propria and is independent of germinal centers.

Conflicting findings have recently been presented as to the sites and sources of B cells that undergo class switch recombination (CSR) to IgA in the gut. In this study we provide compelling evidence in CD40(-/-) mice demonstrating that IgA CSR can be independent of CD40 signaling and germinal center formation and does not occur in the gut lamina propria (LP) itself. We found that CD40(-/-) mice had near normal levels of gut total IgA despite lacking germinal centers and completely failing to raise specific responses against the T cell-dependent Ags cholera toxin and keyhole limpet hemocyanin. The Peyer's patches in CD40(-/-) mice expressed unexpectedly high levels of activation-induced cytidine deaminase mRNA and germline alpha transcripts, but few postswitch circular DNA transcripts, arguing against significant IgA CSR. Moreover and more surprisingly, wild-type mice exhibited no to low IgA CSR in mesenteric lymph nodes or isolated lymphoid follicles. Importantly, both strains failed to demonstrate any of the molecular markers for IgA CSR in the gut LP itself. Whereas all of the classical sites for IgA CSR in the GALT in CD40(-/-) mice appeared severely compromised for IgA CSR, B cells in the peritoneal cavity demonstrated the expression of activation-induced cytidine deaminase mRNA comparable to that of wild-type mice. However, peritoneal cavity B cells in both strains expressed intermediate levels of the germinal center marker GL7 and exhibited no germline alpha transcripts, and only three of 51 mice analyzed showed the presence of postswitch circular DNA transcripts. Taken together, these findings strongly argue for alternative inductive sites for gut IgA CSR against T cell-independent Ags outside of the GALT and the nonorganized LP.