Investigation of the role of podocalyxin in cancer progression and its potential as a cancer therapeutic

The advent of targeted therapies has vastly improved cancer diagnostics and treatments over the last three decades, however, cancer still remains the second leading cause of death worldwide. Importantly, the majority of cancer-related deaths are the result of metastatic disease. This highlights the need to identify biomarkers of tumors at high risk of metastasizing, and to generate targeted therapies against them. Previous studies have demonstrated a strong association between high expression of podocalyxin and decreased patient survival, however, little is known about the role of podocalyxin in promoting cancer progression or its potential as a therapeutic target. Here we perform an in-depth characterization of PODO83, an anti-podocalyxin antibody that we previously demonstrated delays primary tumor growth in murine tumor models. We show that despite its ability to delay primary tumor growth, its primary effect is actually the prevention of metastasis to the lung. We identified the binding epitope of PODO83 on the extracellular juxtamembrane domain of podocalyxin and showed that the antibody recognizes the core podocalyxin protein in both tumors and healthy tissue. Further, using urothelial, breast and ovarian carcinomas tissue microarrays, we present PODO83 as a promising diagnostic tool. Podocalyxin is normally readily expressed in the vascular endothelia and kidney podocytes, representing a source of concern surrounding the use of podocalyxin-based therapies. Here, we identified a novel tumor-restricted glycoepitope on podocalyxin and generated and characterized an antibody (PODO447) to target it. We found that while unconjugated PODO447 does not exert any inhibitory or toxic effect on tumor cells, when used as the targeting arm in a Vedotin antibody-drug conjugate (ADC), the PODO447-ADC specifically targets cancerous cells and increases the survival in pre-clinical models. The work presented in this thesis contributes directly to scientific understanding of the role of podocalyxin in tumor growth and metastasis. Further, we provide pre-clinical evidence supporting the furthered development of the novel podocalyxin antibodies PODO83, and PODO447 as diagnostic and targeted immunotherapies, respectively, to be used in the fight against cancer and to improve patient outcome.Medicine, Faculty ofMedical Genetics, Department ofGraduat

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 (GalNAcβ1) 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

Targeting a Tumor-Specific Epitope on Podocalyxin Increases Survival in Human Tumor Preclinical Models

Podocalyxin (Podxl) is a CD34-related cell surface sialomucin that is normally highly expressed by adult vascular endothelia and kidney podocytes where it plays a key role in blocking adhesion. Importantly, it is also frequently upregulated on a wide array of human tumors and its expression often correlates with poor prognosis. We previously showed that, in xenograft studies, Podxl plays a key role in metastatic disease by making tumor initiating cells more mobile and invasive. Recently, we developed a novel antibody, PODO447, which shows exquisite specificity for a tumor-restricted glycoform of Podxl but does not react with Podxl expressed by normal adult tissue. Here we utilized an array of glycosylation defective cell lines to further define the PODO447 reactive epitope and reveal it as an O-linked core 1 glycan presented in the context of the Podxl peptide backbone. Further, we show that when coupled to monomethyl auristatin E (MMAE) toxic payload, PODO447 functions as a highly specific and effective antibody drug conjugate (ADC) in killing ovarian, pancreatic, glioblastoma and leukemia cell lines in vitro. Finally, we demonstrate PODO447-ADCs are highly effective in targeting human pancreatic and ovarian tumors in xenografted NSG and Nude mouse models. These data reveal PODO447-ADCs as exquisitely tumor-specific and highly efficacious immunotherapeutic reagents for the targeting of human tumors. Thus, PODO447 exhibits the appropriate characteristics for further development as a targeted clinical immunotherapy