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

Abstract

BackgroundThe 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.MethodsSplenic 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.ResultsOne 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.ConclusionsWe 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

“…Previously we showed that PODO447 exhibits high affinity for tumor-specific glycoepitopes using a well-characterized printed glycan array ( 32 , 41 ). One limitation of these arrays, however, is that they present glycostructures in isolation and not in the context of their normal attachment to proteins or lipids ( 41 ).…”

“…Cells were resuspended in 100 μl blocking buffer (FACS buffer, 1 μg/ml of anti-CD16/CD32 (clone 2.4G), 2% rat serum) for 20 min at 4°C in the dark, then spun at 394 g for 4 min and incubated in 100 μl primary antibody (Ab) solution for 30 min at 4°C in the dark. Rabbit-PODO83 ( 16 ) (2 μg/ml); and either rabbit or chimeric PODO447 ( 32 ) (5 μg/ml) were used to detect Podxl. Biotinylated pan-lectenz lectin (1 μg/ml, Lectenz-Bio #SK0501B) was used to detect sialylated complex glycans.…”

“…The 53 kDa Podxl core protein undergoes extensive, tissue-specific N- and O-linked glycosylation leading to a mature protein with an apparent molecular weight of 150-200 kDa ( 8 , 10 , 14 ). Since glycosylation processes are frequently altered in cancer ( 29 – 31 ), we recently engaged in an antibody development campaign to identify antibodies that would recognize tumor specific glycoepitopes on Podxl but not react with Podxl expressed by normal tissue ( 32 ). One resultant antibody, PODO447, exhibits exquisite specificity for a tumor glycoform of Podxl but lacks reactivity with normal adult human tissue.…”

“…One resultant antibody, PODO447, exhibits exquisite specificity for a tumor glycoform of Podxl but lacks reactivity with normal adult human tissue. Furthermore, in a tissue microarray (TMA) screen we showed that this epitope is expressed by over 60% of high grade serous (HGS) ovarian tumors ( 32 ). Thus, this antibody exhibits the appropriate specificity and distribution for further development as a therapeutic, particularly for highly metastatic disease.…”

Revealing the human mucinome

“…Mucin domains also exist in proteins outside of the 21 canonical mucins ( Fig 1A ). For example, CD43 on the surface of leukemia cells selectively interacts with the glyco-immune checkpoint receptor Siglec-7 through its N-terminal mucin domain 16 ; mucin domain-containing splice variants of CD44 (CD44v) serve as cancer cell markers relative to the ubiquitously expressed standard isoform 17 ; CD45 mucin domains act as suppressors of T-cell activation 18 ; mucin domain O-glycosylation on PSGL-1 is required for leukocyte-endothelial interactions 19 ; and aberrant regulation of mucin domains in podocalyxin and SynCAM1 are implicated in a variety of cancers 20,21 . In all of these cases, shared functional attributes of mucin domains impart structural and biophysical properties relevant to their biology.…”