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A Novel Antibody to a Tumor-Restricted Epitope on the Cancer Antigen

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A Novel Antibody to a Tumor-Restricted Epitope on the Cancer Antigen Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001128 on 26 November 2020. Downloaded from PODO447: a novel antibody to a tumor- restricted epitope on the cancer antigen podocalyxin 1 1 2 3 Diana Canals Hernaez , Michael R Hughes, Pamela Dean, Peter Bergqvist, Ismael Samudio,3 Ola Blixt,4 Katharina Wiedemeyer,5 Yicong Li,1 Chris Bond,3 3 5 6 2 1 Eric Cruz, Martin Köbel, Blake Gilks, Calvin D Roskelley, Kelly M McNagny To cite: Canals Hernaez D, ABSTRACT BACKGROUND Hughes MR, Dean P, et al. Background The success of new targeted cancer Ovarian carcinoma (OC) is one of the PODO447: a novel antibody to therapies has been dependent on the identification of a tumor- restricted epitope on leading causes of cancer mortality in tumor- specific antigens. Podocalyxin (Podxl) is upregulated 1 the cancer antigen podocalyxin. women. Frequently diagnosed only at on tumors with high metastatic index and its presence Journal for ImmunoTherapy advanced stages and with a 5- year survival of is associated with poor outcome, thus emerging as an of Cancer 2020;8:e001128. approximately 47%,2 OC is a ‘silent killer’ doi:10.1136/jitc-2020-001128 important prognostic and theragnostic marker in several human cancers. Moreover, in human tumor xenograft that offers few treatment options. Although new diagnostic and treatment options have ► Additional material is models, Podxl expression promotes tumor growth and published online only. To view, metastasis. Although a promising target for immunotherapy, led to a significant improvement in survival please visit the journal online the expression of Podxl on normal vascular endothelia and rates for many cancers over the last 50 (http:// dx. doi. org/ 10. 1136/ jitc- kidney podocytes could hamper efforts to therapeutically years, the 5- year survival for OC has hardly 2020- 001128). target this molecule. Since pathways regulating post- changed since 1980,3 4 highlighting the translational modifications are frequently perturbed in urgent need for new treatment strategies. Accepted 05 November 2020 cancer cells, we sought to produce novel anti- Podxl Although genetic and/or epigenetic antibodies (Abs) that selectively recognize tumor-restricted changes are known to drive tumor develop- glycoepitopes on the extracellular mucin domain of Podxl. ment, dysregulation of protein expression Methods Splenic B cells were isolated from rabbits and altered post- translational modifica- immunized with a Podxl- expressing human tumor cell line. Abs from these B cells were screened for potent reactivity tions are also known to occur. Specifically, to Podxl+ neoplastic cell lines but not Podxl+ primary changes in the patterns of protein glyco- sylation are well known to occur in cancer5 endothelial cells. Transcripts encoding heavy and light http://jitc.bmj.com/ chain variable regions from promising B cells were cloned and, in some cases, are thought to drive and expressed as recombinant proteins. Tumor specificity tumor progression.6 This can be through was assessed using primary normal tissue and an ovarian well- known effects on protein stability cancer tissue microarray (TMA). Mapping of the tumor- and folding; or through modification of restricted epitope was performed using enzyme-trea ted processes including protein trafficking, human tumor cell lines and a glycan array. immune recognition, cell migration, ligand– Results One mAb (PODO447) showed strong reactivity on September 24, 2021 by guest. Protected copyright. receptor interaction, signal transduction with a variety of Podxl+ tumor cell lines but not with 7–11 normal primary human tissue including Podxl+ kidney and cell adhesion. Indeed, glycans can podocytes and most vascular endothelia. Screening of an confer a selective advantage on cells that 12 13 ovarian carcinoma TMA (219 cases) revealed PODO447 facilitates tumor spread. Moreover, reactivity with the majority of tumors, including 65% of cancerous cells undergo reprograming and the high-grade serous histotype. Subsequent biochemical transcriptional changes that greatly impact analyses determined that PODO447 reacts with a their glycome and glycoproteome, leading © Author(s) (or their highly unusual terminal N- acetylgalactosamine beta-1 to overexpression or de novo expression employer(s)) 2020. Re- use (GalNAcβ1) motif predominantly found on the Podxl protein of specific glycoepitopes. Importantly, permitted under CC BY-NC. No core. Finally, Ab–drug conjugates showed specific efficacy these cancer- specific alterations in protein commercial re- use. See rights in killing tumor cells . in vitro glycosylation may also provide a unique and permissions. Published by Conclusions We have generated a novel and exquisitely BMJ. opportunity for clinical intervention with tumor-restricted mAb, PODO447, that recognizes a For numbered affiliations see glycoepitope on Podxl expressed at high levels by a reduced toxicity since these modifications end of article. variety of tumors including the majority of life- threatening are largely tumor- specific. Correspondence to high- grade serous ovarian tumors. Thus, tumor- restricted Podocalyxin (Podxl) is a member of a Professor Kelly M McNagny; PODO447 exhibits the appropriate specificity for further 3- gene family of stem cell sialomucins that kelly@ brc. ubc. ca development as a targeted immunotherapy. includes Podxl, CD34 and endoglycan. Canals Hernaez D, et al. J Immunother Cancer 2020;8:e001128. doi:10.1136/jitc-2020-001128 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001128 on 26 November 2020. Downloaded from These proteins can be distinguished from other cell METHODS surface sialomucins based on a shared genomic orga- Ab production nization (each encoded by a similar, 8- exon, genomic New Zealand White rabbits were immunized with Podxl- locus) and a shared protein domain structure: they expressing human glioblastoma A-172 cells (#CRL-1620, each contain a large, highly glycosylated mucin domain American Tissue Culture Collection (ATCC)). Rabbit followed by a cysteine- bonded globular domain, stalk mAbs were isolated using selected lymphocyte anti- domain, transmembrane domain and a short (approxi- body method (SLAM) technology.35 Briefly, individual mately 70 amino acid) cytoplasmic tail with a consensus B- cell clones whose supernatants reacted with Podxl- C- terminal binding site for PDZ- domain proteins.14–16 expressing MDA- MB-231 cells were selected. These were Although widely expressed during embryonic devel- then screened via ELISA for high reactivity against Podxl opment,15 17 in normal adult tissue Podxl is primarily isolated from human breast cancer MDA-MB-231 and expressed on vascular endothelia,18 19 kidney podo- low reactivity to the human embryonic kidney 293 (HEK cytes15 and a restricted subset of epithelial lumens.15 293) cells. Next, selected clones were screened against Nevertheless, Podxl is also aberrantly expressed by Podxl- expressing MDA- MB-231 and its PODXL- KO coun- a wide variety of cancer types, and this expression is terpart to confirm Podxl specificity. Supernatants were consistently an indicator of poor prognosis.20–30 For also screened for reactivity with Chinese hamster ovary example, in early retrospective studies of breast cancer, (CHO) cells expressing Podxl, and low reactivity to CHO patients with tumors expressing high levels of Podxl cells expressing related family members CD34 (Cd34) and exhibited a greatly reduced disease- specific survival, endoglycan (Podxl2). Binding profiles for tumor versus and multivariant analysis showed Podxl to be a highly- normal cells were generated to enrich for those that pref- 20 significant independent predictor of poor outcome. erentially bound to Podxl on tumor cells. Ab VH and VL Similarly, in OC where Podxl is expressed by the regions were subsequently cloned into expression vectors majority of high- grade serous carcinomas, cell surface containing the constant regions of the rabbit, human, or expression of Podxl correlated with a significant rabbit/human chimeric heavy and light chains, respec- decrease in disease free survival.22 Interestingly, recent tively, in order to generate high-affinity anti- Podxl rabbit, gene silencing experiments suggest that upregulation humanized and rabbit/human chimeric IgG1 Abs. of Podxl is not merely a predictor of poor outcome but also an important player in disease progression by Cell culture enhancing tumor invasion and metastasis.21 29 31 32 In HEK293, human umbilical vein endothelial (HUVEC), summary, because of its cell surface localization and SKOV3, SUM149, PANC-1, A-172, MIAPACA and MDA- its direct role in tumor metastasis, Podxl is a promising MB-231 cells were obtained from ATCC. OV3331 target for cancer immunotherapy. patient tumor- derived EOC cell line was kindly provided As a first foray into exploiting this opportunity, we by Dr. Mes- Masson from the University of Montreal. generated a series of anti- Podxl monoclonal antibodies HEK293, human pancreatic adenocarcinoma (PANC-1, (mAbs) to the extracellular domain of Podxl expressed MIAPACA), A-172 and MDA- MB-231 cells were grown in http://jitc.bmj.com/ on the surface of tumor cells.21 One mAb, PODOC1 Dulbecco’s Modified Eagle’s Me (DMEM, Gibco, #11965- (hereafter designated PODO83), showed a potent 092) supplemented with 10% fetal bovine serum (FBS) ability to restrict primary tumor growth in vivo and to and 10 U/mL penicillin and streptomycin (P/S) (Gibco, block metastatic progression when administered to #15140-122).
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