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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.


INTRODUCTION
Despite dramatic improvements in early detection, diagnosis and treatment, cancer remains one of the leading causes of death worldwide with an estimated 10 million deaths in 2020 (1). Importantly, over 90% of these cancer-related deaths are the result of metastatic disease which remains the most difficult to treat. This is largely due to its diffuse nature and the paucity of effective immuno-and chemotherapeutic agents with the appropriate selectivity for targeting tumor cells while sparing normal healthy tissue (2).
Importantly, in addition to its normal tissue expression pattern, Podxl is also aberrantly expressed by a wide variety of human tumors and its expression is consistently an indicator of poor prognosis (10,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Recent gene silencing and xenograft studies reveal an essential role for Podxl in disease progression through enhancing the mobility and invasiveness of tumor cells and promoting formation of distal metastasis (16,(26)(27)(28). Accordingly, Podxl has garnered increasing attention as a target for immunotherapy and we previously identified a core protein binding antibody (PODO83/PODOC1) that slows primary tumor growth and blocks metastatic disease (16). Nevertheless, further development of Podxl core proteinbinding antibodies as cancer immunotherapeutics has been hampered by concerns over possible toxicity to normal vascular and renal tissue where Podxl is abundantly expressed in healthy adults.
The 53 kDa Podxl core protein undergoes extensive, tissuespecific 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)(30)(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. 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.
In the current work, we evaluate the ability of PODO447 to serve as the targeting arm for therapeutic drug delivery in ADC assays. We find that although this antibody lacks inherent Podxl function blocking activity, it is highly effective in killing a variety of tumor cells in vitro as an ADC. Moreover, we find that PODO447-ADC shows potent efficacy against human tumor cell lines in two different mouse xenograft models. Finally, using a cell-based glycan array we reveal the PODO447-reactive epitope to be a core 1 O-glycan in the context of the Podxl polypeptide. In aggregate, these data reveal PODO447-ADC as a highly specific and efficacious immunotherapeutic agent and highlight its promise for treating high grade human tumors.

In Vitro Antibody Internalization
Sub-confluent cells were harvested and plated in 50 ml per well at the following concentrations: MIA PaCa-2 (8 x 10 3 cells/well), A-172 (8 x 10 3 cells/well) and SKOV3 (1 x 10 4 cells/well). Cells were then incubated overnight at 37°C, in 5% CO 2 . On day 1, coupling of the antibodies was performed by incubation of biotinylated PODO447 or palivizumab control mAb (40) with pHrodo ™ Red Avidin (ThermoFisher, #P35362) at 1:1 molar ratio for 30 min at 4°C. Next, 50 ml of cold pHrodo ™ -mAb mix was added to the cells and the plate was placed in the Incucyte ® . The Incucyte ® ZOOM software was set in "standard" mode with the phase and red channels selected and set to scan every 10 min for 24 hours. The rate of antibody internalization was evaluated through increased fluorescent area, reported as total internalization area (mm 2 /well). Average internalization was calculated, and statistical analyses was performed using two-way ANOVA in GraphPad Prism software.

In Vitro Antibody Cytotoxicity
Cells were plated on 96-well plates in 100ml culture medium and allowed to adhere overnight. SKOV3, MIA PaCa-2, A-172, THP-1 and HUVEC were seeded at 2.5 x 10 3 cells per well; OV3331, TOV3133D and ONS-76 were seeded at 5 x 10 3 cells per well. On day 1, a 5X stock solution of each mAb/ADC concentration to be tested was prepared in a stepwise 1:3 serial dilution series in cell culture medium, and 25ml of each dilution was added to cells in triplicate. Treated cells were cultured at 37°C, 5% CO 2 and high humidity for 144 h. On day 6, thiazolyl blue tetrazolium bromide (MTT) assays were used to determine relative cytotoxicity. Briefly, 100ml of MTT (1mg/ml, Thermo Fisher Scientific, #AC158990010) was added to the cells and left to incubate for 3 h at 37°C. Next, MTT media was removed and 50ml of DMSO was added to the cells and allowed to incubate for 15 min at RT, protected from light and with mild shaking. Absorbance was then read at 570nm. Percent viability was calculated as [1-(absorbance of treated samples/average absorbance of control samples)] x 100. Average relative cytotoxicity was calculated, and statistical analyses were performed using two-way ANOVA (GraphPad Prism software).

Antibody-Dependent Cell-Mediated Cytotoxicity Assay
A-172 (2x10 6 ) cells were stained with 10mM CellTrace ™ Far Red (Invitrogen ™ , #C34572) for 30 min at 37°C. Next, cells were washed 2X and seeded at 2 x 10 3 cells per well on a 96-well plate and allowed to adhere. 16 h after seeding A-172 labelled cells were incubated with 2.5, 0.5, or 0.1 mg/ml of PODO447, and 2.5 mg/ml of control human IgG1 for 15 min at RT. Next, antibodylabelled A-172 cells were co-cultured with human PBMCs at a 7:1 and 3:1 ratio for 1 h at 37°C, 5% CO 2. Cells were then harvested and stained with 1 mg/ml of PI for 5 min at RT. Cell viability was assessed via flow cytometry using a BD LSRFortessa ™ X-20 and analyzed using FlowJo ™ software (BD Biosciences, Ashland).

Mice
Tumor model animal experiments were carried out using 6-12week-old female NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) and NU/J (nude) mice obtained from Jackson's Laboratories (#005557 and #002019, respectively). Animals were bred in a specific, pathogen-free facility and all experiments were carried out under approved University of British Columbia Animal Care committee protocols (ACC protocol #A16-0007 and A20-0042) based on the recommendations of the Canadian Council on Animal Care.

Pre-Clinical
In Vivo Xenograft Models to Assess PODO447-ADC Efficacy MIA PaCa-2 (1x10 6 ) or OV3331 (1x10 6 ) cells were injected subcutaneously into the right flank of NSG or nude mice. Tumor dimensions were measured twice a week and tumor volumes (cm 3 ) were calculated by [(length x width 2 )/2]. Once tumors reached 0.15cm 3 , mice were treated with either PODO447-or palivizumab-Vedotin at concentrations ranging from 4 -2 mg/kg. ADC treatments were administered intravenously every 4 days. Average tumor volume over time was calculated, and statistical analysis was performed using twoway ANOVA test in GraphPad Prism software. Endpoints for survival analyses included animals with tumors exceeding 1 cm 3 or any animals with signs of morbidity reaching a humane endpoint regardless of tumor size. Survival Kaplan-Meier curves were calculated, and statistical analysis was performed using a Log-rank (Mantel-Cox) test in GraphPad Prism software.

PODO447 Binds to a Core 1 Glycostructure on Podxl
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). Thus, while they provide guidance on the types of carbohydrate structures antibodies detect, they lack the full antigenic context for determining their true specificity. Accordingly, to gain further insight into PODO447's specificity in a natural context, we probed its reactivity against a cell-based glycan array composed of isogenic HEK-293 cells, which express endogenous Podxl, with a combination of knockout (KO) and knock-in (KI) glycosyltransferase genes that control the loss or gain of core 1 and core 2 O-glycosylation (33). First, we verified that the glycoengineered HEK-293 cells correctly displayed the predicted O-glycoforms using a variety of lectins and monoclonal antibodies (mAbs) (Supplementary Figure 1). Next, we probed this cell array with PODO447. In contrast to the printed array, PODO447 failed to recognize LacDiNAc glycostructures on HEK-293 cells and, instead, exhibited selective binding for core 1 O-glycans as demonstrated by the lack of Ab recognition after the loss of core 1 structures (Tn, KO C1GALT1), and increased Ab binding after the loss of core 2 Oglycans (T/ST/dST, KO GCNT1) ( Figure 1A). Additionally, loss of a6 (KO ST6GALNACT2/3/4) and a3 (KO ST3GAL1/2) sialylation increased Ab binding suggesting that sialylation, although tolerated, is dispensable for PODO447 binding. Lastly, we also tested PODO447 recognition of cancerassociated STn (KI ST6GALNAC1/KO COSMC) and core 3 O-glycans (KI B3GNT3/KO COSMC) and observed no Ab binding to either of these structures. From these data, we conclude that PODO447 binds preferentially to a nonsialylated core 1 O-glycan, also known as T-antigen (T), a glycomotif that has been previously reported as a cancerassociated glycan epitope (42). Importantly, PODO447 recognizes T-antigen only in the context of the Podxl backbone since PODO447 fails to bind to PODXL-KO HEK-293 cells ( Figure 1A).
To further validate the contribution of Podxl's peptide backbone to the PODO447 epitope, we evaluated the binding of PODO447, PODO83 and peanut agglutinin (PNA) to SKOV3 wild type (WT) and Podxl knock-out (PODXL-KO) cells. The Podxl core protein-binding Ab, PODO83 (32), served as a control for ablation of protein expression, while PNA served as a control for T-antigen expression by all cell surface proteins. We found that PNA bound both SKOV3 WT and SKOV3 PODXL-KO cells suggesting a wide array of T-antigen epitopes are expressed on these cell lines ( Figure 1B). Conversely, PODO447 and PODO83 Abs reacted exclusively with WT but not PODXL-KO cells, confirming the specificity of these Abs for epitopes linked to the Podxl core protein. In addition, we further validated the contribution of the T-antigen to the PODO447 epitope using the human pancreatic CFPAC-1 cell line. This cell line shows heterogeneous expression of the PODO447 epitope (low to high) and a linear correlation between PODO447 and PNA staining. This was further validated by sorting this line into PODO447-positive and -negative CFPAC-1 cell subclones which, correspondingly, showed high and low PNA staining, respectively (Supplementary Figure 2). In summary, these experiments suggest that PODO447 specifically binds a glycopeptide epitope consisting of the core 1 O-glycan in the context of the Podxl polypeptide but does not recognize general core 1 glycans decorating other proteins.

PODO447 Lacks a Direct Effect on Cell Viability
Prompted by previous reports of anti-Podxl Abs with cytotoxic activity against undifferentiated human embryonic stem cells (43), and our own data showing PODO83 blocks metastasis in  vivo (16), we investigated whether PODO447 binding exhibits any effects on cell viability. We performed a long-term (144h) in vitro exposure assay using unconjugated PODO447 or palivizumab, an Ab specific for the F protein of the respiratory syncytial virus (RSV) that served as a control. We used normal human endothelia (HUVEC) cells and an array of human cancer cell lines, including pancreatic (MIA PaCa-2), glioblastoma (A-172) and acute monocytic leukemia (THP-1, AML) lines (Figures 2A-D). Unconjugated PODO447 had no effect on cell viability at any concentration, suggesting that binding of this antibody does not block pathways critical for cancer cell survival and proliferation in vitro.

PODO447 Induces Cellular Toxicity Via ADCC
We next evaluated if binding of PODO447 induces antibodydependent cell-mediated cytotoxicity (ADCC). We first incubated A-172 cells with 0.1, 0.5 and 2.5 mg/ml of PODO447 or 2.5 mg/ml of palivizumab control antibody. Next, we cocultured the antibody-bound cells with human peripheral blood mononuclear cells (PBMCs) at 7:1 and 3.5:1 ratio of effector (PBMCs) to target (A-172) cells and assessed A-172 cell viability by flow cytometry. We found that PODO447 induced a modest, dose-dependent, level of ADCC activity on A-172 cells at both effector to target cell ratios ( Figure 2E). From these results we conclude that unconjugated PODO447 can affect tumor cell viability via ADCC.

Evaluation of PODO447 Antibody-Drug Conjugates
Since unconjugated PODO447 did not directly affect cancer cell survival and only showed a low level of ADCC activity, we next investigated the potential use of PODO447 as the targeting arm for an ADC. We selected monomethyl auristatin E (MMAE) as the toxic payload since it is a well-studied tubulin polymerization inhibitor that, when internalized into cells, exhibits potent and broad anti-tumor activity (44). MMAE was conjugated to either PODO447 or palivizumab control. Coupling was accomplished using a valine-citrulline (val-cit) proteolytically cleavable linker, resulting in "Vedotin" (Val-Cit-PABC-MMAE)-ADCs ( Figure 3A). Linker stability was independently assessed by ReachBio Research Labs (RRL, www.reachbio.com) using an in vitro neutrophil differentiation killing assay, where extracellular proteolysis of the cleavable linker results in neutrophil cytotoxicity (45). RRL found that PODO447-ADC behaved very similarly to palivizumab control-ADC in the presence of neutrophils (Supplementary Figure 3A), thus suggesting both antibodies induce similar low levels of off-target toxicity as a result of non-specific extracellular cleavage.
Since these ADCs are dependent on internalization into acid compartments for cleavage and toxicity, we first tracked the ability of membrane-bound PODO447 to be internalized by tumor cells. Biotinylated PODO447 or palivizumab control Ab were conjugated to pHrodo ™ Red Avidin (Invitrogen ™ ), a pH sensitive dye that becomes fluorescent in acidic intracellular environments. Importantly, neither MMAE nor biotin conjugation altered the binding profile of PODO447 (Supplementary Figures 3B, C). We monitored antibody internalization by SKOV3, A-172 and MIA PaCa-2 cultures over 24 hours ( Figure 3B) and found that PODO447 was robustly internalized by all cell lines tested. In contrast, palivizumab control Ab exhibited negligible background  internalization. Thus, PODO447-ADC is competent to deliver a cytotoxic payload to the intracellular compartment of Podxlexpressing tumor cells ( Figure 3B).
To evaluate PODO447-ADC cytotoxic activity, we selected a panel of Podxl-expressing human cancer cell lines, including SKOV3 (ovarian, ascites-derived), MIA PaCa-2 (pancreatic, primary tumor-derived), A-172 (glioblastoma, primary tumorderived), ONS-76 (medulloblastoma, primary tumor-derived), THP-1 (acute monocytic leukemia), and patient tumor-derived TOV3133D and OV3331 [ovarian, primary solid tumor-and ascites-derived, respectively (35, 36)] ( Figure 4A; Supplementary  Figure 4). Cells were treated with serial dilutions of PODO447-or palivizumab-ADC for 6 days and then evaluated for cell viability. Although we saw a significant but modest toxicity (~40%) on ONS-76 cells at 2500 ng/ml, we observed potent cytotoxicity against THP-1 and A-172 cells with an IC 50 of 31 ng/ml and 93 ng/ml, respectively, and over 90% cytotoxicity at 278 ng/ml and higher PODO447-Vedotin doses. While the cytotoxic effect of PODO447-ADC on TOV3133D cells was modest (IC 50 of 833 ng/ml), we observed a potent cytotoxic effect in OV3331 cells with an IC 50 of 278 ng/ml and 80% cytotoxicity at 833 ng/ml. In contrast, no significant toxicity was observed against any cell lines treated with control palivizumab-ADC. Importantly, PODO447-ADC treatment of HUVEC cells, which express the Podxl core protein but not the PODO447 epitope, led to no significant toxicity. In summary, these data suggest that PODO447-ADC exhibits specific toxicity against a broad array of Podxl-expressing tumor cell lines and is well tolerated by cells expressing the native Podxl polypeptide but lacking the tumor-specific PODO447 glycoepitope.
To further confirm that PODO447-ADC cytotoxicity is linked to binding of the PODO447 epitope in the context of the Podxl core protein, we assessed in vitro killing of WT and PODXL-KO SKOV3 and MIA PaCa-2 cell lines (32) ( Figure 4B). PODO447-ADC led to a 3-fold reduction in the viability of SKOV3 WT cells and an 8-fold reduction in the viability of MIA PaCa-2 WT cells compared to control ADCtreated cells. In both cell types, this cytotoxic effect was highly specific as we did not observe a reduction in cell viability of palivizumab-ADC treated cells, or in PODXL-KO control cells treated with PODO447-ADC ( Figure 4B). We conclude that PODO447 is effective in delivering toxic payloads to tumor cells expressing an appropriately glycosylated form of Podxl.

PODO447-ADC Leads to Regression of Xenografted Human Ovarian and Pancreatic Tumors
To further evaluate the anti-tumor activity of PODO447-ADC, we next evaluated its efficacy against human tumors xenografted into immunocompromised NOD.Cg-Prkdc scid Il2rgtmWji/SzJ (NSG) mice. These mice bear mutations in genes essential for the development of T, B and natural killer (NK) cells, rendering them unable to reject xenografts and are, thus, a widely used model to evaluate human tumor growth and anti-tumor therapeutics. Mice were injected subcutaneously with MIA PaCa-2 cells, which have previously been shown to grow rapidly in immunocompromised mice (46). Once tumors reached a size of 0.15 cm 3 , we began bi-weekly intravenous administration of PODO447-ADC at a dose of 4 mg/kg ( Figure 5A). In order to monitor off-target toxicity, we used palivizumab-ADC as our control treatment. We found that four doses of PODO447-ADC led to complete tumor regression and significantly increased survival of these mice compared to control animals treated with palivizumab-ADC which exhibited only a minor and transient dip in tumor growth at the time of injection likely due to a low level of non-specific toxin uptake by tumors ( Figure 5A, PODO447-ADC-treated median survival > 78 days vs. control-ADCtreated = 46 days, c 2 = 20.69). Similar results were obtained at a 2 mg/kg dose of PODO447-ADC, which also led to a dramatic increase in the length of survival of tumor bearing mice (PODO447-ADC-treated median survival > 56 days vs. control-ADC-treated = 43 days, c 2 = 22.61). In addition, to further evaluate the effects of ADC treatment on the PODO447 epitope and PODXL core protein expression in vivo, we isolated similar sized tumors from all treatment groups and evaluated them histologically for PODO447 or PODO83 (core protein) epitope expression. While virtually all cells of the palivizumab-ADCtreated tumors continued to express the PODO447 and PODO83 epitopes, in PODO447-ADC treated animals these epitopes were virtually absent in the 4 mg/kg treatment group and restricted to a core population of the cells in the center of tumors in the 2mg/kg cohort, possibly reflecting lower exposure of these core tumor cells to the ADC (Supplementary Figure 5). This intriguing result suggests that tumors that survive PODO447-ADC treatment do so by downregulating Podxl protein expression rather than simply altering the expression of the glycoepitope. Moreover, the much slower growth of PODO447-ADC treated tumors is consistent with our previous studies where shRNAmediated dampening of Podxl expression in breast cancer cells suppressed tumor growth in mouse xenografts (16).
Because PODO447 recognizes the majority of high grade serous (HGS) ovarian carcinomas (32), we performed a similar experiment with the patient-derived HGS ovarian carcinoma line OV3331 (36). Again, bi-weekly intravenous injections of PODO447-ADC at 3 mg/kg dose, for a total of four doses, led to tumor regression and a striking increase in survival compared to control-ADC-treated group ( Figure 5B, PODO447-ADC-treated median survival = 167 days vs. control-ADC-treated = 82 days, c 2 = 5.70). Importantly, there were no signs of adverse events or weight loss in any of the treatment groups. Thus, PODO447-ADC has a potent anti-tumor effect in vivo suggesting that PODO447-based therapeutics could prove beneficial for patients with Podxl-positive tumors.

Enhanced PODO447-ADC Efficacy at Lower Doses in Animals With Functional B and NK Cells
Despite genetic differences, previous studies have noted that the efficacy of therapeutic Abs and ADCs and the level of off-target cytotoxicity varies widely depending on the degree of immunocompetence of the mouse model (47). Accordingly, we examined the effectiveness of PODO447-ADC in a second human tumor xenograft model. Specifically, we chose Nude mice, which bear a spontaneous chromosomal deletion of the Foxn1 gene  essential for the development of an appropriate thymic niche for T cell development. As a result, these mice are severely depleted of T cells but remain competent for the production of B and NK cells. Once again, we observed a strong therapeutic effect at similar doses of PODO447-ADC confirming our previous results. Treatment with PODO447-ADC at 4mg/kg/dose resulted in complete shortterm tumor regression in all mice, and long-term regression in 4 out of 7 mice (no tumor relapse present at 100 days, Figure 6A, PODO447-ADC-treated median survival > 93 days vs. control-ADC-treated = 35 days, c 2 = 5.22). In contrast, control ADC at 4mg/kg had no significant effect on tumor burden, nor did it induce a transient dip in tumor growth. Additionally, we tested PODO447-ADC at 2 mg/kg/dose. While we observed a trend toward tumor regression in mice treated with PODO447-ADC compared to control, this did not reach statistical significance likely due to half of the control-treated mice reaching their humane endpoint very early ( Figure 6B). Nevertheless, we found that treatment with 2 mg/ kg of PODO447-ADC significantly increased OS and resulted in complete remission in 1 out of 4 mice as assessed at 125 days after initial treatment ( Figure 6B, PODO447-ADC-treated median survival > 43 days vs. control-ADC-treated = 30.5 days, c 2 = 6.80). One intriguing explanation for this subtle difference between Nude and NSG mouse models is that Nude, but not NSG mice, have functional antibody secreting B cells and relatively normal levels of endogenous immunoglobulin in their serum which would be predicted to block non-specific antibody binding by tumor cells. Regardless, the data suggest that in two different xenograft models PODO447-ADC exhibits effective tumor killing at clinically relevant immunotherapeutic Ab doses.

DISCUSSION
Although novel immunotherapeutics (antibody-and cell-based) are providing an ever-increasing array of approaches to cancer treatments, several barriers continue to limit their broad-spectrum use, including failure to effectively target late-stage metastatic disease and safety concerns over targeting normal healthy tissues expressing reactive antigenic epitopes (toxicity). Accordingly, there has been a sustained effort to identify rare tumor-specific antigens expressed by a wide range of cancers that can effectively target molecules critical to late-stage disease progression. We find that the PODO447 Ab fulfills these key criteria.
In previous work, we and others have shown that the Podxl core protein is expressed by a wide variety of cancers and is consistently linked to poor outcome (10, 14-17, 32, 48). Although the new PODO447 Ab does not bind to all Podxlexpressing tumors, it does detect many including the majority of high grade serous ovarian tumors, highlighting its therapeutic potential (32). Here we developed this concept further and showed that, when used as the targeting arm of an ADC, PODO447 is highly effective at killing a range of tumor types including human glioblastoma, acute myelomonocytic leukemia, ovarian and pancreatic lines while sparing normal tissue as exemplified by the lack of cytotoxicity on HUVEC cells. Thus, PODO447-ADC could potentially be developed as a broadspectrum immunotherapeutic to human cancers.
Our previous survey of normal human tissue revealed PODO447 to be exquisitely tumor-specific and lack reactivity with normal kidney podocytes or vascular endothelia, which abundantly express the Podxl core protein (32). This conclusion is further supported by the glycoepitope mapping data presented here, which shows that the PODO447 Ab selectively reacts with a cancer-associated core 1 O-glycostructure in the context of the Podxl polypeptide. A priori, this alleviates concerns regarding toxicity of this Ab on normal healthy tissue expressing Podxl and this is further supported by our data showing that PODO447-ADC fails to exhibit any substantial toxicity against normal HUVECs or against human cell lines where the Podxl gene has been inactivated, while simultaneously exhibiting dose-dependent killing of a broad spectrum of Podxl-positive tumor targets at clinically relevant concentrations in vitro. Finally, we provide key proof-of-concept data that PODO447-ADCs are effective in vivo and provide longterm tumor clearance in two immunodeficient animal models.
The finding that the residual tumors that survive PODO447-ADC treatment have downregulated not only the PODO447 epitope but also expression of the Podxl core protein is noteworthy and encouraging for several reason. First, it suggests that it may be difficult for PODO447+ human tumors to "escape" antibody treatment by simply altering the pattern of PODO447 glycosylation, a general concern for therapeutic strategies targeting these post-translational modifications. Secondly, it suggests that the enzymes that generate the PODO447 glycoepitope may be playing an important functional role for tumor behavior that is difficult to modify. Lastly, it is consistent with previous studies showing that ablation of Podxl in tumor lines cripples the ability of these cells to form tumors in xenografted mice, particularly during the metastatic phase of the disease (16,27). Indeed, these reports highlight a critical role for Podxl in the subset of tumor cells responsible for tumorsphere formation in vitro and for colonization and establishment of tumors in specific niches in vivo, two key properties of tumor initiating cells (TIC). Thus, even in tumors where a minor subset of cells express Podxl, these data suggest that targeting this Podxl-expressing TIC population could target cells with the highest potential for the establishment of distal metastasis. Accordingly, targeting these cells could well have an outsized effect on disease progression. It will now be important to determine whether the PODO447 glycoepitope shows even greater specificity for tumor cells with TIC potential, Notably, there is an ever-expanding array of antibody-based immunotherapeutic platforms gaining traction in treatment of neoplastic disease. Although our analyses here was limited to the use of PODO447 as an ADC, given its unusual tumor specificity, it holds equal promise for development as a radio-immunotherapeutic reagent for tumor imaging and killing, a bi-specific antibody for recruitment of T cells to tumors, or as a targeting arm for chimeric antigen receptor (CAR-T)-based therapeutic. Indeed, preliminary data suggest PODO447 is suitable for several of these additional applications (in preparation) and highlight the importance of antibodies with this rare specificity for future development.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
All animal experiments were carried out under approved University of British Columbia Animal Care committee protocols (ACC protocol #A16-0007 and A20-0042) based on the recommendations of the Canadian Council on Animal Care. This study was performed in consideration with the Tri-Council Policy Statement of Ethical Conduct for Research involving Humans (TCPS 2).

AUTHOR CONTRIBUTIONS
DCH designed and performed most experiments, analyzed and interpreted data, and was the primary author of this manuscript. MRH assisted with experimental design. YL assisted in flow cytometry and cytotoxicity assays. IMR provided the HEK-293based glycan array data. PD created the SKOV3 and MIA PaCa-2 PODXL-KO cell lines and assisted in flow cytometry assays. JB provided the CFPAC-1 flow cytometry data. EMB created the CFPAC-1 sub-clones. IS provided the A172 ADCC data. AM provided and characterized the patient-derived ovarian carcinoma cell lines. HC, YN, and OB assisted with the glycan array experiments and analysis. CDR and KMM were key contributors to the experimental concept and design. All authors contributed to the article and approved the submitted version.