HEK-293 WT, HUVEC, SKOV3, A-172, MIA PaCa-2, THP-1 and MDA-MB-231 cells were obtained from the American Tissue Culture Collection (ATCC). Glycosylation-mutant HEK-293 cells (33, 34) and patient tumor-derived epithelial high grade serous ovarian OV3331 and TOV3133D cancer cell lines (35, 36) were previously described. ONS-76 medulloblastoma cell line was generously provided by Dr. Sorensen from the University of British Columbia. PODO447-positive and PODO447-negative CFPAC-1 cells were generated by sorting parental pancreatic CFPAC-1 cells (metastatic tumor-derived) with or without PODO447 expression. HEK-293 WT and isogenic cells were grown in DMEM (Gibco, #11965-092) supplemented with 10% FBS and 2mM GlutaMAX (Gibco, #35050061). CFPAC-1 cells were grown in IMDM (Gibco, #12440053) supplemented with 10% FBS. MIA PaCa-2, A-172, and MDA-MB-231 cells were grown in DMEM (Gibco, #11965-092) supplemented with 10% FBS and 10 U/ml penicillin and streptomycin (P/S) (Gibco, #15140-122). Human umbilical vein endothelial (HUVEC) cells were harvested from donor umbilical cords (Human Ethics no. H10-00643), grown in Endothelial Cell Growth Medium-2 Bulletkit™ (LONZA, #CC-3162), and used between passages 2 and 8. Human ovarian cancer SKOV3 cells were grown in DMEM F-12 with 15 mM HEPES (Sigma, #D6421) supplemented with 10% FBS, 0.2 mM L-glutamine (Gibco, #25030-081) and 10 U/ml P/S. OV3331 and TOV3133D cells were grown in complete OSE medium (36). ONS-76 cells were grown in RPMI (Gibco, #11875093) supplemented with 10% FBS. All cell lines were maintained at 37°C, 5% CO₂ and high humidity.

Cells were washed 1X with Ca²⁺- and Mg²⁺-free HBSS (Gibco, #14170-112), incubated for 1-2 min at 37°C in a 0.25% trypsin solution, quenched with complete growth media, then centrifuged for 4 min at 394g, washed 2X with FACS buffer (PBS, 2 mM EDTA, 5% FBS, 0.05% sodium azide) and transferred to a 96 well ‘v’ bottom plate. 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 394g 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. 3C9 (37), 5F4 (38) and TKH2 (39) antibodies were used to detect core 1, Tn and STn glycostructures, respectively. Wisteria Floribunda lectin (WFA, 0.25 μg/ml, Vector Laboratories, #B-1355-2) was used to detect α/β N-acetylgalactosamine (α/β-GalNAc). Erythrina Cristagalli lectin (ECL, 1 μg/ml, Vector Laboratories, #B-1145-5) was used to detect galactose, lactose and N-acetylgalactosamine. Peanut agglutinin (PNA, 5μg/ml, Vector Laboratories #B1075-5; ThermoFisher #L32459) was used to detect T-antigen. Rabbit-IgG (5 μg/ml, Vector Laboratories, #I-1000-5) and mouse/human-chimeric palivizumab (5 μg/ml, National Research Council, NRC) were used as isotype controls. Next, cells were washed 3X with FACS buffer and resuspended in 100 μl of secondary Ab solution (Alexa Fluor 647 (AF647) donkey-anti-rabbit (2 μg/ml, Invitrogen, #A31573); AF647 goat-anti-human (2 μg/ml, Jackson ImmunoResearch Laboratories, #109-605-098); Brilliant Violet 450 (BV450) Streptavidin (2 μg/ml)) for 30 min at 4°C in the dark. Cells were washed 2X with FACS buffer and resuspended in FACS buffer containing propidium iodide (PI) (0.5 μg/ml, Life Technologies, #P3566). High-throughput flow cytometry data was acquired using an SA3800 spectral analyzer. The remaining flow cytometry data was acquired using a BD LSRII and analyzed using FlowJo™ software (BD Biosciences, Ashland).

Sub-confluent cells were harvested and plated in 50 μl per well at the following concentrations: MIA PaCa-2 (8 x 10³ cells/well), A-172 (8 x 10³ cells/well) and SKOV3 (1 x 10⁴ cells/well). Cells were then incubated overnight at 37°C, in 5% CO₂. 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 μl 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 (μm²/well). Average internalization was calculated, and statistical analyses was performed using two-way ANOVA in GraphPad Prism software.

Cells were plated on 96-well plates in 100μl culture medium and allowed to adhere overnight. SKOV3, MIA PaCa-2, A-172, THP-1 and HUVEC were seeded at 2.5 x 10³ cells per well; OV3331, TOV3133D and ONS-76 were seeded at 5 x 10³ 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 25μl of each dilution was added to cells in triplicate. Treated cells were cultured at 37°C, 5% CO₂ and high humidity for 144 h. On day 6, thiazolyl blue tetrazolium bromide (MTT) assays were used to determine relative cytotoxicity. Briefly, 100μl 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 50μl 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).

A-172 (2x10⁶) cells were stained with 10μM CellTrace™ Far Red (Invitrogen™, #C34572) for 30 min at 37°C. Next, cells were washed 2X and seeded at 2 x 10³ 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 μg/ml of PODO447, and 2.5 μg/ml of control human IgG1 for 15 min at RT. Next, antibody-labelled 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 μg/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).

Human cord blood CD34+ cells (1x10⁶ cells/well, STEMCELL Technologies, # 70008.2) were seeded in 200 µL of complete growth medium [X-Vivo™ 15 (Lonza, #BE02-060F), rhIL-3 (20 ng/mL, Peprotech, #200-03), rhIL-6 (20 ng/mL, Peprotech, #200-06), rhSCF (100 ng/mL, Peprotech, #AF-300-07) and rhFlt-3L (100 ng/mL, Peprotech, #300-19)] in round-bottom 96-well plates and cultured at 37°C in 5% CO₂. On day 3, cells were washed 2X with X-Vivo™ 15 medium and incubated in X-Vivo™ 15 medium supplemented with rhSCF (50 ng/mL), rhFlt-3L (100 ng/mL), rh-IL3 (5 ng/mL), rhGM-CSF (5 ng/mL, Peprotech, #300-03) rhG-CSF (5 ng/mL, Peprotech, #300-23) at 37°C in 5% CO₂ for 4 days. On day 7, cells were then washed 2X and incubated in X-Vivo™ 15 medium supplemented with rhIL-3 (5 ng/mL) and rhG-CSF (30 ng/mL) for an additional 4 days. On day 11, cells were washed 2X and cultured in X-Vivo™ 15 medium supplemented with rhG-CSF (30 ng/mL) and either PODO447- or palivizumab-Vedotin at concentrations ranging from 0.01 – 10 µg/mL for an additional 6 days. On day 17, cells were harvested and prepared for flow cytometric analysis, as follows. Cells were blocked with 10% FBS and 20 µg/mL human IgG at 4°C for 10 minutes. Following blocking, cells were stained with anti-CD66b antibody (Biolegend, #305106) and DAPI (Biolegend, #422801) at 4°C for 20 minutes. Cells were then washed 2X and resuspended in FACS buffer. Flow cytometry data was acquired using a Beckman Coulter CytoFlex and analyzed using FlowJo™ software (BD Biosciences, Ashland) This assay was performed by ReachBio Research Labs, Seattle, USA (www.reachbio.com). The effect of ADCs on neutrophil differentiation is represented by percentage of CD66b+ cells within the viable cell population.

Tumor model animal experiments were carried out using 6-12-week-old female NOD.Cg-Prkdc ^scid Il2rg^{t m1Wjl}/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.

MIA PaCa-2 (1x10⁶) or OV3331 (1x10⁶) cells were injected subcutaneously into the right flank of NSG or nude mice. Tumor dimensions were measured twice a week and tumor volumes (cm³) were calculated by [(length x width²)/2]. Once tumors reached 0.15cm³, 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 two-way ANOVA test in GraphPad Prism software. Endpoints for survival analyses included animals with tumors exceeding 1 cm³ 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.

Tumor slides were stained with either rabbit-PODO83 (5 μg/ml), Rbt/Hu-chimeric PODO447 (1 μg/ml), Mo/Hu-chimeric pavilizumab control (1 μg/ml) or rabbit IgG control (5 μg/ml, Vector laboratories, #I-1000-5). Briefly, slides were deparaffinized and rehydrated in 100% xylene (3X, 5 min), 100% ethanol (2X, 3 min), 95% ethanol (1X, 3 min), 70% ethanol (1X, 3 min) and distilled water (1X, 3 min). Antigen retrieval was performed by heating slides in citrate buffer at 90°C for 40 min. Slides were washed in PBS (3X, 5min), incubated for 30 min in blocking solution (PBS, 5% donkey serum, 0.5% BSA, 0.3% Triton X-100), and incubated in primary Ab solution overnight at 4°C. Slides were then washed with TBST (3X, 15 min), incubated with either anti-rabbit-BIOT (2 μg/ml, SouthernBiotech, #6440-06) or anti-human-BIOT (2 μg/ml, Jackson ImmunoResearch, #209-065-098) in blocking solution for 30 min at RT, and washed again with TBST (4X, 15 min). Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 40 min at RT prior to rinsing 3X with PBS. Signal was amplified using Vectastain® Elite® ABC HRP Kit (Vector Laboratories, #PK-6100) according to manufacturer’s instructions, and then washed with PBS (3X, 5min). Signal was visualized using a DAB peroxidase (HRP) Substrate Kit (Vector Laboratories, #SK-4100) following the manufacturer’s recommendations. Each slide was incubated with DAB solution for 30-40 sec until strong signal appeared, then the reaction was stopped by dilution with PBS. Slides were counterstained with Harris Hematoxylin (2.5 min, Fisher HealthCare™ PROTOCOL™, #23-245651), rinsed 1X with distilled water (30 sec), incubated with differentiator solution (30 sec, 1% HCl in 70% EtOH) and rinsed with distilled water (30 sec). Staining was fixed in Bluing Solution (0.1% sodium bicarbonate) for 30 sec and rinsed in distilled water (1X, 30 sec) prior to dehydration in graded alcohols and xylene. Slides were mounted using Permount solution (Fisher Scientific, #SP15100) and subsequently analyzed for Podxl staining.

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 O-glycans (T/ST/dST, KO GCNT1) ( Figure 1A ). Additionally, loss of α6 (KO ST6GALNACT2/3/4) and α3 (KO ST3GAL1/2) sialylation increased Ab binding suggesting that sialylation, although tolerated, is dispensable for PODO447 binding. Lastly, we also tested PODO447 recognition of cancer-associated 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 non-sialylated core 1 O-glycan, also known as T-antigen (T), a glycomotif that has been previously reported as a cancer-associated 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 glyco-peptide 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.

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.

We next evaluated if binding of PODO447 induces antibody-dependent cell-mediated cytotoxicity (ADCC). We first incubated A-172 cells with 0.1, 0.5 and 2.5 μg/ml of PODO447 or 2.5 μg/ml of palivizumab control antibody. Next, we co-cultured 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.

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 Podxl-expressing 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 tumor-derived), 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 IC₅₀ 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 (IC₅₀ of 833 ng/ml), we observed a potent cytotoxic effect in OV3331 cells with an IC₅₀ 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 ADC-treated 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.

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 cm³, 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-ADC-treated = 46 days, χ² = 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, χ² = 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-ADC-treated 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 shRNA-mediated 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, χ² = 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.

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 short-term 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, χ² = 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, χ² = 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.