Fig 2: GPC2 is a MYCN-regulated oncogene in SCLC(A) GPC1–GPC6 expression across normal lung tissue (N; n = 26) and SCLC (T; n = 31 SCLC tumors and 22 SCLC cell lines).29(B) Plot of GPC2 molecules/cell in neuroblastoma and SCLC cell lines.(C and D) GPC2 (C) and pan-Myc (D) western blot in neuroblastoma and SCLC cell lines.(E and F) GPC2 histogram (E) and GPC2 versus SCLC stem cell marker (F) flow cytometry plots of the H526 xenograft.(G) Fold-change mean fluorescent intensity (MFI) of SCLC surface molecules in GPC2UltraHi versus GPC2Hi cells in the H526 xenograft.(H) Percent input plot of MYCN ChIP PCR in the SCLC H526 cell line. GABRG2 is a non-MYCN-targeted gene.(I and J) MYCN and GPC2 RNA quantification (left, 72 h and right, 96 h; I) and western blot (J) of H526 cells following MYCN and GPC2 depletion.(K) Western blot of SCLC cells following GPC2 depletion.(L) SCLC cell growth plot following GPC2 depletion.Data in (A) represent mean ± SEM expression for each glypican. Data in (B), (G), and (L) represent mean ± SEM of 3–4, 2–4, and 2–3 independent experiments, respectively. Data in (H) and (I) represent mean ± SEM from a representative experiment done in technical triplicate with each experiment being done at least two independent times. NB-EbC1, SMS-SAN, and SH-SY5Y cells in (B)–(D) are representative neuroblastoma cells. C in (I)–(L) represents control non-targeting short hairpin RNA (shRNA); 2 and 4 in (I)–(L) represent unique GPC2-targeting shRNAs; and 1, 5, 6, and 7 in (I) and (J) represent unique MYCN-targeting shRNAs.*p < 0.05; **p < 0.001; ***p < 0.0001; ns, not significant. See also Figure S1.

Fig 3: D3-GPC2-PBD significantly extends the survival of mice with neuroblastoma COG-N-421x PDXs and SK-N-AS xenografts(A) COG-N-421x PDX volumes after treatment with D3-GPC2-PBD (n = 8–9 mice/cohort; represented as mean ± SEM).(B) PFS analysis of treatment arms in (A). Median survival of 3.6 weeks for vehicle, 6.6 weeks for 0.25 mg/kg ADC, 11.6 weeks for 0.5 mg/kg ADC, and >14.1 weeks for 1 and 3 mg/kg ADC treatment cohorts.(C) IHC of COG-N-421x PDXs 5 days after treatment with 1 mg/kg D3-GPC2-PBD.(D) Quantification of relative ?H2AX, cleaved caspase-3, or cleaved PARP IHC staining after D3-GPC2-PBD treatment.(E) Locally advanced COG-N-421x PDX volumes after treatment with D3-GPC2-PBD (n = 4 mice/cohort; starting mean tumor volumes = 0.75–0.77 cm3).(F) PFS of treatment arms in (E). Median survival of 2.3 weeks for vehicle, 5.4 weeks for 3 mg/kg ADC, and >14.6 weeks for 1 mg/kg ADC × 4 treatment cohorts.(G) Neuroblastoma SK-N-AS xenograft volumes after treatment with D3-GPC2-PBD (n = 10 mice/cohort; represented as mean ± SEM).(H) PFS of treatment arms in (G). Median survival of 3.2 weeks for vehicle; 14.0 weeks for 0.5 mg/kg ADC; and >14.3 weeks for 1 mg/kg ADC, 1 mg/kg ADC × 4, and 3 mg/kg ADC treatment cohorts.(I) IHC of SK-N-AS xenografts 5 days after treatment with 1 mg/kg D3-GPC2-PBD.(J) Quantification of relative ?H2AX, cleaved caspase-3, or cleaved PARP IHC staining after D3-GPC2-PBD treatment.Scale bars: 200 µm in (C) and (I). Blue arrows in (A), (B), and (E)–(H) represent initial ADC dose, and red arrows in (E)–(H) indicate subsequent 3 ADC doses for the 1 mg/kg ADC × 4 treatment cohort.**p < 0.001; ***p < 0.0001.See also Figure S7.

Fig 4: D3-GPC2-PBD induces specific, potent, and durable eradication of neuroblastomas by inducing DNA damage and apoptosis without GPC2 loss(A) NB-1643 PDX tumor volumes after treatment with D3-GPC2-PBD (n = 8–9 mice/cohort; represented as mean ± SEM).(B) PFS analysis of treatment arms in (A). Median survival of 3.4 weeks for vehicle; 3.1 weeks for D3-GPC2-IgG1; and >20.9 weeks for 1 mg/kg ADC, 1 mg/kg ADC × 4, and 3 mg/kg ADC treatment cohorts.(C) Locally advanced NB-1643 PDX tumor volumes after treatment with D3-GPC2-PBD (starting mean tumor volumes = 0.98–1.01 cm3).(D) PFS analysis of treatment arms in (C). Median survival of 1.3 weeks for vehicle and >14.3 weeks for 1 mg/kg ADC × 4 and 3 mg/kg ADC treatment cohorts.(E) Immunohistochemistry (IHC) of locally advanced NB-1643 PDX tumors 4 days after treatment with 3 mg/kg D3-GPC2-PBD.(F) Quantification of relative ?H2AX, cleaved caspase-3, or cleaved PARP IHC staining after D3-GPC2-PBD treatment shown in (E) and Figures S7E and S7F.(G) CHLA-79 xenograft volumes after treatment with D3-GPC2-PBD (n = 9 mice/cohort; represented as mean ± SEM).(H) PFS analysis of treatment arms in (G). Median survival of 3.6 weeks for vehicle; 9 weeks for 0.5 mg/kg ADC; 10.4 weeks for 1 mg/kg ADC/50× IgG1; and >14.3 weeks for 1 mg/kg ADC, 1 mg/kg ADC × 4, and 3 mg/kg ADC cohorts.(I) D3-GPC2-PBD ADC re-treatment (1 mg/kg; indicated with red arrow) of 2 tumors initially treated with 1 mg/kg of ADC in (G). Five additional mice in the initial 1 mg/kg ADC treatment cohort with stable tumor regression were also followed in parallel to the ADC re-treated mice.(J) H&E (top) and GPC2 IHC (bottom) of indicated tumor (*) from (I).(K) High-power GPC2 IHC of indicated tumor (*) from (I).(L) CHLA-79 xenograft volumes after treatment with free PBD (n = 5 mice/cohort; represented as mean ± SEM).(M) PFS analysis of treatment arms in (L). Median survival of 4.0 weeks for vehicle versus 3.4 weeks for free PBD.Scale bars: 200 µm in (E) and (K) and 800 µm (top) and 900 µm (bottom) in (J). Blue arrows in (A)–(D), (G), (H), (L), and (M) represent initial ADC dose and red arrows in (A)–(D), (G), and (H) indicate subsequent 3 ADC doses for the 1 mg/kg ADC × 4 treatment cohort and in (I) indicate the ADC rechallenge dose given to 2 of 7 remaining mice on the CHLA-79 1 mg/kg ADC treatment arm.cCaspase-3, cleaved caspase-3; cPARP, cleaved PARP.***p < 0.0001; ns, not significant.See also Figure S7.

Fig 5: Neuroblastoma stem cells have highly enriched GPC2 expression(A and B) GPC2 histograms (A) and GPC2 versus CD133, CD338, CD117, and GD2 flow cytometry plots (B) of neuroblastoma PDXs/xenografts.(C) Fold-change GPC2 MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma PDXs/xenografts.(D) Fold-change stem cell marker MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma PDXs/xenografts.(E and F) Gpc2/GPC2 histograms (E) and Gpc2/GPC2 versus CD133, CD338, CD117, and GD2 flow cytometry plots (F) of murine (9464D) and human (SK-N-AS) neuroblastoma cell lines.(G) Fold-change stem cell marker MFI plot of GPC2UltraHi versus GPC2Hi cells in neuroblastoma cell lines.(H) GPC2 (left) and CD133 (right) histograms in SK-N-AS cells after D3-GPC2-PBD ADC treatment.(I) Quantification of CD133- and GPC2-positive SK-N-AS cells after D3-GPC2-PBD ADC treatment (left, ADC × 1; right, ADC × 2).(J) GPC2 western blot of SK-N-SH/NBL-SRPMI/NB cells.(K) Relative growth of SK-N-SH/NBL-SRPMI/NB cells 4 days after treatment with the D3-GPC2-PBD ADC.(L) Fold-change MFI plot of SH-SY5Y-EmptyNB and -GPC2RPMI/NB isogenic cells.Data in (C) and (D) are represented as mean ± SEM of PDXs/xenografts analyzed 2–3 independent times with 4 technical replicates each time for each PDX/xenograft in (C). Data in (G), (I), and (L) are represented as mean ± SEM of experiments done 2–3, 2, and 5 times, respectively. Data in (K) represent mean ± SEM from a representative experiment done in technical triplicate at least two independent times.Neg, negative.*p < 0.05; **p < 0.001; ***p < 0.0001. See also Figure S2.