Antibody-Drug Conjugate Efficacy in Neuroblastoma: Role of Payload, Resistance Mechanisms, Target Density, and Antibody Internalization

Abstract

Antibody-drug conjugates (ADC) are a targeted cancer therapy that utilize the specificity of antibodies to deliver potent drugs selectively to tumors. Here we define the complex interaction among factors that dictate ADC efficacy in neuroblastoma by testing both a comprehensive panel of ADC payloads in a diverse set of neuroblastoma cell lines and utilizing the glypican 2 (GPC2)-targeting D3-GPC2-PBD ADC to study the role of target antigen density and antibody internalization in ADC efficacy in neuroblastoma. We first find that DNA binding drugs are significantly more cytotoxic to neuroblastomas than payloads that bind tubulin or inhibit DNA topoisomerase 1. We additionally show that neuroblastomas with high expression of the ABCB1 drug transporter or that harbor a TP53 mutation are significantly more resistant to tubulin and DNA/DNA topoisomerase 1 binding payloads, respectively. Next, we utilized the GPC2-specific D3-GPC2-IgG1 antibody to show that neuroblastomas internalize this antibody/GPC2 complex at significantly different rates and that these antibody internalization kinetics correlate significantly with GPC2 cell surface density. However, sensitivity to pyrrolobenzodiazepine (PBD) dimers primarily dictated sensitivity to the corresponding D3-GPC2-PBD ADC, overall having a larger influence on ADC efficacy than GPC2 cell surface density or antibody internalization. Finally, we utilized GPC2 isogenic Kelly neuroblastoma cells with different levels of cell surface GPC2 expression to define the threshold of target density required for ADC efficacy. Taken together, DNA binding ADC payloads should be prioritized for development for neuroblastoma given their superior efficacy and considering that ADC payload sensitivity is a major determinant of ADC efficacy.

Figure 1.

DNA interacting ADCs payloads are significantly more cytotoxic to neuroblastomas. A, Heatmap showing the log IC₅₀ values for each ADC payload tested in 11 unique neuroblastoma cell lines. Payloads ranked from left to right by the cell line panel median log IC₅₀ value. B, Plot showing median ADC payload IC₅₀ value grouped by drug family across the panel of neuroblastoma cell lines. Horizontal line in each group denotes mean of each payload family. C, Western blot analysis of the neuroblastoma SMS-SAN and NB69 cell lines 72 hours after treatment with 50 and 200 pmol/L of duocarmycin SA, N-acetyl-calicheamicin γ1, PBD dimer, or PNU-159682 DNA binding ADC payloads. D, Plot showing payload class median IC₅₀ for each neuroblastoma cell line. IC₅₀s in A, B, and D represent summary data from at least two independent experiments. NAC, N-acetyl-calicheamicin γ1; Duo SA, duocarmycin SA; Dol 10, monomethyl dolastatin 10; MMAE, monomethyl auristatin E; cPARP, cleaved PARP; cCaspase-3, cleaved caspase-3 (*, P < 0.05).

Figure 2.

ABCB1 overexpression and TP53 mutation impart selective resistance to specific ADC payloads in neuroblastoma cells. A, Heatmap summarizing expression of common drug transporter and resistance genes across the neuroblastoma cell line panel. B, ABCB1 and BCL2 western blot of neuroblastoma cell line panel. C (left), Plots showing IC₅₀ data in ABCB1 low versus high expressing neuroblastoma cell line cohorts for DNA (top), tubulin (middle), and DNA topoisomerase 1 (bottom) interacting payloads. NB-EbC1, NB69, NBL-S, and IMR-5 represent ABCB1 high expressing cell lines as indicated in B. C (middle), Plots showing IC₅₀ data in TP53 mutant versus wild-type (WT) neuroblastoma cell line cohorts for DNA (top), tubulin (middle), and DNA topoisomerase 1 (bottom) interacting payloads. SK-N-AS, NB-SD, and NGP represent TP53 mutated cell lines as indicated in Table 2. C (right), Plots showing IC₅₀ data in MYCN amplified (amp) versus MYCN non-amplified (NA) neuroblastoma cell line cohorts for DNA (top), tubulin (middle), and DNA topoisomerase 1 (bottom) interacting payloads. NB-SD, SMS-SAN, NB-1643, LA-N-5, CHP-134, NGP, and IMR-5 represent MYCN amplified cell lines as indicated in Table 2. D, NB69 DM1 (top) and MMAE (bottom) IC₅₀s with and without co-treatment with the ABCB1 inhibitor tariquidar. E, Nb-EbC1 DM1 (top) and MMAE (bottom) IC₅₀s with and without co-treatment with the ABCB1 inhibitor tariquidar. IC₅₀ data in C represent the mean of at least 2 biological replicates. Horizontal lines in C represent cell line cohort median value. Data in D and E represent the mean ± SEM of at least three biological replicates. FPKM, Fragments Per Kilobase of transcript per Million mapped reads; MYCN amp, MYCN amplified cell lines; MYCN NA, MYCN non-amplified cell lines; WT, wild-type (*, P < 0.05; **, P < 0.01; ***, P < 0.001). See also Supplementary Fig. S1.

Figure 3.

Cell surface antigen density and antibody internalization are other factors involved in ADC efficacy. A, Representative GPC2 flow cytometry histograms for 11 neuroblastoma cell lines. B, Summary of neuroblastoma cell line GPC2 cell surface densities. C, Plot of D3-GPC2-IgG1-Red internalization kinetics for 11 neuroblastoma cell lines. D, Summary of neuroblastoma cell line D3-GPC2-IgG1-Red internalization AUC from C. E, Plot of relative neuroblastoma cell growth after treatment with increasing concentrations of the GPC2 ADC D3-GPC2-PBD. F, Summary of neuroblastoma cell line D3-GPC2-PBD ADC IC₅₀s. G, Plot of D3-GPC2-IgG1-Red internalization AUC versus GPC2 cell surface density for the 11 neuroblastoma cell lines (r = 0.75, P = 0.01). H, Plot of D3-GPC2-IgG1-Red internalization AUC versus D3-GPC2-PBD ADC IC₅₀ for the 11 neuroblastoma cell lines (r = −0.20, P = 0.55). I, Plot of D3-GPC2-PBD ADC IC₅₀ versus PBD dimer IC₅₀ for the 11 neuroblastoma cell lines (r = 0.79, P = 0.006). Relative D3-GPC2-IgG1-Red internalization for each cell line indicated. Very high D3-GPC2-IgG1 internalizing cell lines represent a D3-GPC2-IgG1-Red AUC of >3 million, high represents an AUC of 2 to 3 million, moderate represents an AUC of 1 to 2 million, and low represents an AUC of <1 million. Data in B and F represent mean ± SEM of at least two independent experiments and data in A, C, D, and E represent data from a representative experiment repeated at least two independent times. See also Supplementary Fig. S1.

Figure 4.

Defining the GPC2 cell surface density required for D3-GPC2-PBD efficacy. A, Representative GPC2 flow cytometry histograms for GPC2 isogenic Kelly cell line panel. B, Summary of GPC2 cell surface density for GPC2 isogenic Kelly cell line panel. C, Plot of relative GPC2 isogenic Kelly cell line growth after treatment with increasing concentrations of the D3-GPC2-PBD ADC. D, Plot of the D3-GPC2-PBD ADC IC₅₀ versus GPC2 cell surface density for the GPC2 isogenic Kelly cell line panel. E, Plot of relative GPC2 isogenic Kelly cell line growth after treatment with increasing concentrations of free PBD across a range of different GPC2 cell surface densities (GPC2 molecules/cell noted in parentheses in legend). F, Plot of PBD and D3-GPC2-PBD ADC IC₅₀s for GPC2 isogenic Kelly cell lines across a range of different GPC2 cell surface densities (GPC2 molecules/cell noted in parentheses on the x-axis). Data in B, D, and F represent mean ± SEM of at least two independent experiments. For D, SEM is indicated for both the ADC IC₅₀ and GPC2 molecule per cell values. Data in A, C, and E are representative data from experiments done at least two independent times. US, unstained.