Research ArticleCancer

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma

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Science Translational Medicine  13 Mar 2019:
Vol. 11, Issue 483, eaau9732
DOI: 10.1126/scitranslmed.aau9732
  • Fig. 1 ALK is widely expressed in neuroblastoma.

    (A) Cell surface ALK was quantified in a representative panel of nine neuroblastoma-derived cell lines expressing amplified (NB-1), wild-type (NB-1691 and NBL-S), or mutated ALK [SH-SY5Y, F1174L; COG-N-453, F1174L; NB-1643, R1275Q; SMS-SAN, F1174L; and Felix, F1245C (indicated with red symbols)]. Data are shown as means ± SD (n = 2 to 8). (B) ALK expression was determined by TMA analysis of 32 different neuroblastoma PDX models. Expression was quantified via determination of H-scores, which takes into consideration the staining intensity in conjunction with the percentage of cells staining positively. (C) Representative H-scores and staining patterns are shown in a subset of samples: SK-N-AS (null), COG-N-424x (moderate), NBL-S (moderate), COG-N-453x (moderate), and Felix-PDX (strong). Scale bars, 200 μm. (D) A fraction of mouse-depleted homogenates was subjected to immunoblotting analysis to determine ALK expression. β-Actin was used as a loading control. (E) ALK bands visualized in (D) were quantitated using densitometry analysis and expressed as the ratio of ALK signal over β-actin signal. Data are presented as means ± SD (n = 3). (F) Flow cytometry analysis was performed in mouse-depleted dissociated PDXs, and fluorescence was quantitated on the basis of MESF using standard beads. Data are presented as means ± SD (n = 2).

  • Fig. 2 CDX-0125-TEI efficiently binds to cell surface ALK and is internalized.

    (A) CDX-0125-TEI binds to both mouse (Neuro-2a) and human ALK (NB-1) with picomolar affinity and does not bind to SK-N-AS cells, an ALK-null line. Experiments were performed three independent times. Data are presented as means ± SEM (n = 3). (B) Representative images of immunofluorescence experiments showing localization of CDX-0125-TEI at the cell membrane (30 min at 4°C, left) and at the cytoplasm of NB-1 cells (4 hours at 37°C, right). Scale bars, 50 μm. (C) Quantitative internalization of CDX-0125 in NB-1, Felix, NBL-S, SK-N-SH, IMR-32, and SK-N-AS cells over a 60-min time course. Data are presented as mean values (n = 2 to 3). (D) Schematic illustration of the CDX-0125-TEI molecule. The TEI NMS-P528 bearing a minor groove binding moiety (MGBM) is released after peptidic cleavage by lysosomal proteases and spacer self-immolation, resulting in the removal of the protective moiety and release of the toxin.

  • Fig. 3 CDX-0125-TEI is cytotoxic to ALK-expressing neuroblastoma cells in vitro.

    Neuroblastoma cells expressing wild-type ALK (NB-1 and IMR-32) and mutated ALK (SK-N-SH) or with no detectable ALK expression (SK-N-AS) were treated with increasing doses of CDX-0125-TEI, a control IgG1 antibody conjugated with TEI (IgG1-TEI), or the free payload (NMS-P528). Titration of CDX-0125-TEI induced cytotoxic activity in all ALK-expressing models, with IC50 values in the picomolar range and independent of ALK mutation status or number of cell surface ALK receptors. By contrast, no measurable IC50 value could be derived in the ALK-negative cell line SK-N-AS. Free NMS-P528 elicited complete cell killing at sub-picomolar concentrations in all cell lines tested. Quoted IC50 and ALK expression values reflect calculated means and SEM (n = 3) from at least three independent experiments.

  • Fig. 4 CDX-0125-TEI induces apoptosis.

    (A) Apoptosis was quantified by annexin V–FITC (fluorescein isothiocyanate) and analyzed by FACS (fluorescence-activated cell sorting) in neuroblastoma cells (SK-N-AS, COG-N-453, NBL-S, Felix, and NB-1) treated with CDX-0125, CDX-0125-TEI, NMS-P528, or IgG1 control at their predetermined IC50 values. Values from a representative experiment are shown. Experiments were performed three independent times. (B) NBL-S and (C) COG-N-453 were subjected to immunoblot analysis and probed for the apoptosis marker cleaved caspase-3 and GAPDH as a loading control. Each bracket represents three independent samples. WT, wild type.

  • Fig. 5 CDX-0125-TEI induces antitumor activity in ALK mutant PDX models of neuroblastoma.

    The in vivo efficacy of CDX-0125-TEI was evaluated in female CB17 severe combined immunodeficient (SCID) mice bearing (A) Felix-PDX (ALK F1245C) and (B) COG-N-453x (ALK F1174L). Upon enrollment, mice (n = 10 per group) were intraperitoneally injected with the following drugs: CDX-0125-TEI [1 mg/kg (yellow), 3 mg/kg (light blue), or 10 mg/kg (red)], IgG control (10 mg/kg) (black), or CDX-0125 (10 mg/kg) (dark blue) on day 0 of enrollment and on day 7, indicated by arrows. Tumor volume was measured twice a week until tumors reached ≥3 cm3 (left graphs). Kaplan-Meier curves (middle graphs) were compared using a log-rank test and expressed as percentage of survival. Mouse body weight was measured twice a week and expressed as percentage of initial body weight (right graphs).

  • Fig. 6 CDX-0125-TEI induces antitumor activity in ALK wild-type models of neuroblastoma.

    The in vivo efficacy of CDX-0125-TEI was evaluated in female CB17 SCID mice bearing (A) COG-N-424x (ALK wild-type) and (B) NBL-S (ALK wild-type). Upon enrollment, mice (n = 10 per group) were intraperitoneally injected with 15 mg/kg of one of the following drugs: CDX-0125-TEI (magenta), IgG control (black), or CDX-0125 (blue) on day 0 of enrollment and on day 7, indicated by arrows. Tumor volume was measured twice a week until tumors reached ≥3 cm3 (left graphs). Kaplan-Meier curves (right graphs) were compared using a log-rank test and expressed as percentage of survival.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/483/eaau9732/DC1

    Fig. S1. Unconjugated anti-ALK mAbs do not elicit cytotoxic activity alone or as mediators of ADCC.

    Fig. S2. ALK is expressed in neuroblastoma but not in normal pediatric tissues.

    Fig. S3. The DNA-alkylating CDX-0125-TEI is more potent than the CDX-0125-tubulin inhibitor.

    Fig. S4. CDX-0125-TEI induces single-strand DNA damage.

    Fig. S5. CDX-0125-TEI down-regulates phosphorylated ALK in treated PDXs and cell line xenografts.

    Fig. S6. Weekly dosing with CDX-0125-TEI shows potent but transient antitumor activity in the ALK wild-type NBL-S xenograft model.

    Table S1. List of screened ALK mAbs.

    Table S2. Statistical analysis for in vivo efficacy studies in Felix, NBL-S, and COG-N-453x.

    Table S3. Statistical analysis for in vivo efficacy studies in COG-N-424x and NBL-S.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Unconjugated anti-ALK mAbs do not elicit cytotoxic activity alone or as mediators of ADCC.
    • Fig. S2. ALK is expressed in neuroblastoma but not in normal pediatric tissues.
    • Fig. S3. The DNA-alkylating CDX-0125-TEI is more potent than the CDX-0125-tubulin inhibitor.
    • Fig. S4. CDX-0125-TEI induces single-strand DNA damage.
    • Fig. S5. CDX-0125-TEI down-regulates phosphorylated ALK in treated PDXs and cell line xenografts.
    • Fig. S6. Weekly dosing with CDX-0125-TEI shows potent but transient antitumor activity in the ALK wild-type NBL-S xenograft model.
    • Table S1. List of screened ALK mAbs.
    • Table S2. Statistical analysis for in vivo efficacy studies in Felix, NBL-S, and COG-N-453x.
    • Table S3. Statistical analysis for in vivo efficacy studies in COG-N-424x and NBL-S.

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    Other Supplementary Material for this manuscript includes the following:

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