Research ArticleCancer

A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo

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Science Translational Medicine  26 Aug 2015:
Vol. 7, Issue 302, pp. 302ra136
DOI: 10.1126/scitranslmed.aac9459
  • Fig. 1. Elevated expression of DLL3 mRNA in SCLC.

    (A) DLL3 transcripts conveyed as reads per kilobase per million reads mapped to annotated exons (RPKM_Transcript) in normal tissues (NL tissues) and SCLC and LCNEC PDXs. (B) Relative expression of DLL3 in NL tissues, primary SCLC biopsy specimens (SCLC), and SCLC and LCNEC PDX, as measured by quantitative PCR. (C and D) Relative expression of ASCL1 (C) and NEUROD1 (D) versus DLL3 in SCLC (blue diamond) and LCNEC (red triangle) PDX, as measured by quantitative PCR. (E) DLL3 transcripts (RPKM_Transcript) in normal lung, primary SCLC tumors, and SCLC cell lines (CL). (F) Quantile normalized log2 intensity values of DLL3 mRNA in NL tissues and PDX lines assessed by microarray. Horizontal bars represent the geometric mean. Normal tissues included in each expression metric are detailed in table S1.

  • Fig. 2. Characterization of DLL3-specific and species cross-reactive monoclonal antibodies.

    (A) SC16 shows equivalent binding to human, cyno, and rat DLL3 expressed on the surface of HEK-293T cells. (B) SC16 reacts only with DLL3 and not related family members DLL1 or DLL4. (C) DLL3 protein was detected in SCLC and LCNEC PDX by ELISA. Horizontal bars represent the mean. Normal tissue samples and the amounts of DLL3 protein detected are detailed in table S1. (D) IHC of two SCLC (LU64, H-score = 96; LU149, H-score = 134) and one LCNEC (LU37, H-score = 147) PDX, as well as primary SCLC (H-score = 170 and 200) and LCNEC (H-score = 160) tumors. Scale bars, 20 μm. (E) DLL3 membrane expression as measured by IHC in normal lung tissue and primary tumors including lung squamous cell (NSCLC-SqCC), lung adenocarcinoma (NSCLC-Adeno), LCNEC, and naïve and recurrent/refractory (R/R) SCLC. Horizontal bars represent the mean. (F) Surface DLL3 expression on SCLC LU149 and LCNEC LU37 PDX tumor cells assessed by flow cytometry with phycoerythrin (PE)–conjugated anti-DLL3 (black line) or IgG1 isotype control (gray-filled) antibodies. MESF, mean equivalents of soluble fluorescein.

  • Fig. 3. Characterization of DLL3-mediated internalization and cytotoxicity.

    (A) Schematic of SC16LD6.5. (B) Demonstration of SC16LD6.5 and IgG1LD6.5 localization (red) in HEK-293T.hDLL3 cells engineered to express red fluorescent protein (RFP)–SLP-1 (false color displayed as green) in late endosomes. Colocalization is indicated by yellow/orange. Scale bars, 25 μm. (C to G) In vitro cytotoxicity of SC16, IgG1LD6.5, D6.5, and SC16LD6.5 upon incubation with (C) HEK-293T, (D) HEK-293T.hDLL3, (E) LU64 PDX, (F) LU37 PDX expressing endogenous DLL3, or (G) LU37 PDX lacking DLL3 expression (LU37.D3hp) after shRNA-mediated knockdown. mAb, monoclonal antibody.

  • Fig. 4. Demonstration of in vivo efficacy with SC16LD6.5.

    (A to F) Mice bearing SCLC LU64 (A and D), SCLC LU86 (B and E), or LCNEC LU37 (C and F) PDX tumors were treated with IgG1LD6.5 or SC16LD6.5 (1 mg/kg) (A to C) on a Q4D×3 regimen, or vehicle (saline) or SOC chemotherapy (D to F). (G) DLL3 surface expression quantified by IHC (H-score) correlated with dTTP in 10 SCLC and 1 LCNEC PDX model. (H and I) Mice bearing SCLC LU64 PDX tumors were treated with C/E and, upon tumor recurrence (35 days after initial C/E treatment), were randomized and treated again either with (H) IgG1LD6.5 or SC16LD6.5 (1 mg/kg) on a Q4D×3 regimen or with (I) vehicle or C/E.

  • Fig. 5. Elimination of TIC by SC16LD6.5.

    (A) The frequency of no tumor growth after serial transplantation of SCLC LU64 PDX tumor cells in limiting dilutions is shown for IgGLD6.5 (black triangles) and SC16LD6.5 (red circles) cohorts. (B) The frequency of no tumor growth after serial transplantation of SCLC LU64 PDX tumor cells in limiting dilutions is shown for the naïve (gray diamonds) and C/E (blue triangles) cohorts. (C) The frequency of TICs was estimated by Poisson distribution statistics using tumor growth frequencies within each cohort.

  • Table 1. In vivo efficacy of SC16LD6.5 and TIC frequency in SCLC and LCNEC PDX.

    %TGI, percent tumor growth inhibition; Q4D×3, once every 4 days for a total of three doses; N.D., not determined; SD, single dose. SOC for SCLC: cisplatin (5 mg/kg) SD on day 0 and etoposide (8 mg/kg) on days 0, 1, and 2; LCNEC: cisplatin (5 mg/kg) SD. AJCC, American Joint Committee on Cancer.

    Tumor typePDXAJCC
    stage
    Untreated TIC
    frequency*
    SOCDLL3
    protein
    (ng/mg)
    SC16LD6.5
    %TGI
    (dTTP;
    days)
    Dose level
    (mg/kg)
    Regimen%TGI
    (dTTP;
    days)
    SCLCLU64IV1:18986 (18)4.251Q4D×3100 (133)
    LU73IIIa1:13686 (28)2.771Q4D×377 (32)
    LU80IV1:14382 (14)0.601Q4D×355 (4)
    LU86IV1:3139 (0)3.051Q4D×380 (32)
    LU95IV1:6056 (14)6.521Q4D×395 (115)
    LU100Ia1:166100 (64)0.0011Q4D×396 (3)
    LU117IV1:38899 (25)2.951Q4D×399 (137)
    LU124IIIb1:27185 (14)3.231Q4D×372 (35)
    LU129IVN.D.84 (32)N.D.1Q4D×395 (106)
    LU149IV1:20793 (21)2.711Q4D×399 (142)
    Mean81 (23)87 (74)
    SEM6 (5)5 (18)
    Median86 (17)95 (71)
    LCNECLU37IIb1:1660 (4)5.191Q4D×397 (132)
    LU240IIb1:3127 (0)28.572SD95 (42)
    Mean44 (2)96 (87)
    SEM17 (2)1 (45)
    Median44 (2)96 (87)

    *Determined by implanting various cell doses (2 to 1000 cells) of dissociated cells from passage 2 to 5 PDX into mice.

    †IgG1.LD6.5 data not available; dTTP value represents TTP.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/302/302ra136/DC1

      Materials and Methods

      Fig. S1. Elevated expression of DLL3 mRNA in SCLC.

      Fig. S2. Specificity of anti-DLL3 IHC antibody.

      Fig. S3. DLL3 protein expression by IHC in representative samples from tissue microarrays.

      Fig. S4. In vitro plasma stability of SC16LD6.5.

      Fig. S5. Enumeration of cells with localization of human antibody in the late endosome.

      Fig. S6. DLL3 knockdown confirmation by flow cytometry.

      Fig. S7. In vivo efficacy of SC16LD6.5, naked SC16 antibody, and free toxin, D6.5.

      Fig. S8. In vivo tolerability of SC16LD6.5.

      Fig. S9. Elimination of TICs by SC16LD6.5.

      Fig. S10. Off-target toxicities observed in nonhuman primates.

      Table S1. DLL3 normal tissue expression.

      Table S2. DLL3 microarray expression in PDX.

      Table S3. Biacore affinity characterization of SC16 and SC16LD6.5 binding to human, cyno, and rat DLL3.

      Table S4. LU64 TIC frequency determination.

      Table S5. DLL3 whole transcriptome metrics (provided as an Excel file).

    • Supplementary Material for:

      A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo

      Laura R. Saunders, Alexander J. Bankovich, Wade C. Anderson, Monette A. Aujay, Sheila Bheddah, KristenAnn Black, Radhika Desai, Paul A. Escarpe, Johannes Hampl, Amy Laysang, David Liu, Javier Lopez-Molina, Milly Milton, Albert Park, Marybeth A. Pysz, Hui Shao, Brian Slingerland, Michael Torgov, Samuel A. Williams, Orit Foord, Philip Howard, Jacek Jassem, Andrzej Badzio, Piotr Czapiewski, David H. Harpole, Afshin Dowlati, Pierre P. Massion, William D. Travis, M. Catherine Pietanza, J. T. Poirier, Charles M. Rudin, Robert A. Stull, Scott J. Dylla*

      *Corresponding author. E-mail: scott.dylla{at}stemcentrx.com

      Published 26 August 2015, Sci. Transl. Med. 7, 302ra136 (2015)
      DOI: 10.1126/scitranslmed.aac9459

      This PDF file includes:

      • Materials and Methods
      • Fig. S1. Elevated expression of DLL3 mRNA in SCLC.
      • Fig. S2. Specificity of anti-DLL3 IHC antibody.
      • Fig. S3. DLL3 protein expression by IHC in representative samples from tissue microarrays.
      • Fig. S4. In vitro plasma stability of SC16LD6.5.
      • Fig. S5. Enumeration of cells with localization of human antibody in the late endosome.
      • Fig. S6. DLL3 knockdown confirmation by flow cytometry.
      • Fig. S7. In vivo efficacy of SC16LD6.5, naked SC16 antibody, and free toxin, D6.5.
      • Fig. S8. In vivo tolerability of SC16LD6.5.
      • Fig. S9. Elimination of TICs by SC16LD6.5.
      • Fig. S10. Off-target toxicities observed in nonhuman primates.
      • Table S1. DLL3 normal tissue expression.
      • Table S2. DLL3 microarray expression in PDX.
      • Table S3. Biacore affinity characterization of SC16 and SC16LD6.5 binding to human, cyno, and rat DLL3.
      • Table S4. LU64 TIC frequency determination.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S5. DLL3 whole transcriptome metrics (provided as an Excel file).

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