Research ArticleCancer

A Mucin 16 bispecific T cell–engaging antibody for the treatment of ovarian cancer

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Science Translational Medicine  19 Jun 2019:
Vol. 11, Issue 497, eaau7534
DOI: 10.1126/scitranslmed.aau7534
  • Fig. 1 REGN4018 binds to human and monkey CD3 and MUC16.

    (A) Schematic of bispecific antibody format. Heavy-chain variable regions of different specificities are in red and green. Red asterisk indicates the Fc mutation used for purification. (B) REGN4018 binding to CD3 on human and cynomolgus monkey T cells (CD2+CD16) shown by flow cytometry (two replicates). (C) The number of MUC16 epitopes per cell on the cell lines is reported as antibody binding capacity. (D) Schematic of MUC16 including the “membrane-proximal region” where the antibody binds. (E) Binding of REGN4018 to human and cynomolgus monkey MUC16 on a range of tumor cell lines shown by flow cytometry. Isotype control, green; REGN4018, blue (two replicates).

  • Fig. 2 REGN4018 activates T cells in the presence of target and induces redirected T cell–mediated cytotoxicity in the presence of soluble CA-125 in vitro.

    (A) Jurkat/NFAT-Luc cells were incubated with OVCAR-3 cells (left), or Jurkat/NFAT-Luc cells expressing cynoCD3 (human CD3 deleted) were incubated with ID8-VEGF/cynoMUC16 cells (right) and serial dilutions of REGN4018 (blue squares) or a CD3-binding control (gray triangles). Relative luminescence units (RLU) are plotted. T cells and target cells were incubated with various concentrations of REGN4018 or CD3-binding control for 48 hours. Human (left) and monkey (right) T cell killing of OVCAR-3 cells are shown. (B) Assays performed in triplicate wells and plotted as means ± SD (n = 3). (C) At 48 hours, human and monkey T cells (CD2+CD16 and then CD4+ or CD8+) were examined for PD-1 expression by flow cytometry. (D) Cytokines were measured at 48 hours with anti-CD3/anti-CD28 used as a positive control. Assays performed in triplicate and plotted as means ± SD (performed twice). Significance measured by unpaired test in comparison to CD3-binding control (*P < 0.05, **P < 0.01, and ***P < 0.001). (E) Human T cell killing of ID8-VEGF/huMUC16Δ (left), ID8-VEGF/cynoMUC16 (middle), or ID8-VEGF cells (right). (F) Flow cytometry measurements of REGN4018 (blue) or anti-MUC16 clone 3A5 (black) binding alone or in the presence of CA-125 or a truncated MUC16. (G) Killing of OVCAR-3 cells by human PBMCs in the presence of REGN4018 with increasing concentrations of CA-125 (left) or truncated MUC16 (right) (performed twice).

  • Fig. 3 REGN4018 activates T cells and reduces tumor burden in a xenogenic tumor model in a dose-dependent manner.

    (A) OVCAR-3/Luc cells were implanted into NOD SCID gamma (NSG) mice pre-engrafted with PBMCs and intraperitoneally treated with the indicated doses of REGN4018 or control antibodies on days 6, 10, 13, 16, and 21 after tumor implantation (four to five mice per group; left) and day 7 (five mice per group; right). Values represent group median with the associated confidence interval (range). *P < 0.05 and **P < 0.01 compared to nonbinding control by Mann-Whitney test. (B) BLI showing tumor burden in mice. (C) Serum from day 23 after tumor implantation was examined for CA-125 concentrations. (D) Cytokines from serum were examined at 4 hours after first dose. **P < 0.01 compared to nonbinding control by Mann-Whitney test. (E) CD8 T cells were examined for CD25, PD-1, and granzyme B 48 hours after administration of CD3-binding control or REGN4018 by flow cytometry; dotted line shows fluorescence minus one (FMO). MFI, median fluorescence intensity. (F) Spleen and ascites were examined for human T cells at end of the study by flow cytometry.

  • Fig. 4 REGN4018 does not induce any systemic cytokine response or histopathology in genetically engineered mice.

    (A) Schema of humanization of membrane-proximal region of MUC16. (B) Expression of the human MUC16 in various tissues from HuT mice (left) and mouse MUC16 (right), relative to expression in the trachea, was measured by TaqMan. (C) Left: IHC analysis of MUC16 expression on OVCAR-3 and HT29 cells. Right: IHC analysis of MUC16 expression in the trachea, stomach, and ovary from wild-type (WT) or HuT mice. (D) Flow cytometry analysis of TCR Vß usage in CD4 (left) and CD8 (right) T cells of wild-type (white) and huMUC16huCD3 (gray striped) mice (five mice per group, two replicates). (E) Total numbers of T cells from spleens of wild-type (white) or HuT (gray striped) mice were determined by flow cytometry. To examine activation of T cells, mice were dosed with REGN4018 (10 mg/kg; four to five mice per group, three replicates). T cell numbers in blood were examined 4 hours after dosing with phosphate-buffered saline (PBS; black), CD3-binding control (gray), REGN4018 (blue), or OKT3 (red). (F) T cells were identified as CD45+/CD90.2+CD19/CD4+ or CD8+. (G) Serum cytokines were examined 4 hours after intravenous administration of CD3-binding control (gray), REGN4018 (blue), or OKT3 (red) by meso scale discovery (MSD). (H) Hematoxylin and eosin (H&E)–stained sections of the trachea, stomach, and ovary from wild-type or HuT mice 5 days after administration of REGN4018.

  • Fig. 5 REGN4018 accumulates in target-bearing tissues including the spleen, LNs, and MUC16-expressing tumors.

    WT mice or HuT mice for MUC16 and CD3 were administered with 89Zr-labeled REGN4018 (0.5 mg/kg) or anti-MUC16 parental antibody to track binding in vivo (n = 3 mice per group). (A) Images show analysis from 6 days after dosing. 89Zr-labeled nonbinding control antibody or REGN4018 was administered at 0.5 mg/kg to mice bearing ID8-VEGF/huMUC16Δ tumors implanted 20 days previously. (C) Anti-MUC16 parental antibody (10 mg/kg; second from the right) or CD3-binding control antibody (10 mg/kg; right) was administered at same time as 89Zr-labeled REGN4018 (n = 4 mice per group). Images show analysis from 6 days after dosing. (B and D) Graphs show the percentage of injected dose per gram of tissue (% ID/g), shown as means ± SD.

  • Fig. 6 REGN4018 induces antitumor efficacy in immunocompetent HuT mice.

    (A) HuT mice implanted with ID8-VEGF/huMUC16Δ cells subcutaneously were treated intraperitoneally with 100 μg of REGN4018 or CD3-binding control on days 0, 4, 7, 10, and 13 after implantation (left; n = 5 mice per group, two individual studies) or days 10, 14, 17, 21, and 24 after implantation (right). Mean tumor volume shown ± SEM (n = 5 to 6 mice per group, two individual studies). Significance measured by two-way analysis of variance (ANOVA) (**P < 0.01). HuT mice implanted with ID8-VEGF/huMUC16Δ cells were intraperitoneally treated with REGN4018 (5 mg/kg) or CD3-binding control from day 3. (B) Survival curves represent euthanasia upon 20% weight gain (n = 5 to 12 mice per group, four replicates). Significance measured by Gehan-Breslow-Wilcoxon test (****P < 0.0001). (C) Histograms show expression of MUC16 on tumor cells at the end of the study (blue, anti-MUC16; green, secondary alone; and gray, no antibody). (D) CD4 and CD8 T cells were examined for Ki67 and granzyme B (GZM B) 48 hours after administration of CD3-binding control or REGN4018 by flow cytometry (n = 4 mice per group, two individual studies). Significance measured by ANOVA (***P < 0.001, ****P < 0.0001 between ascites samples and #P < 0.05 between spleens and ascites samples in control groups). n.s., not significant.

  • Fig. 7 The addition of PD-1 blockade enhances REGN4018 efficacy.

    (A) PD-1 expression on CD4 and CD8 T cells in the peritoneal cavity of tumor-bearing mice is shown by flow cytometry. T cells were examined 32 days after implantation when tumor growth was evident (n = 5 mice per group with three replicates). (B) Flow cytometry of PD-L1 expression on ID8-VEGF/huMUC16Δ cells ex vivo. PD-L1 was examined at the end of the study in four separate studies. (C) HuT mice implanted with ID8-VEGF/huMUC16Δ cells were treated with REGN4018 (5 mg/kg) or CD3-binding control, and T cells in the peritoneal cavity were examined for PD-1 expression 48 hours later (four mice per group, two replicates). (C) Significance measured by ANOVA (**P < 0.01 between ascites samples and ###P < 0.001 between spleens and ascites samples in control groups). HuT mice implanted with ID8-VEGF/huMUC16Δ cells were intraperitoneally treated with REGN4018 (5 mg/kg) or CD3-binding control in combination with anti–PD-1 (5 mg/kg, each). Survival curves represent euthanasia upon 20% weight gain (10 to 12 mice per group). (D) Significance measured by Gehan-Breslow-Wilcoxon test compared to CD3-binding control + isotype (***P = 0.0005, ****P < 0.0001, ##P = 0.0252). Three studies were completed.

  • Fig. 8 Toxicokinetics and safety assessment in cynomolgus monkeys.

    Cynomolgus monkeys were administered five weekly doses of REGN4018 at 0.01, 0.1, and 1 mg/kg. (A) Concentration-time profiles are characterized by a brief distribution phase, followed by a single elimination phase, shown as means ± SD (n = 12 up to day 35, then n = 6 thereafter). LLOQ, lower limit of quantification. (B) Serum CRP over time. (C) Serum IL-6 concentration over time. T cell numbers were examined by flow cytometry, shown as means ± SD (n = 12 until day 36, then n = 6 thereafter). (D) Necropsies were performed on day 35 after first dose or day 119 after first dose. (E) Tissues were examined for MUC16 expression by IHC (left) in control-treated animals, and H&E staining was performed at the end of the dosing period (week 5) and at end of recovery (week 12; right). Several organs from the high-dose (1.0 mg/kg) group are shown.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/497/eaau7534/DC1

    Materials and Methods

    Fig. S1. REGN4018 lacks antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity functions.

    Fig. S2. PD-1 and CD25 are up-regulated in response to both REGN4018 and anti-CD3/anti-CD28 stimulation.

    Fig. S3. REGN4018 binds CA-125 to a much lower degree than anti-MUC16 clone 3A5.

    Fig. S4. REGN4018 did not induce any weight loss in animals with detectable drug concentrations.

    Fig. S5. MUC16 expression is maintained in vivo after REGN4018 treatment.

    Fig. S6. BLI at baseline correlates with complete tumor clearance.

    Fig. S7. Human T cells are maintained after transfer of human PBMCs and can respond to anti-CD3 or REGN4018.

    Fig. S8. Antitumor efficacy and cytokine induction by REGN4018 require T cells and MUC16-expressing OVCAR-3/Luc cells.

    Fig. S9. PD-1 blockade enhances efficacy of suboptimal REGN4018 in the xenogenic OVCAR-3 model.

    Fig. S10. MUC16 expression in the lung from cynomolgus monkey.

    Data file S1. Primary data.

    References (4246)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. REGN4018 lacks antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity functions.
    • Fig. S2. PD-1 and CD25 are up-regulated in response to both REGN4018 and anti-CD3/anti-CD28 stimulation.
    • Fig. S3. REGN4018 binds CA-125 to a much lower degree than anti-MUC16 clone 3A5.
    • Fig. S4. REGN4018 did not induce any weight loss in animals with detectable drug concentrations.
    • Fig. S5. MUC16 expression is maintained in vivo after REGN4018 treatment.
    • Fig. S6. BLI at baseline correlates with complete tumor clearance.
    • Fig. S7. Human T cells are maintained after transfer of human PBMCs and can respond to anti-CD3 or REGN4018.
    • Fig. S8. Antitumor efficacy and cytokine induction by REGN4018 require T cells and MUC16-expressing OVCAR-3/Luc cells.
    • Fig. S9. PD-1 blockade enhances efficacy of suboptimal REGN4018 in the xenogenic OVCAR-3 model.
    • Fig. S10. MUC16 expression in the lung from cynomolgus monkey.
    • References (4246)

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    Other Supplementary Material for this manuscript includes the following:

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