Research ArticleCancer

p95HER2–T cell bispecific antibody for breast cancer treatment

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Science Translational Medicine  03 Oct 2018:
Vol. 10, Issue 461, eaat1445
DOI: 10.1126/scitranslmed.aat1445
  • Fig. 1 Expression of p95HER2 in normal tissues.

    (A) Schematic illustrating the specificity of the anti-p95HER2 antibodies used in this study. (B) Immunohistochemical analyses of the expression of HER2 and p95HER2 in the indicated human epithelia. (C) Lysates from the indicated normal human tissues were analyzed by Western blot with antibodies against the C terminus of HER2. As a reference for the electrophoretic migration of HER2 and p95HER2, we used lysates from a PDX expressing HER2 and p95HER2 (PDX67). Ponceau staining is shown as loading control (LC).

  • Fig. 2 Characterization of p95HER2-TCB.

    (A) Schematic showing the structure of p95HER2-TCB. (B) Median fluorescence intensity (MFI) of binding of p95HER2-TCB to human Jurkat cells and PBMCs (left) or MCF10A cells expressing empty vector or the same vector encoding HER2 and/or p95HER2 (right) (n = 3 expressed as means ± SD, measured by flow cytometry). (C) The same MCF10A cells as in (B) (right) expressing the indicated constructs were analyzed by Western blotting with antibodies directed against the C terminus of HER2. (D) MFI of binding of p95HER2-TCB to control MCF10A cells or the same cells overexpressing full-length HER2 (n = 3 expressed as means ± SD). (E) Lysates from MCF10A cells transfected with wild-type (WT) HER2 or HER2 bearing an M611A mutation were analyzed by Western blotting with anti-HER2 antibodies. Ponceau staining is shown as loading control (LC). (F) The same cells as in (E) were analyzed by flow cytometry with the indicated antibodies (n = 3 expressed as means ± SD). (G) Schematic drawing illustrating the coculture experiments to monitor the activity of p95HER2-TCB. (H and I) MCF10A cells transfected with the indicated vectors were incubated with PBMCs and different concentrations of p95HER2-TCB for 48 hours. Then, the expression of the activation marker CD25 on CD8+ cells was assessed by flow cytometry (H), and cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release (I) (n = 3 expressed as means ± SD). (J) MCF10A p95HER2 cells were incubated with PBMCs and 10 nM p95HER2-TCB, and the production of the indicated factors was determined by enzyme-linked immunosorbent assay (n = 3 expressed as means ± SD). (D, F, and J) *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

  • Fig. 3 Effect of p95HER2-TCB on tumor growth in vivo.

    (A) Schematic drawing illustrating humanization of mice with PBMCs and orthotopic implantation of MCF7 p95HER2 cells. (B) Mice were treated with vehicle (control) or nontargeting TCB (1 mg/kg) or p95HER2-TCB (1 mg/kg) (arrows). Tumor volumes, expressed as means ± SD, are shown (n ≥ 7 per group). (C) Percentage of circulating human CD45+, relative to total leukocytes, at the end of the experiment shown in (B). (D) Cells positive for cytokeratin were quantified in tumors by immunohistochemistry (IHC). (E) Percentages of circulating (blood) and intratumoral (tumor) human CD8+ at the end of the experiment shown in (B). Percentages in blood were calculated by flow cytometry and are relative to total leukocytes. Percentages in tumors were calculated by IHC and are relative to tumor cells. (F) Schematic drawing illustrating humanization of mice with CD34+ cells and intracranial injection of MCF7 p95HER2/luciferase cells. (G) Percentages of circulating human CD45+ cells, relative to total leukocytes, were determined 5 months after injection of CD34+ cells into NSG mice. (H and I) Intracranial tumor growth was monitored by assessing bioluminescence (H). Results (I) are expressed as means ± SD (n ≥ 4 per group). *P < 0.05, **P < 0.01, ***P < 0.001, two-way analysis of variance (ANOVA) and Bonferroni correction (B and I) and two-tailed t test (C to E). In all cases, we compared the group treated with vehicle with the group treated with p95HER2-TCB. n.s., not significant.

  • Fig. 4 Effect of HER2-TCB and p95HER2-TCB on nontransformed cells.

    MCF10A cells, NECs from reduction mammoplasties (A), or human cardiomyocytes (B) were cultured with PBMCs from healthy donors for 48 hours, and the effect of p95HER2-TCB was analyzed as in Fig. 2 (n = 3 expressed as means ± SD).

  • Fig. 5 Effect of p95HER2 expression on the efficacy of p95HER2-TCB.

    (A) p95HER2 expression in tumor samples and corresponding PDXs was determined by IHC with specific antibodies. (B) Schematic drawing illustrating the coculture experiments. (C) Cells from PDXs 118 and 67 (negative and positive for p95HER2, respectively) were cocultured in the presence of the indicated concentrations of p95HER2-TCB, with Jurkat cells expressing a luciferase-based reporter for the activation of the TCR. Relative luminescence units (RLU) were assessed and normalized to nontargeting TCB (n = 6 expressed as means ± SD). (D) Cultures from the indicated PDXs were analyzed as in (C) with 10 nM p95HER2-TCB. Statistics compare the results obtained with the different cultures with those obtained with the p95HER2-negative PDX118 (n ≥ 3 expressed as means ± SD). (E) Results obtained in (D) were plotted against the amount of p95HER2 determined using a quantitative IHC-based assay (20). (C and D) *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

  • Fig. 6 Effect of p95HER2-TCB on different PDX-based models.

    (A) In vitro, carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled primary cultures from PDX173 were incubated with PBMCs from the same patient (matched) or a healthy volunteer (nonmatched) and treated with vehicle or p95HER2-TCB. CFSE-positive cells were counted by flow cytometry. In vivo, NSG mice carrying PDX173 were monitored until tumors reached ~200 mm3 (shadowed). Then, matched or nonmatched PBMCs were transferred. Mice were treated with vehicle or p95HER2-TCB (1 mg/kg) (arrows). Means ± SD are shown (n ≥ 3 per group). At the end of the experiment, the percentages of circulating human CD45+ cells, relative to total leukocytes, were determined. (B) Schematic drawing illustrating the in vivo experiment shown in (C) to (E). (C) CD34+ cells obtained from human cord blood were injected into 5-week-old NSG mice. After 5 months, the percentages of circulating human CD45+ cells, relative to total leukocytes, were determined. (D) PDX173 was implanted into mice analyzed in (C). These mice were treated with vehicle or p95HER2-TCB, and tumor volume was monitored. Volumes were normalized to the volume at day 0 of treatment. (E) Percentages of circulating (blood, relative to total leukocytes) and intratumoral (tumor) human CD8+ cells at the end of the experiment shown in (D). The expression of the activation marker CD25 was also analyzed in intratumor CD8+ lymphocytes. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA and Bonferroni correction (A, middle, and D) and two-tailed t test (A, left).

  • Fig. 7 Effect of p95HER2-TCB on the growth of PDXs positive and negative for p95HER2.

    (Left) Primary cultures from PDX251G (p95HER2-positive) and PDX445 (p95HER2-negative) were treated as described in Fig. 6A with nonmatched PBMCs. (Middle) In vivo analyses were performed as described in Fig. 6A. Means ± SD are shown (n ≥ 3 per group). (Right) Percentages of circulating human CD45+ cells, relative to total leukocytes, were determined at the end of the experiment. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test (left) and two-way ANOVA and Bonferroni correction (middle).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/461/eaat1445/DC1

    Fig. S1. Expression of p95HER2 in normal tissues.

    Fig. S2. Identification and characterization of the epitope recognized by the monoclonal antibody used to generate p95HER2-TCB.

    Fig. S3. Affinities of the original anti-p95HER2 monoclonal antibody and the anti-p95HER2 TCB.

    Fig. S4. Effect of anti-p95HER2 and HER2-TCB on cultures of MCF7 p95HER2 cells with or without PBMCs.

    Fig. S5. Effect of anti-p95HER2 on the activation of CD4+ and CD8+ cells in cocultures of MCF10A cells transfected with HER2 and/or p95HER2 and PBMCs.

    Fig. S6. Generation of proliferating MCF7 cells expressing p95HER2.

    Fig. S7. Effects of PBMCs from different donors and of p95HER2-TCB on the growth of MCF7 p95HER2 cells as xenografts.

    Fig. S8. Effect of p95HER2-TCB on an in vitro model of BBB.

    Fig. S9. Effect of p95HER2-TCB on parental MCF7 cells and on MCF7 cells transfected with HER2.

    Fig. S10. Expression of p95HER2 in different PDXs.

    Fig. S11. Cytokeratin expression and lymphocyte infiltration in PDXs treated with p95HER2-TCB in vivo.

    Table S1. Primary data (provided as an Excel file).

  • The PDF file includes:

    • Fig. S1. Expression of p95HER2 in normal tissues.
    • Fig. S2. Identification and characterization of the epitope recognized by the monoclonal antibody used to generate p95HER2-TCB.
    • Fig. S3. Affinities of the original anti-p95HER2 monoclonal antibody and the anti-p95HER2 TCB.
    • Fig. S4. Effect of anti-p95HER2 and HER2-TCB on cultures of MCF7 p95HER2 cells with or without PBMCs.
    • Fig. S5. Effect of anti-p95HER2 on the activation of CD4+ and CD8+ cells in cocultures of MCF10A cells transfected with HER2 and/or p95HER2 and PBMCs.
    • Fig. S6. Generation of proliferating MCF7 cells expressing p95HER2.
    • Fig. S7. Effects of PBMCs from different donors and of p95HER2-TCB on the growth of MCF7 p95HER2 cells as xenografts.
    • Fig. S8. Effect of p95HER2-TCB on an in vitro model of BBB.
    • Fig. S9. Effect of p95HER2-TCB on parental MCF7 cells and on MCF7 cells transfected with HER2.
    • Fig. S10. Expression of p95HER2 in different PDXs.
    • Fig. S11. Cytokeratin expression and lymphocyte infiltration in PDXs treated with p95HER2-TCB in vivo.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Primary data (provided as an Excel file).

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