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A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells

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Abstract

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2,3,4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.

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Fig. 1: Contribution of different arms of the trispecific antibody to HER2 on immune activation in vitro.
Fig. 2: Effect of the HER2 trispecific antibody on tumour growth in a humanized mouse model.
Fig. 3: The HER2 trispecific antibody-stimulated CD4+ T cell inhibited cell cycle progression and/or proliferation in HER2+ breast and gastric cancer cells.
Fig. 4: HER2 trispecific antibody-stimulated CD4+ T cell inhibited cell cycle progression and/or proliferation and stimulated pro-inflammatory pathways in the breast cancer cell line HCC1954.
Fig. 5: Immune effects and cell binding of the HER2 trispecific antibody after dosing in NHPs.

Data availability

Source data used to generate graphs for Figs. 1, 2, 4 and 5, along with Extended Data Figs. 1, 2 and 4, are provided with this paper in spreadsheet format (in its Supplementary Information files). Further data that support the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank C. Lawendowski for excellent programme management; T. Schmidt, T. Bouquin, S. Rao, S. Sidhu, B. Thurberg, K. Klinger, J. Darbyshire, C. Dangler, Z. Jayyosi and C. J. Wei for organizational support; and J. Kingsbury, S. Kathuria, N. Maestrali, S. Somarriba, E. Deschamps, N. Couteault and L. Maton for technical support.

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Authors and Affiliations

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Contributions

Z.-y.Y., E.S., Z.X., E.R., R.W., V.C.-R., E.-J.P., P.K., R.V. and G.J.N. designed the research. Z.-y.Y., E.S., Z.X., E.R., R.V., V.C.-R., P.P., E.-J.P., P.K., C.B., B.Z. L.P., D.W. and G.J.N. led the research. E.S. and Z.X. designed and performed the experiments for the in vitro cellular assays. P.P. and G.U. designed and performed the imaging experiments. E.S., Z.-y.Y., V.C.-R., B.O., L.C. and D.S.B. designed and performed the mouse study experiments. B.Z., H.Q., M.L., G.D. and A.H. designed and performed the experiments to characterize antibodies. Z.S. performed the experiments to characterize tumour cell lines. E.S., P.K., A.L., E.-J.P., R.V. and A.G. designed, analysed or performed the NHP studies. Z.-y.Y., E.S., Z.X., L.W., V.C.-R., E.-J.P., R.V., A.L., M.S.-N., P.P. and G.J.N. analysed the data. Z.-y.Y., E.S., Z.X., R.W., V.C.-R., A.L., R.V. and G.J.N. wrote the paper. All authors reviewed and approved the final manuscript for submission and are accountable for the accuracy and integrity of the manuscript. The research conducted in this study by the authors has been used in the development of a therapeutic drug against tumours currently in a phase I clinical trial that started in August 2021 (ClinicalTrials.gov identifier NCT05013554).

Corresponding authors

Correspondence to Lily Pao, Zhi-yong Yang or Gary J. Nabel.

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Competing interests

All authors are or were employees of Sanofi while engaged in this research project and may hold shares and/or stock options in the company. ModeX employees performed additional informatic evaluation of data, graphic illustration and writing at ModeX. Sanofi develops and manufactures cancer treatment medicines. ModeX is a private biotechnology company that develops multispecific antibodies for the treatment of cancers and viral diseases. G.J.N. formerly served as chief scientific officer of Sanofi. G.J.N., Z.-y.Y., E.R., R.W., E.S., L.W., Z.X., C.B. and H.Q. are listed on intellectual properties developed and owned by Sanofi related to development of novel cancer treatments.

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Extended data figures and tables

Extended Data Fig. 1 The effect of HER2 trispecific Ab on human T cell proliferation and cytotoxic granzyme expression in vitro.

a, Surface expression of early T cell activation marker CD69 after one day stimulation by the HER2 trispecific Ab, the indicated single binding site inactivating mutants, or triple mutants, at 100 nM. The data are from a single experiment with PBMC from 3 donors. b, T cells were stimulated in the presence of the indicated mutants or wild type trispecific antibodies as described in Methods to determine the effects of CD28 co-stimulation on T cell proliferation in vitro. Lines in representative graph from one PBMC donor indicate fold change in cell numbers from Day 0 of viable T cells in vitro; 2 other PBMC donors demonstrated similar results. c, Stimulation of cytotoxic granzyme was measured in primary T cells in the presence of HCC1954 tumor cells and the trispecific Ab or indicated single mutant or triple mutant negative control (1 nM). Intracellular flow staining was used to determine percentage of CD8+ cells expressing Granzyme B. The representative graph is from a single experiment with one PBMC donor performed in triplicate; a second experiment with same donor and two experiments with a second PBMC donor demonstrated similar results.

Source data

Extended Data Fig. 2 Cytolysis of BT20 and MDA-MB-468 breast tumor cell lines by T cells incubated with the HER2 trispecific Ab and mutants and correlation in target cell lysis with HER2 density on tumor cell lines.

a, Cytolysis of HER2-expressing breast cell line BT20, and HER2-negative breast cell line MDA-MB-468, were assessed with HER2 trispecific Ab in vitro using CD8+ as effector cells (E:T = 3:1). The crucial contribution of anti-HER2 arm of the trispecific Ab is demonstrated with the single binding site inactivating mutant. b, Correlation in target cell lysis with HER2 density on tumor cell lines. Additional breast tumor cell lines, along with gastric tumor cells lines, (HCC1954, MDAMB453, HCC1143, MDAMB231, OE.19, GSU, and MKN-45) were measured for HER2 surface protein expression using QIFI kit (Dako, Denmark) as described in Methods. Cytolysis of the tumor targets with HER2 trispecific Ab was assessed in vitro using CD8+ effector cells. The EC50 for target cytolysis was calculated for each tumor cell line (left) and the maximum percentage of dead target cells was also calculated for each tumor cell line (right).

Source data

Extended Data Fig. 3 HER2 trispecific Ab stimulated CD8+ T cell did not inhibit cell cycle progression/proliferation in multiple HER2 expressing cancer cell lines but upregulated proinflammatory pathways.

a, The antitumor effect of HER2 trispecific Ab against multiple tumor cell lines was evaluated with human CD8+ T cells as effector cells (E:T = 5:1). Breast cancer cell lines HCC1937, HCC70, BT549 and gastric cancer cell line OE19 were used as targets. After 1 day of incubation, the tumor cell lines did not exhibit cell cycle arrest at the G1/S stage in cells that remained alive based on flow cytometry. (left), Scatter plots of RNAs significantly upregulated (≥ 5-fold, red), or downregulated (≤ 5-fold, green), in HER2 trispecific Ab treated target cells compared to the control in presence of CD8+ effector cells. b, (right), Enrichment analysis of gene sets in tumor cells after HER2 trispecific Ab treatment compared to control in the presence of CD8+ effector cells. Red bar = upregulation.

Extended Data Fig. 4 Inflammatory cytokines mediated T cell lysis of HER2+ tumor cells with the HER2 trispecific Ab.

Sorted CD4+ or CD8+ T cell subsets were obtained from ex vivo expanded human CD3+ T cells, as previously described, to be used as effectors against HER2+ breast cancer cell line (E:T = 3:1 (all subsets)) with HER2 Trispecific Ab. Neutralizing antibodies against TNF-α (5 µg/mL, R&D systems), IFN-α (5 µg/mL, PBL), or IFN-γ (5 µg/mL, R&D systems) were added to tumor lysis assay against MDA-MB-453 tumor cells using CD4 or CD8 T cells as effectors (a). A representative graph is shown. The tumor lysis assay using neutralizing anti- TNF-α Ab was verified with CD4 T cells as effectors from different PBMC donor against HER2-expressing breast cancer cell lines MDA-MB-453 (b, left) or ZR-75-1 (b, right). The representative graphs are from two experiments using a single PBMC donor each.

Source data

Extended Data Fig. 5 Time lapse of Her2 Tri-specific antibody induced T-cell mediated killing of target HER2+ MDA-MB-453 breast cancer cells.

HER2+ MDA-MB-453 cells were labeled with CellVue Maroon dye (Invitrogen) and combined with PBMCs in 12-well plates. HER2 trispecific Ab was added at 1 µg/ml concentration. Time lapse imaging was conducted for 24 h using a confocal microscope equipped with an environmental chamber which maintains cultures at 37 °C, 5% CO2.

Extended Data Table 1 Characterization of HER2 trispecific Ab
Extended Data Table 2 Clinical observations and chemistry after single and multiple doses of trispecific Ab in NHPs
Extended Data Table 3 Serum pharmacokinetics after HER2 trispecific Ab treatment in NHPs

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Seung, E., Xing, Z., Wu, L. et al. A trispecific antibody targeting HER2 and T cells inhibits breast cancer growth via CD4 cells. Nature 603, 328–334 (2022). https://doi.org/10.1038/s41586-022-04439-0

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