Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer

Abstract

The human epidermal growth factor receptor 2 (HER2) has been targeted as a breast cancer-associated antigen by immunotherapeutical approaches based on HER2-directed monoclonal antibodies and cancer vaccines. We describe the adoptive transfer of autologous HER2-specific T-lymphocyte clones to a patient with metastatic HER2-overexpressing breast cancer. The HLA/multimer-based monitoring of the transferred T lymphocytes revealed that the T cells rapidly disappeared from the peripheral blood. The imaging studies indicated that the T cells accumulated in the bone marrow (BM) and migrated to the liver, but were unable to penetrate into the solid metastases. The disseminated tumor cells in the BM disappeared after the completion of adoptive T-cell therapy. This study suggests the therapeutic potential for HER2-specific T cells for eliminating disseminated HER2-positive tumor cells and proposes the combination of T cell-based therapies with strategies targeting the tumor stroma to improve T-cell infiltration into solid tumors.

Fig. 1

Specificity, avidity and function of HLA-A2-restricted, HER2-specific CTL clones. a Following expansion, the antigen-specificity of three CTL clones (CTL 3, 16, 45) was documented by A2/HER2_369–377 tetramer staining (filled histogram). Non-binding A2/Melan-A_26–35A27L multimer was used as negative control (open histogram). b The tumor recognition by CTL clones (second expansion) was documented by using the ovarian cancer cell line SKOV3tA2 (filled square) as target cell line. The HLA-A2⁺ HER2⁻ K562tA2 cell line (filled triangle) and the HLA-A2⁻ HER2⁺ SKOV3 cell line (filled circle) were used as negative controls. c Peptide avidity of CTL clones (third expansion) was determined by lysis of T2 cells pulsed with graded amounts of HER2_369–377 peptide (filled circle) at a fixed E:T ratio of 60:1. T2 cells loaded with HIVpol_476–484 peptide (open circle) were used as negative control. Half (50%) maximal target lysis occurred between 1 nM (10⁻⁹ M) and 10 nM (10⁻⁸ M) of peptide HER2_369–377

Fig. 2

HER2 expression of liver metastases and disseminated tumor cells in the BM. a The mononuclear cells from the BM of patient #1 revealed disseminated tumor cells that also overexpressed HER2 as documented by immunocytochemical analysis (A085-HercepTest^R). b The liver metastasis of patient #1 overexpressed HER2 (Score 3±) as determined by immunohistochemical analysis (A085-HercepTest^R)

Fig. 3

Frequency analyses of HER2-specific T cells in the peripheral blood and the BM prior to and after adoptive transfer. a PBMCs of the patient were isolated before and at indicated time points after adoptive T-cell transfer and then stained with A2/HER2_369–377 multimers and anti-CD8 antibody in the presence of PI at 4°C. Frequencies of A2/HER2_369–377-specific T cells were documented by analyzing 1,782,715 events prior to transfer; 1,051,196 events at 1 h; 811,238 events at 4 h; 1,718,357 events at 24 h; and 1,652,305 events at 48 h after transfer. Indicated frequencies refer to CD8⁺ T cells. b Frequency of HER2-specific T cells in the BM of the patient was measured prior to the first T-cell transfer and 24 h after the fifth transfer using A2/HER2_369–377 multimers. Staining was performed in the presence of anti-CD8 antibody and PI at 4°C. Dot plots are gated on PI-negative cells. The frequencies of A2/HER2_369–377-specific T cells were analyzed by counting 889,608 events prior to transfer and 1,961,744 events after the last transfer. Indicated frequencies refer to CD8⁺ T cells

Fig. 4

Systemic distribution of adoptively transferred HER2-specific T cells. Prior to transfer, the T cells of clone 45 were labeled with ¹¹¹In in order to monitor the migration in vivo. a Whole body camera images were obtained from the patient at 4, 24 and 48 h after infusion. At 4 h, the uptake of labeled T cells was detected in the lung, spleen, liver and BM. After 24–48 h, the lung activity cleared whereas activity in the spleen, liver and BM remained stable. b Comparable sections of MRI, ¹¹¹In single photon emission computed tomography and [¹⁸F] fluorodeoxyglucose positron emission tomography. The arrow points to the 3 × 5 cm measuring liver metastases located in segment S8. The metastasis displays high FDG-uptake indicating viable tumor tissue. However, accumulation of ¹¹¹In-labeled T cells is lower than in surrounding normal liver tissue (c). Migration kinetics of the ¹¹¹In-labeled T cells to the lung (filled diamond), the liver (filled square) and the spleen (filled triangle). d Over a time period of 96 h about 0.1% of the infused CTLs was detected in the metastases located in the liver segment S8

Fig. 5

Evaluation of disseminated tumor cells in the BM prior to and after transfer of HER2-specific T cells. BM-derived MNCs were stained with a cytokeratin-directed antibody. a Prior to T-cell transfer cytokeratin-positive tumor cells were detected at a frequency of 256 in 10⁶ MNCs. b Disseminated tumor cells were not visible 24 h after the fifth T-cell transfer (0/10⁶ MNCs)