Gene Therapy (2001) 8, 891–895 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt BRIEF COMMUNICATION T cells engrafted with a recombinant anti-CD30 receptor target autologous CD30+ cutaneous lymphoma cells A Hombach1, JM Muche2, M Gerken1, S Gellrich2, C Heuser1, C Pohl3, W Sterry2 and H Abken1 1Department I of Internal Medicine, Laboratory Tumor Genetics, University of Cologne; 2St Elisabeth-Krankenhaus Ko¨ln-Hohenlind, Cologne; and 3Department of Dermatology and Allergy, University Hospital Charite´, Humboldt University Berlin, Berlin, Germany T cells can be directed to antigen-specific, MHC-inde- Peripheral blood-derived T cells from the lymphoma patient pendent target cell lysis by grafting with a recombinant were retrovirally engrafted with a recombinant anti-CD30- receptor with antibody-like specificity. Here, we asked scFv-␥ receptor. Upon cocultivation with autologous CD30+ whether T cells from the peripheral blood of a patient with lymphoma cells, grafted T cells increase IFN-␥ secretion and cutaneous T cell lymphoma can be recruited for an immune lyse specifically lymphoma cells with high efficiency, even at response against autologous tumor cells. Lymphoma cells an effector to target cell ratio of as low as 1:20. Our data with a CD3− CD4+ CD30+ phenotype and clonal TCR-V␤7 demonstrate that the recombinant anti-CD30-␥ receptor rearrangement were isolated from a cutaneous lesion. The overcomes T cell tolerance for tumor cells and directs T cells lymphoma lesion additionally harbored CD3+ CD25+ acti- specifically against autologous lymphoma cells. Gene Ther- vated normal T cells despite ongoing tumor progression. apy (2001) 8, 891–895. Keywords: cutaneous T cell lymphoma; adoptive immunotherapy; chimeric receptor, CD30 T cells can be equipped with pre-defined binding speci- CTLs, that were successfully generated by peptid vacci- ficity by grafting with a recombinant receptor that har- nation, did not induce significant tumor regression.7 On bors an antibody-derived binding domain and an intra- the other hand, the cellular anergy seems to be restricted cellular signalling domain for cellular activation. The to tumor-specific CTLs because viral antigens induce a chimeric receptor strategy combines the universal normal cellular immune response in these patients.6 properties of antibodies for specific antigen recognition From this situation arises the question of whether T with highly efficient cellular activation upon antigen-spe- cells from the peripheral blood of a tumor patient whose cific receptor crosslinking. To test this strategy for use immune functions are not yet substantially affected by in the cellular immunotherapy of malignant diseases, a the malignant disease can be recruited for an immune number of in vitro and in vivo systems have been applied. response against the autologous tumor cells. To address Most of these systems have in common that cells of estab- this issue we engrafted peripheral blood T cells of a lished tumor cell lines were utilized as a model system patient suffering from a CD30+ cutaneous T cell lym- (Refs 1–3, for review see Ref. 4). On the other hand, it phoma with a recombinant receptor molecule that has was recently demonstrated that primary, in situ growing binding specificity for the CD30 antigen. The antigen- tumors and transplanted tumor cell lines differ substan- binding site of the receptor is composed of an anti-CD30 tially in their ability to induce a cellular antitumor single chain antibody fragment (HRS3-scFv) that was response and that tumor cell lines may poorly reflect the fused to the transmembrane and intracellular domains of clinical situation.5 Particularily, in situ growing autolog- the high affinity IgE receptor signalling chain (Fc⑀RI␥). ous tumor cells are partially or completely tolerized by The CD30 target-antigen is a 120 kDa glycoprotein of the the immune system and an efficient antitumor response TNF/nerve growth factor receptor family8 that is pre- is frequently suppressed during tumor progression. dominantly expressed on Hodgkin- and some non-Hodg- Accordingly, in the case of melanoma substantial popu- kin lymphomas and on virus-infected lymphocytes. A lations of tumor-specific CD8+ cytotoxic T cells (CTLs) small subpopulation of normal activated lymphocytes in could be detected despite tumor progression. Tumor- lymphatic tissues, predominantly around B cell follicles specific CTLs, however, were demonstrated to be func- and the edge of germinal centers in lymph nodes and tionally anergized6 and, moreover, melanoma-specific spleen, express CD30 whereas resting lymphocytes do not.9 It was suggested that CD30 may be involved in the negative selection of thymocytes.10 However, CD30-lack- ing mice do not show an altered primary or secondary B Correspondence: H Abken, Lab. Tumor Genetics, Dept I of Internal Medi- 10 cine, University of Cologne, Josef-Stelzmann-Str. 9, D-50931 Cologne, cell and T cell response, respectively. Due to its highly Germany restricted expression pattern on malignant and virus- Received 21 December 2000; accepted 19 March 2001 infected cells and on a small proportion of activated T Autologous T cell targeting of cutaneous lymphoma A Hombach et al 892 and B cells, the CD30 antigen is a suitable target for an the CD3− CD4+ CD30+ phenotype could not be detected antibody-based immunotherapy. While the binding in the patient’s peripheral blood either by flow cytometry specificity and the functional activity of the recombinant or by clone-specific PCR (data not shown). We conclude anti-CD30-␥ immunoreceptor was previously shown in that the lymphoma lesion harbors one predominant cell detail,11 we demonstrate here that receptor-grafted per- clone specified by clonal V␤7–J␤2.7 TCR rearrangement ipheral blood T cells of a patient with a cutaneous T cell and a CD3− CD4+ CD30+ phenotype suggesting that this lymphoma are specifically activated against autologous cell population represents the malignant cell clone. More- tumor cells mediating a MHC-independent, CD30- over, due to the lack of CD3 expression, the lymphoma specific cellular immune response that is highly effective cells differ phenotypically from infiltrating normal CD3+ even at very low effector to target cell ratios. T lymphocytes. To determine whether the latter represent Suspension cells were isolated from a biopsy of a activated or resting T cells we monitored the number of cutaneous T cell lymphoma lesion. Flow cytometric IL-2 receptor (CD25) expressing CD3+ cells present within analysis revealed a predominant cell population with a the lymphoma lesion. Strikingly, about 23% of normal CD3− CD4+ CD30+ phenotype in addition to low numbers CD3+ T cells derived from the lymphoma biopsy express of normal cells (Figure 1a, b). These CD3− CD4+ CD30+ the IL-2 receptor (Figure 1c, d) indicating the presence of cells clonally express the TCR V␤7 chain as demonstrated a high number of activated T cells. by clone-specific V␤7-PCR (data not shown). Although We asked whether anti-CD30-␥ immunoreceptor- present in high numbers in the biopsy material, cells of grafted T cells from the lymphoma patient’s peripheral blood are specifically activated against autologous CD30+ lymphoma cells. Therefore, the patient’s peripheral blood lymphocytes and for comparison six healthy donors were engrafted by retroviral gene transfer with the recombi- nant anti-CD30 receptor HRS3-scFv-␥ and the anti-CEA receptor BW431/26-scFv-Fc-␥, respectively. Recombinant receptor-expressing T cells were identified by flow cyto- metry utilizing receptor-specific anti-idiotypic and anti- CD3 mAbs. Retroviral transduction of the lymphoma patient’s peripheral blood T cells resulted in about 5% of T cells expressing the recombinant receptor whereas the transduction rates of healthy donors were in the range of 4% to 40% (Table 1). To test whether anti-CD30-␥ recep- tor-expressing T cells are specifically activated by CD30+ lymphoma cells we cocultivated anti-CEA- and anti- CD30-␥ receptor-grafted allogeneic and autologous T cells, respectively, with lymphoma cells for 72 h at an initial cell ratio of 1:1. The cell culture supernatants were harvested and analyzed for the presence of IFN-␥ by ELISA. Cocultivation of both HRS3-scFv-␥ receptor- grafted allogeneic (Figure 2a) and autologous (Figure 2b) T cells with CD30+ lymphoma cells resulted in increased IFN-␥ secretion indicating CD30-specific cellular acti- vation of grafted T cells. In contrast, neither cocultivation Figure 1 Immunophenotyping of cells isolated from the cutaneous lym- of nontransduced or anti-CEA-␥ receptor-grafted allo- phoma lesion and the peripheral blood. Cells of a cutaneous T cell lym- geneic or autologous T cells with CD30+ lymphoma cells phoma were derived from a 37-year-old male, who developed a tumor nod- ule on the left lower leg. The diagnosis of a primary cutaneous CD30- nor incubation of receptor-grafted T cells without lym- positive anaplastic large cell lymphoma was established according to the phoma cells resulted in increased IFN-␥ secretion. It is EORTC classification.12 Immunohistochemistry revealed large anaplastic noteworthy that cells obtained from the lymphoma tumor cells with a CD30+ CD4+ CD3− and V␤7+ phenotype (data not lesion, that represent predominantly CD3− CD4+ CD30+ shown). Staging by CT scan excluded an extracutaneous manifestation of lymphoma cells, secreted substantial amounts of IFN-␥ the lymphoma. X-ray therapy was administered as initial therapy (total (Figure 2). dose 40 Gy). After a total dose of 20 Gy, perilesional multiple tumor nod- We next analyzed whether anti-CD30-␥ receptor- ules with up to 2 cm in diameter occurred. Biopsies were taken from these + nodules and tumor cells were prepared as previously described.13 Primary grafted autologous T cells deplete CD30 lymphoma cells cells of the cutaneous T cell lymphoma biopsy were cultured in RPMI in a cocultivation assay. Therefore, receptor-grafted and 1640 medium supplemented with 10% (v/v) FCS, 100 U/ml penicillin, nontransduced peripheral blood T cells from the lym- ␮ ␮ + 100 g/ml streptomycin (all Life Technologies), 200 g/ml Meropenem phoma patient were co-incubated with autologous CD30 (Gru¨nenthal, Stolberg, Germany), and 400 U/ml IL-2 (Endogen).