Cytotoxic T cell response against the chimeric ETV6-AML1 protein in childhood acute lymphoblastic leukemia

214

T cells in cancer

Tuesday, 24 June 1997 - Oral presentations

IO.51 0.2 1 CTL-mediated immunity against human paplllomavirus type 10 E7+ncoded epitopes presented by HLA42

IO.51 0.4 1 Cytotoxic T cell response against the chimeric ETV&AMLl protein in chlldhood acute tymphoblastic leukemia

ME. Ressing ‘, R.M.P. Brancfll, J.H.de Jong ‘, K. Kono’, R. Kiessling*, R.K. Potku14,G. Kente?, J.E.Tdmbos3, R. Cffrfngai, W.M. Kast4, C.J.M. Melief ‘. ’ Deptof /mmunohematokyy & B/ood Bank, University Hospiral Leklen, The Netherlands, 3Dept of Gynaecok?gKUnivarsity Hospital Leiden, The Netherlands, *Karolinska Institute, Stockholm, Sweden, 4Cardinal Bemadin Cancer Center, Loyola University of Chicago, Maywood, IL, USA

P. Yotnda ‘, F. Garcia ‘, M. Peuchmaur *, F. Lemonnier ‘, E. Vilmer3, P. Langlade-Demoyen ‘. ’ Immunologic Cellulaire Anti-Vim/e, lnstitut Pasteur Paris, France, *labomtoire d’anapathoiogie, H6pital Robert Dabre, Fmnce, 3serviie d’H6metologie. H6pital Robert Dabre, France

Most cervical carcinoma cells constitutively express human papillomavtrus type 16 (HPVIG) E6 and E7 oncoproteins. These proteins are, therefore, attractive targets for CTL-based immunotherapy. Using immunization protocols, we have identified 3 HPV16 E7-encoded CTL epitopes presented by HLAA2 that are immunogenic both in vitm to PBMC of healthy donors and in viva in HLAA2 transgenic mice. In the HLA-A2 transgenic mice different vaccination strategies are currently investigated for the induction of T cell responses and protection against a lethal dose of HPVl Mransfonned syngeneic cells. Furthermore, we have screened the blood of healthy donors and patients with HPVI 6 positive cervical lesions for T cell functionality and reactivity. CD% chain expression and cytokine production were reduced in patients when compared to donors, suggesting a decrease in immune functionality. Specific CTL reactivity was tested against 2 HPVl6 E7-encoded epitopes and against a control influenza virus matrix epitope. Despite their lesion or therapeutic interventions, functional influenza virus-specific CTL responses were detected in the majority of cervical carcinoma patients. Moreover, in patients, occasional memory CTL responses were detected against one HPVl6 E7-encoded peptlde, thus providing evidence for natural CTL immunity against HPVIG in these patients. Combined, these findings have resulted in a clinical trial for vaccination with HPVIG-encoded peptides in order to induce or augment CTL responses against cervical carcinoma, which is currently in progress at our institute.

IO.5.10.3

] Therapeutic benefit of adjuvant active specific immunotherapy (A.S.I.) with autologous tumor-vaccines in patlents with colon cancer. Results of a randomlsed fase III study

A.M.E. Claessen ?aA. Stam’, E. Bloemena, H. Gall*, J.B. Vennorken *, R.J.Scheper’, H.M. Pinedo*,C.J.L.M. Meijer’. ‘DaptofPatholog)! Free Uniwsity Hospital, Amstanlam, The Netherlands, 2Dept of Medical Oncol~ Free University Hospital, Amsterdam, The Netherlands Introduction: From 1967 till 1996,254 patients with colon cancer (Dukes’B2,s and C) were entered into a prospectively randomised multicenter study of A.S.I. with an autologous tumor cell-BCG vaccine. Main objective of this study was to investigate whether adjuvant A.S.I. could improve disease free status and survival of colon carcinoma patients. In connection with this study different colon-tumorassociated antigens (CTAA), identified by monocional-antibodies derived from B-cells of these immunized patients, were tested for their ability to induce an immune response in vaccinated patients (Bloemena et al Cancer Res 53,456; 1993). This should provide information on relevant CTAA with potential for formulation into a generic vaccine. Materlair & Methods: 254 Patients with a Dukes& en C colon carcinoma were randomized into two groups: one treated with surgical resection of the tumor (standard treatment; n = 126) the other one with surgical resection followed by A.S.I. with (irradiated) autologous tumor cells (n = 126). Autologous vaccines were prepared as described by Peters et al (Cancer Res 39, 1363; 1979) and given intradermally. Patients received 4 vaccines in total. The first two (days 30 and 37 postsurgery) contained 10’ irradiated (200 Gy) tumor cells and 10’ BCG (Tlce) organisms. The last two vaccines (on days 44 and 160 postsurgery) contained irradiated tumor cells only. At several time points during vaccination patients were skin tested with the different CTAA. Both delayed type hypersensitivity (DTH) reactions (24.46 hr) and in vitro antigen specific T-cell responses were measured. Reeulte: All immunized patients showed DTH reactivity to the vaccine (induration and etythema), which in general showed an increase at the 4th vaccination. Toxicity of the vaccination consisted of local ulceration at the vaccination site due to BCG (in nearly all patients), and lymphadenopathy and fever/chills during the first 24 hours in nearly 50% of the patients. At a median follow up off 3.5 years a significant difference with regard to disease free interval and disease free survival is observed in favour of the vaccine treated group. Cf the 10 patients tested, 7 showed DTH response to a mixture of CTAA 26A32-50W-32K. These responses correlated with the in vitro proliferative T-cell responses to CTAA 26A32-32K. Conclusion: The study shows that A.S.I. with autologous tumor vaccines is effective as adjuvant treatment of colon cancer. Additionally the study shows that CTAA 26A32-32K antigen which belongs to the family of annexines, might contain an important tumor-related T-cell epitope. Whether this antigen is suitable for the preparation of a genetic vaccine remains to be studied.

Introduction: Cytotoxic T lymphocytes (CTL) are potent effecters of the cellular immune response which could provide long term protective immunity if induced by appropriate antitumor vaccines. CTL recognize 6-14 amino acid long pep tides processed intracellularly and presented by MHC class I molecules. A well charactertsed example of a potential tumor antigen in childhood pm-6 Acute Lymphoblastic Leukemia (ALL) results from the t(12;21) banslocation leading to the fusion of the ETV6 and AMLI genes. This translocation is observed in over 25% of patients. Results and Conclusions: In this study we have examined whether the chimertc ETVG-AMLl protein could serve as a tumor specific antigen in HLAA2.1 individuals. We have identified a peptide, encoded by the fusion region of the ETVGAMLI protein, that binds to the HLAA2.1 molecule and induces specific primary CTL in PBL from healthy donors. We have shown that lymphocytes within bone marrow (BM) at the time of diagnosis are involved in the immune response to AU cells. The CTL from patients’ BM lyse HtAA2.1 tumor cells expressing the fusion protein peptide. Furthenore, after pdyclonal stimulation of BM lymphocytes from one patient we isolated several CTL clones specific for the ETVG-AMLI peptide, indicating natural in vivo immunization in some patients.

0.5.10.5

Diversity of the cytotoxic melanoma-specific immune response: Some CTL recognize autoloaous fresh tumor’cells and not tumor ceg lines -

E. Dufour, C. Flament, G. Carcelain, C. Gaudin, V. Scott, M.F. Avrtl, F. Fauns. INSERM U333, IGR 94305 Villejug Francs In the present work, in order to analyze the heterogeneity of the tumor-specific cytotoxic immune response, a large number of T cell clones were generated from the infiltrate of a tumor-proximal invaded lymph node and two kinds of melanoma-specific CD6+ CTL clones were derived. Functional analyses of TIL were mostly performed in cytotoxicity assays toward either tumor cells (single cell suspension and tumor cell lines) or peptide-loaded EBV transformed cells. TCR repertoire was studied by CDR3 size analysis with fluorescent TCR beta primers using the Immunoscope software. The majority ofT cell clones (about a hundred) are characterized by a specific lysis of the autologous tumor cell lines. Among 34 of the latter clones, HLA A2 molecule and MART-l 27-35 peptlde have been shown to play a predominant role in tumor recognition. The other kind of tumor-specific CTL (1 oligoclonal CTL line and 2 different CTL clones) did not lyse the autologous melanoma cell lines but lysed the < autologous tumor cells. Functional analysis of these two CD6+ CTL clones have shown that recognition of the fresh autofogous tumor cells was MHC class l-dependent. These 2 T cell clones did not display cytolytic activity toward NK targets, toward autologous peripheral monocytes, activated T cells and transformed B cells and toward the few allogeneic cultured and uncultured melanoma cells we tested. TCR repertoire analysis have shown that one of these CTL clones was significantly detectable in situ among tumor infiltrating lymphocytes (from both primary and metastatic lesions) while not among PBMC. Such melanoma-specific lymphocytes which could not have been picked out through conventional screening procedures using tumor cell lines, could potentially play a role in tumor rejection. These results suggest that the immune response analyzed toward melanoma cell lines does not totally reflect the “in situ” immune status. Supported by ARC grant # 1260.

I 0.5.10.6

I TumorspecifIc CD8+ and CD4+ Tcells are induced using an IL2-producing melanoma cell line

C.A. Aamoudse, A. van Elsas, W.T.Weeda, C.E. van der Minne, P.I. Schrier. Dept.of Clinical Oncology, University Hospital Leiden PO. box 9600,230O RC Lekfan, The Netherlands Introductton: Melanoma is supposed to be an immunogenic tumor and several antigens that code for a T-cell epitope have been cloned from melanoma cell lines by now. Most of these studies concerned CD6+ T-cells and only little is known about T-helper reactivity against melanoma. Existing tumors might be the result of insufficient immune responses in viva. Cytokine transfection of tumor cells improves their immunogenicity and therefore the lL2-transfected melanoma cell line 516112.14was tested for tt.simmunogenicity in vitro. Materials and Methods: An autologous Mixed Lymphocyte-Tissue Culture was performed with PBMC and the lL2-transfected melanoma cell line