RESEARCH ARTICLE T-cell engineering by a chimeric T-cell receptor with antibody-type specificity for the HIV-1 gp120 S Masiero 1 , C Del Vecchio 1 , R Gavioli 2 , G Mattiuzzo 1 , MG Cusi 3 , L Micheli 3 , F Gennari 1 , A Siccardi 4 , WA Marasco 5 , G Palu ` 1 and C Parolin 1 1 Department of Histology, Microbiology and Medical Biotechnologies, Section of Microbiology and Virology, University of Padova, Padova, Italy; 2 Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy; 3 Department of Molecular Biology, Virology Section, University of Siena, Siena, Italy; 4 Department of Biology and Genetics, University of Milano, Milano, Italy; and 5 Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA, USA Immune-based approaches of cell therapy against viral pathogens such as the human immunodeficiency virus type 1 (HIV-1) could be of primary importance for the control of this viral infection. Here, we designed a chimeric cell surface receptor (105TCR) to provide primary human T-lymphocytes with antibody-type specificity for the HIV-1 envelope glyco- protein. This receptor includes the single chain Fv domain of the neutralizing anti-gp120 human monoclonal antibody F105, CD8a hinge and the transmembrane and the cytoplasmic domains of TCRz. Our results show that 105TCR is expressed at the cellular surface and is capable of recognizing the HIV-1 envelope glycoprotein inducing highly efficient effector T-cell responses, including extra- cellular signal-regulated kinase phosphorylation and cytokine secretion. Moreover, human primary CD8+ T-lymphocytes transduced by oncoretroviral and lentiviral vectors containing the 105TCR gene are able to mediate in vitro-specific cytolysis of envelope-expressing cells and HIV-1-infected CD4+ T-lymphocytes. These findings suggest that 105TCR is particularly suited for in vivo efficacy studies. Gene Therapy (2005) 12, 299–310. doi:10.1038/sj.gt.3302413 Published online 21 October 2004 Keywords: adoptive immunotherapy; chimeric TCR; HIV-1 envelope glycoprotein; CD8+ cell engineering Introduction The CD8+ cellular cytotoxic response specifically directed toward human immunodeficiency virus type 1 (HIV-1)-infected cells plays a critical role in the control of HIV-1 infection and is likely to be a major component of the host immune response both in acute and chronic infection. 1–4 Genetic approches offer a potential means to enhance immune recognition and elimination of infected cells. Our group has previously proposed a new strategy of adoptive immunotherapy based on ex vivo transfer of autologous CD4 T-cell lines, resistant to HIV-1, which recognize specific antigen of HIV-1 and opportunistic infectious agents. 5–7 However, the HLA-restricted nature of viral antigen recognition by the T-cell receptor limits the application of adoptive immunotherapy strategies to MHC-matched individuals. To overcome this problem, one promising strategy is to engineer T-lymphocytes genetically to express artificial TCRs that direct cytotoxi- city toward viral antigens. 8–10 Artificial receptors typically comprise a viral antigen-specific recognition element derived from a single-chain antibody variable fragment (scFv). 11,12 When used to reprogram T-cell specificity, such fusion receptors allow MHC-independent recogni- tion of antigen. 8,10,13 ScFv-based TCRs are engineered to contain a signaling domain that delivers an activation stimulus. 14 Several scFv-based chimeric TCR (cTCR) have been developed that contain the TCRz cytoplasmatic domain. This domain is capable of inducing a potent activation signal in the absence of the remaining components of TCR-CD3 complex, thus activating cyto- lytic functions. 15 It has been demonstrated that chimeric receptors containing the TCRz chain and the CD8a hinge are not associated with endogenous subunits of the TCR complex. 16–18 Therefore, generation of unpredictable and deleterious immune specificity is not likely to occur. Roberts et al 19 have developed two classes of HIV- specific chimeric receptors, in which the antigen binding domain is comprised of either CD4 or a single chain specific for the envelope glycoprotein gp41, character- ized by the ability of activating a number of T-cell effector functions in response to target cells. In vitro studies and clinical trials have confirmed the feasibility and safety of adoptive therapy with CD4+ 20,21 or CD4+ and CD8+ 22,23 T cells modified with cTCR containing the human CD4 molecule as the extracellular domain. In turn, these engineered T cells were endowed with antiviral activity. Indeed, a sustained decrease of replication-competent virus cultured from blood, as well as of HIV-DNA isolated from rectal mucosa, was achieved. These data are encouraging and provide evidence that the cTCR strategy is potentially useful as an immunotherapeutic approach in vivo. Received 11 May 2004; accepted 5 September 2004; published online 21 October 2004 Correspondence: Professor G Palu ` and Professor C Parolin, Department of Histology, Microbiology and Medical Biotechnologies, Section of Micro- biology and Virology, University of Padova, Via A Gabelli 63, Padova 35121, Italy Gene Therapy (2005) 12, 299–310 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt