Autocrine costimulation: Tumor-specific CD28-mediated costimulation of T cells by in situ production of a bifunctional B7–anti-CEA diabody fusion protein Bele ´n Blanco, 1 Philipp Holliger, 2 and Luis A ´ lvarez-Vallina 1 1 Department of Immunology, Hospital Universitario Clı ´nica Puerta de Hierro, Madrid, Spain; and 2 MRC Laboratory of Molecular Biology, Cambridge CB2 2QH, UK. T cells require two distinct signals for optimal activation, an antigen - specific signal, provided by engagement of the T - cell receptor ( TCR ) and a second costimulatory signal mediated by engagement of CD28 with members of the B7 family. Although infiltrating T cells are present in many malignancies, they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and / or costimulatory signals. Here we describe a novel strategy for the in situ activation of tumor - specific T cells. We genetically modified T cells to secrete a bifunctional fusion protein, comprising the extracellular portion of B7 - 1 fused to an anticarcinoembryonic antigen ( CEA ) diabody. In coculture with CEA + tumor cells autocrine and paracrine secretion of B7 - CEA provided a potent tumor - specific costimulatory signal to T cells in combination with a recombinant CEAÂCD3 bispecific diabody. B7 - CEA was also found to strongly enhance survival and tumor - specific activation of T cells expressing an anti - CEA TCR – based chimeric immune receptor ( CIR ) both when expressed in cis by the T cells themselves as well as in trans, when added to the culture medium. In the absence of costimulatory signals provided by the tumor, our strategy allows T cells to ‘‘arm themselves’’ by the production of tumor - specific costimulatory proteins. Sustained in situ production of such molecules, like the B7 - diabody fusion protein may create a favorable local environment for the activation and proliferation of tumor - reactive T cells and increase the tumoricidal activity of immunotherapeutic approaches targeting the TCR pathway. Cancer Gene Therapy (2002) 9, 275 – 281 DOI: 10.1038 / sj / cgt / 7700438 Keywords: cancer; tumor - specific costimulation; gene therapy; immunotherapy T cells recognize antigen as peptide fragments presented in the context of major histocompatibility complex (MHC) molecules at the surface of cells. 1 However, although T-cell antigen receptor (TCR) signal transduction may be sufficient to activate antigen -primed T cells, under most circumstances TCR ligation alone is not sufficient to initiate an immune response. 2,3 For optimal activation of naive T cells, subsequent autocrine - driven clonal expansion, and induction of effector functions, additional costimulatory signals are needed. 4 The interaction between the B7 family members (B7-1; CD80, B7-2; CD86) on antigen-present- ing cells (APC) with CD28 on T cells has been shown to play a key role in providing costimulation for the activation of T-cell effector functions. 5 Engagement of TCR in the absence of costimulation may result in the induction of specific T- cell unresponsiveness or anergy. Many tumors lack, have lost, or down-regulated surface expression of B7 and/or MHC and, thus, avoid T- cell responses. 6,7 These observations have prompted strat- egies to avoid the induction of anergy in T cells. One strategy has been to provide B7 on the surface of tumor cells by ex vivo transfection with the B7 gene. 8 In animal models, the transfection with the B7 gene enhanced tumor immunoge- nicity, although it had little or no effect on the immunoge- nicity of poorly or nonimmunogenic tumors. 8,9 Moreover, the provision of costimulation in vivo by genetically modified tumor cells is limited to a small fraction of the total tumor burden, and the reinfusion of ex vivo gene - modified autologous tumor cells may present practical and ethical problems. Instead of transfecting tumor cells ex vivo with costimu- latory molecules, T-cell costimulation can be provided by antibody-based targeting. For example, we have shown that chimeric single - chain antibody fragment (scFv)-CD28 receptors could be stably expressed as functional cell surface receptors on T cells, and that antigen - specific costimulatory signals synergized with signals mediated through the native TCR or TCR -based chimeric immune receptors (CIRs) to produce maximal levels of IL-2. 10,11 In the absence of natural costimulatory molecules, tumor - specific CD28 signals enhanced survival and proliferation of human primary T cells. 12 However, the major disadvantage of CIR - based approaches is that tumor - specific primary 13 and/ or costimulatory signals 10,14 are restricted to gene - modified T cells. 15 Received December 10, 2001. Address correspondence and reprint requests to: Dr Luis A ´ lvarez - Vallina, Department of Immunology, Hospital Universitario Clı ´nica Puerta de Hierro, San Martı ´n de Porres, 4, 28035 Madrid, Spain. E- mail: lalvarezv@hpth.insalud.es Cancer Gene Therapy (2002) 9, 275 – 281 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com/ cgt