Efficient Delivery of T Cell Epitopes to APC by Use of MHC Class II-Specific Troybodies 1 Elin Lunde, 2,3 * Karoline H. Western, 2 * Ingunn B. Rasmussen, 2† Inger Sandlie, † and Bjarne Bogen* A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91–101 2 315 , which is presented by I-E d ; 110 –120 hemagglutinin (I-E d ); 323–339 OVA (I-A d ); and 46 – 61 hen egg lysozyme (I-A k ). We denote such APC-specific, epitope-containing Ab “Troybodies.” When mixed with APC, all four class II-specific Troybodies were 1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000–10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100 –100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development. The Journal of Immunology, 2002, 168: 2154 –2162. A central event in the development of an adaptive immune response is the activation of CD4 + T cells. In vaccine development, strategies that increase their activation are therefore attractive. CD4 + T cells become activated when they recognize peptides presented on MHC class II molecules. Thus, an approach to augment their activation has been to increase the num- ber of MHC-peptide complexes on the APC (1–5). This has tra- ditionally been accomplished by complexing antigenic proteins to Ab that are specific for surface markers on APC. Such targeting of Ag to APC has been shown to increase presentation and T cell activation (1–5). However, large Ag-Ab conjugates may have dis- advantages, such as reduced tissue penetration, short serum t 1/2 , batch to batch variability, and a low number of different Ag that can be conjugated to a single Ab. New strategies have therefore been developed that are based on the finding that Ig are themselves processed intracellularly, so that Ig-derived peptides are presented on MHC class II molecules (6 – 9). Accordingly, various T cell epitopes have been introduced into the complementarity-determining region (CDR) 4 2 and 3 of Ab, which is part of the Ag binding site (10, 11). A major drawback of this strategy is loss of Ab specificity. Circumventing this problem, Baier et al. (12) fused T cell epitopes to the C termini of IgD- and MHC class II-specific Fabs. The T cell activation potential of these molecules, however, was lower than that of Ab-Ag complexes (2– 4). The reduction could be due to the fact that Fabs do not allow for bivalent binding and cross-linking of target molecules. Also, peptides tailing an Ab fragment may be prone to degradation (13). We have chosen to make rAb that have antigenic peptides in- tegrated into their C regions in such a way that they do not disrupt the Ig structure. In addition, these Ab have been equipped with V regions specific for APC. When the rAb are internalized and de- graded by the APC, the T cell epitopes can be loaded onto MHC molecules and presented to T cells. In this study, we introduce the term Troybodies for such Ab, because their effect is comparable to that of the Trojan horse: when they enter a cell (the city of Troy) by receptor-mediated endocytosis (a gate in the city wall), their T cell epitopes (soldiers) are released. We have previously made Troybodies with specificity for IgD. When compared with a pep- tide-containing Ab with irrelevant specificity, the IgD-specific Troybodies were far more efficient at priming B cells for T cell activation (14). Previous studies have demonstrated that targeting of conven- tional Ag-Ab complexes to MHC class II has a positive effect on activation of specific CD4 + T cells (2, 3). In the present work, we describe the construction of MHC class II-specific Troybodies with either of four different model T cell epitopes embedded in their C regions. We show that targeting to APC by use of these Ab results in enhanced Ag presentation and T cell activation in vitro as well as in vivo. The current class II-specific Troybodies have the advantage that all the different professional APC expressing MHC class II molecules may be targeted. Indeed, enhanced presentation is obtained using B cells, dendritic cells (DC), and macrophages as APC in the assays. *Institute of Immunology, University of Oslo, National Hospital, Oslo, Norway; and † Department of Biology, Division of Molecular Cell Biology, University of Oslo, Oslo, Norway Received for publication July 6, 2001. Accepted for publication December 18, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Research Council of Norway and the Norwegian Cancer Society. 2 E.L., K.H.W., and I.B.R. contributed equally to this work 3 Address correspondence and reprint requests to Dr. Elin Lunde at the current ad- dress: Department of Biology, Division of Molecular Cell Biology, University of Oslo, P. O. Box 1050 Blindern, N-0316 Oslo, Norway. E-mail address: elunde@ bio.uio.no 4 Abbreviations used in this paper: CDR, complementarity-determining region; DC, dendritic cell; FSC, forward light scatter; HA, hemagglutinin; HEL, hen egg ly- sozyme; NIP, 5 iodo-4 hydroxy-3 nitrophenacetyl. Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00