Soluble recombinant MHC‐peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single‐chain human MHC (scMHC)‐peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA‐A2 in the presence of tumor‐associated or viral peptides. The scMHC molecule was composed of β2‐microglobulin connected to the first three domains of the HLA‐A2 heavy chain through a 15‐amino acid flexible linker. Highly purified scMHC‐peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART‐1 or a viral peptide derived from HTLV‐1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA‐A2‐restricted peptides. We also generated scMHC‐peptide tetramers, which were biologically functional; they induced a peptide‐specific CTL clone to be activated and secrete IFN‐γ, and were able to stain specifically CTL lines. Such recombinant soluble scMHC‐peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen‐specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

A cancer immunotherapy strategy is described herein that combines the advantage of the well established tumor targeting capabilities of high-affinity recombinant fragments of Abs with the known efficient, specific, and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC-peptide complexes. Structurally, it consists of a previously uncharacterized class of recombinant chimerical molecules created by the genetic fusion of single-chain (sc) Fv Ab fragments, specific for tumor cell surface antigens, to monomeric scHLA-A2 complexes containing immunodominant tumor-or viralspecific peptides. The fusion protein can induce very efficiently tumor cell lysis, regardless of the expression of self peptide-MHC complexes. Moreover, these molecules exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results suggest that recombinant scFv-MHCpeptide fusion molecules could represent an approach to immunotherapy, bridging Ab and T lymphocyte attack on cancer cells. C urrent cancer immunotherapy strategies typically employ two arms of the natural immune system: humoral and cellular. In the first, systemic injection of high-affinity mAbs directed against cell surface tumor-associated antigens has demonstrated statistically significant antitumor activities in clinical trials (1-3). Antitumor Abs that carry effector payloads such as toxins (immunotoxins) or cytokines are also potent molecules currently being tested in various clinical trials (4, 5). The second major approach for specific cancer immunotherapy consists of the potentiation of the cellular arm of the immune system, mainly through CD8 ϩ cytotoxic T lymphocytes (CTLs). Two major strategies are currently being used: (i) active immunization of patients with antigens known to be recognized by T lymphocytes and to activate them (6-8) and (ii) adoptive transfer therapies that enable the selection and activation of highly reactive T cell subpopulations with improved antitumor activities (9). Clinical studies using MHC tetramer staining have demonstrated T lymphocyte responses against the immunizing tumor antigens in the course of vaccination. However, these promising clinical trials using active immunization have suffered from a relatively low percentage of tumor remissions and a lack of correlation between clinical and T lymphocyte responses to the vaccine (9, 10). Furthermore, in using this approach there is the potential risk of selecting tumor cell variants that have undergone HLA loss (11). The adoptive transfer approach has recently demonstrated impressive results (12, 13). Regression of metastatic melanoma was reported in patients undergoing adoptive transfer protocols with highly selected tumor-reactive T cells directed against overexpressed self-derived differentiation antigens after a nonmyeloablative conditioning regimen (12, 13). The efficiency of such T cell-based immunotherapy approaches may be limited by the absence or low expression of either MHC molecul...

Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain beta-2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R alpha subunit-specific humanized antibody, anti-Tac. The scFv-MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.

CTLs act as the effector arm of the cell-mediated immune system to kill undesirable cells. Two processes regulate these effector cells to prevent self reactivity: a thymic selection process that eliminates autoreactive clones and a multistage activation or priming process that endows them with a license to kill cognate target cells. Hitherto no subsequent regulatory restrictions have been ascribed for properly primed and activated CTLs that are licensed to kill. In this study we show that CTLs possess a novel postpriming regulatory mechanism(s) that influences the outcome of their encounter with cognate target cells. This mechanism gauges the degree of Ag density, whereupon reaching a certain threshold significant changes occur that induce anergy in the effector T cells. The biological consequences of this Ag-induced postpriming control includes alterations in the expression of cell surface molecules that control immunological synapse activity and cytokine profiles and induce retarded cell proliferation. Most profound is genome-wide microarray analysis that demonstrates changes in the expression of genes related to membrane potential, TCR signal transduction, energy metabolism, and cell cycle control. Thus, a discernible and unique gene expression signature for anergy as a response to high Ag density has been observed. Consequently, activated T cells possess properties of a self-referential sensory organ. These studies identify a new postpriming control mechanism of CTL with anergenic-like properties. This mechanism extends our understanding of the control of immune function and regulation such as peripheral tolerance, viral infections, antitumor immune responses, hypersensitivity, and autoimmunity.