RESUMO
Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.
Assuntos
Apresentação de Antígeno , Membrana Celular/imunologia , Membrana Celular/metabolismo , Antígenos HLA-A/imunologia , Antígenos HLA-A/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Supressora de Tumor p53/imunologia , Proteína Supressora de Tumor p53/metabolismo , Idoso , Animais , Células CHO , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/química , Cricetinae , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Feminino , Antígenos HLA-A/química , Antígeno HLA-A2 , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Nus , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Solubilidade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/químicaRESUMO
We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264-272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector cell conjugation. The protein also has potent antitumor effects in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion proteins represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by the conventional Ab-based approach.
Assuntos
Imunoglobulina G/genética , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes de Fusão/imunologia , Anticorpos Antineoplásicos/química , Antígenos de Neoplasias/imunologia , Linhagem Celular , Citotoxicidade Imunológica , Dimerização , Antígeno HLA-A2/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteína Supressora de Tumor p53/imunologiaRESUMO
Antibody-based targeted immunotherapy has shown promise as an approach to treat cancer. However, many known tumor-associated antigens are not expressed as integral membrane proteins and cannot be utilized as targets for antibody-based therapeutics. In order to expand the limited target range of antibodies, we have constructed a soluble single-chain T-cell receptor (TCR) fusion protein designated 264scTCR/IL-2. This fusion protein is comprised of a three-domain HLA-A2-restricted TCR specific for a peptide epitope of the human p53 tumor suppressor protein, which is overexpressed in a broad range of human malignancies. The 264scTCR/IL-2 fusion protein has been expressed at high levels in mammalian cells, and milligram quantities have been purified. MHC-restricted antigen-specific binding properties are maintained in the single-chain, three-domain TCR portion of the fusion protein, and the IL-2 portion retains bioactivity similar to that of free recombinant IL-2. Moreover, this fusion protein is capable of conjugating target and effector cells, remains intact in the blood and substantially increases the half life of the IL-2 portion of the molecule. Finally, the 264scTCR/IL-2 fusion protein can be used to stain tumor cells and is capable of reducing lung metastases in an experimental model of metastasis. Thus, TCR-based fusion proteins may provide a novel class of targeted immunotherapeutics for cancer.
