guanosine-monophosphate and Kidney-Neoplasms

Topics: Adenosine Monophosphate; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Deoxyribonucleotides; Gene Expression Regulation; Gluconeogenesis; Guanosine Monophosphate; Humans; Inosine Monophosphate; Isoxazoles; Kidney Neoplasms; Liver Neoplasms; Liver Regeneration; Models, Biological; Neoplasms; Purines; Pyrimidines; Ribonucleotides

Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes.. A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients.. The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses.. We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.

Topics: Adoptive Transfer; Carbonic Anhydrases; Carcinoma, Renal Cell; Cell Proliferation; Clinical Protocols; Gene Dosage; Genetic Therapy; Green Fluorescent Proteins; Guanosine Monophosphate; Humans; Immunoglobulin Variable Region; Kidney Neoplasms; Lymphocyte Activation; Neoplasm Metastasis; Recombinant Fusion Proteins; Retroviridae; Single-Chain Antibodies; T-Lymphocytes; Transduction, Genetic; Transgenes

The behavior of the activities of GMP synthetase (xanthosine-5'-phosphate: L-glutamine amino-ligase(AMP-forming),EC 6.3.5.2) and GMP kinase (ATP: (d)GMP phosphotransferase,EC 2.7.4.8) was elucidated in cytosol preparations of rat tissues, including fetal, neonatal and regenerating liver, in a transplantable kidney tumor, and in a spectrum of 11 hepatomas of different growth rates. GMP kinase activity was 60-fold or more higher than GMP synthetase activity in all of the examined tissues. GMP synthetase activity was increased in all hepatomas and in the kidney tumor, compared to control tissues, reaching 5.5-fold the normal liver values in the most rapidly growing hepatoma. This increase correlated with the tumor growth rates. GMP kinase activity showed no consistent pattern of alteration in the tumors. In both fetal and neonatal rat liver the activity of GMP synthetase was 2.5-times higher than in livers of adult rats, but GMP kinase activity did not change markedly during liver development. After partial hepatectomy GMP synthetase activity was elevated, reaching a peak of 155% of the sham-operated control values by 36 h after the operation. GMP kinase activity was not affected by partial hepatectomy. After 3 days starvation hepatic GMP kinase activity decreased slightly faster than total cytosol protein, while GMP synthetase activity was preferentially maintained. These results indicate that GMP synthetase activity was linked with cellular proliferation in differentiating, regenerating and neoplastic tissues.

Topics: Animals; Carbon-Nitrogen Ligases; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Carcinoma; Glutamine; Guanosine Monophosphate; Guanylate Kinases; Kidney Neoplasms; Ligases; Liver; Liver Neoplasms, Experimental; Liver Regeneration; Male; Neoplasms, Experimental; Nucleoside-Phosphate Kinase; Phosphotransferases; Rats; Tissue Distribution