RESUMEN
Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Complejo CD3/genética , Antígeno Carcinoembrionario/inmunología , Neoplasias/terapia , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Anticuerpos Biespecíficos/genética , Complejo CD3/inmunología , Citotoxicidad Inmunológica , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/inmunología , Humanos , Inmunoterapia/métodos , Células Jurkat , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Péptidos/genética , Péptidos/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Doxorubicin is an antineoplastic agent whose clinical administration is limited by dose-dependent irreversible cardiomyopathy. Doxorubicin inhibits the rate of DNA synthesis in cultured rat myocardial cells after 1 h incubation with 16 microM, as is demonstrated by a decreased incorporation of [methyl-3H]thymidine. An analogue of doxorubicin, 4'-epi-doxorubicin, also inhibits the rate of DNA synthesis within 1 h after treatment with 16 microM, to the same extent as doxorubicin-treatment of myocardial cells. Furthermore, similarity between doxorubicin and 4'-epi-doxorubicin in their effect on the myocardial thymidylate pool was also demonstrated by a significantly decreased incorporation of total [methyl-3H]thymidine. The effect of doxorubicin on the rate of DNA synthesis in cultured rat skeletal muscle cells treated for 1 h with 16 microM was quantitatively the same as in myocardial cells. Light microscopy of doxorubicin- and 4'-epi-doxorubicin-treated myocardial cells and doxorubicin-treated skeletal muscle cells showed distinct nucleolar fragmentation and revealed no differences between the two drugs in their effect on either myocardial or skeletal muscle cells. Electron microscopy of myocardial cells following doxorubicin treatment showed increased nuclear pleomorphism and invaginations, along with a striking and distinctive clumping of nuclear chromatin. Furthermore, an apparent high density of the mitochondria due to an increased matrix volume and a concomitant decrease in the intermembrane compartment were observed. The results of this study indicate that doxorubicin-induced inhibition of cardiac DNA synthesis in cultured myocardial cells is nonpredictive of cardiotoxicity. The mechanism is at least bimodal, and the apparent minor toxicity of 4'-epi-doxorubicin compared with that of doxorubicin in clinical trials cannot be distinguished by a difference in the inhibition of DNA synthesis in the rat heart.