A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes by Anja.

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A recombinant bispecific single-chain antibody, CD19 × CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes by Anja Löffler, Peter Kufer, Ralf Lutterbüse, Florian Zettl, Peter T. Daniel, Jan M. Schwenkenbecher, Gert Riethmüller, Bernd Dörken, and Ralf C. Bargou Blood Volume 95(6):2098-2103 March 15, 2000 ©2000 by American Society of Hematology

Purification and binding specificities of bscCD19 × CD3 Purification and binding specificities of bscCD19 × CD3.(A) Western blot analysis of the purified bscCD19 × CD3 construct detected by anti-HisTag antibodies. Purification and binding specificities of bscCD19 × CD3.(A) Western blot analysis of the purified bscCD19 × CD3 construct detected by anti-HisTag antibodies. The molecular mass (kd) is indicated on the left; the μg protein applied on the top. (B) FACS analysis with the bscCD19 × CD3 on different CD19-positive B-cell lines (Daudi, Blin-1, BJAB, Raji, and SKW6.4), on CD3-positive Jurkat cells and primary human PBLs, and on the CD3- and CD19-negative plasmacytoma cell line L363. Broken lines are negative controls with the secondary antibody anti–HisTag-FITC alone. Anja Löffler et al. Blood 2000;95:2098-2103 ©2000 by American Society of Hematology

T-cell proliferation and cytotoxic activity of bscCD19 × CD3 T-cell proliferation and cytotoxic activity of bscCD19 × CD3.(A) Standard 3H-thymidine proliferation assay with unstimulated primary human PBMCs before and after depletion of B cells by immunomagnetic beads. T-cell proliferation and cytotoxic activity of bscCD19 × CD3.(A) Standard 3H-thymidine proliferation assay with unstimulated primary human PBMCs before and after depletion of B cells by immunomagnetic beads. (B) Cytotoxicity of bscCD19 × CD3 in a51Cr-release assay with primary human PBLs and different B-cell lines (BJAB, Raji, Blin-1, SKW6.4, and Daudi) in the presence of 60 units per mL IL-2; the E-T ratio was 10:1 and the incubation time was 4 hours. The SD in all triplicates was below 5%. Anja Löffler et al. Blood 2000;95:2098-2103 ©2000 by American Society of Hematology

CD-19 specificity of bscCD19 × CD3–mediated cytotoxicity CD-19 specificity of bscCD19 × CD3–mediated cytotoxicity.(A) Inhibition of bscCD19 × CD3-mediated cytotoxicity of unstimulated primary human PBLs against CD19+ target cell lines by parental anti-CD19 antibody (HD37) in a standard51Cr-release assay (E-T rati... CD-19 specificity of bscCD19 × CD3–mediated cytotoxicity.(A) Inhibition of bscCD19 × CD3-mediated cytotoxicity of unstimulated primary human PBLs against CD19+ target cell lines by parental anti-CD19 antibody (HD37) in a standard51Cr-release assay (E-T ratio 20:1; incubation time 4 hours; target cells Blin-1). (B) Inhibition of bscCD19 × CD3-mediated cytotoxicity of unstimulated primary human PBLs against CD19+ target cell lines by anti-CD3 antibody (OKT-3) in a standard 51Cr-release assay (E-T ratio 20:1; incubation time 4 hours; target cells Blin-1). (C)51Cr-release assay with unstimulated primary human PBLs against CD19+ Daudi cell line and CD19− plasmacytoma cell lines NCI and L363 at different concentrations of bscCD19 × CD3 (E-T ratio 20:1; incubation time 8 hours). Anja Löffler et al. Blood 2000;95:2098-2103 ©2000 by American Society of Hematology

Role of T cell subsets and E-T ratio in the cytotoxic effect Role of T cell subsets and E-T ratio in the cytotoxic effect.(A) 51Cr-release assay with isolated primary human CD8+ and CD4+ T cells against cell line Blin-1 at different concentrations of bscCD19 × CD3 (E-T ratio 20:1; incubation time 4 hours). Role of T cell subsets and E-T ratio in the cytotoxic effect.(A) 51Cr-release assay with isolated primary human CD8+ and CD4+ T cells against cell line Blin-1 at different concentrations of bscCD19 × CD3 (E-T ratio 20:1; incubation time 4 hours). (B)51Cr-release assay with unstimulated primary human PBLs against CD19+ cell line Blin-1, with titration of bscCD19 × CD3 at different E-T ratios and incubation time of 4 hours. Anja Löffler et al. Blood 2000;95:2098-2103 ©2000 by American Society of Hematology