Our previous studies showed that anti-CD3 activated T cells (ATC) from peripheral blood mononuclear cells could be expanded in interleukin-2 (IL-2) for 14 days and armed with anti-CD3 x anti-Her2/neu (Her2Bi)[

J Hemat & Stem Cell Res
], anti-CD3 x anti-CD20 (CD20Bi)[
Exp Hemat
], or anti-CD3 x anti-EGFR [
Clin Cancer Res
] bispecific antibody (BiAb)and can kill Her2/neu, CD20, and EGFR+ tumor targets, respectively. In this study, we asked whether anti-CD3 activated cord blood T cells (CBATC) could be expanded and targeted with Her2Bi and CD20Bi to tumors or hematologic malignancies for infusions after cord blood stem cell transplant (CBSCT). CB mononuclear cells were activated with anti-CD3 (20 ng/ml) and expanded for 14 days in IL-2 (100 IU/ml). CBATC were armed with Her2Bi or CD20Bi and tested for specific cytotoxicity directed SK-BR-3, Raji, or B9C targets and cytokine secretion or IFNγ EliSpots after binding to tumor cells. Our results show the mean expansion of CBATC to be 43-fold (n=8) after 14 days of culture. By the end of culture, the proportions of CD8+ and CD4+ were 82% and 18%, respectively. The proportion of cells expressing CD19 or CD20 did not exceed 6.3%, CD56+ cells were <3.6% and CD3-CD16+CD56+ cells was <0.7%. Cells positive for CD4+CD25+ or CD8+ CD25+ were 4.2% or 7.1%, respectively (n=2). Specific cytotoxicity was optimized when CBATC were armed with 50 ng/106 cells of Her2Bi or CD20Bi (arming dose ranged from 5, 50, and 500 ng/106 cells); arming significantly increased cytotoxicity of the armed CBATC over that seen for unarmed CBATC. Cytotoxicity peaked between days 12 and 14 for both BiAbs. The ability of CD20Bi armed ATC to produce Elispots for IFNγ were tested by arming ATC the CD20Bi after 0, 8, 11, and 13 days of culture peaked on day 8. Only CD20+ targets induced Elispots and day 8 armed ATC exhibited peak numbers (2,200 Elispots(ranged from 1,700 to 1,800 on day 8)/106 armed ATC plated). At an effector/target ratio (E:T) of 25:1, the mean cytotoxicity of CBATC armed with Her2Bi or CD20Bi was 60% (n=4) and 35% (n=1), respectively. In an extended culture to day 47, mean cytotoxicity for Her2Bi-armed CBATC was 36% at an E/T of 25:1 compared to 4.35% for unarmed CBATC. Unarmed CBATC did not kill Daudi targets. Armed CBATC mediated both specific cytotoxicity and secreted IFN-γ as measured by ELISA or EliSpots. Both fresh and frozen CB could be used in the assays. In a clinical application, specific cytotoxicity of armed CBATC could be used to augment anti-tumor and anti-lymphoma effects after CBSCT.

Author notes

Disclosure: Ownership Interests: Lawrence Lum is founder of Transtarget. Membership Information: Lawrence Lum on speaker’s bureau for Berlex. Off Label Use: Use of IL-2 and GM-CSF.