T cells armed with anti-CD3 x anti-CD20 bispecific antibody enhance killing of CD20+ malignant B cells and bypass complement-mediated rituximab resistance in vitro.
OBJECTIVE
Resistance to rituximab, a chimeric monoclonal antibody that binds to CD20, is a major limitation for the successful treatment of patients with non-Hodgkin lymphoma and other CD20+ B-cell malignancies. To circumvent rituximab resistance in these patient populations, we have constructed a bispecific antibody (BiAb), anti-CD3 x anti-CD20 (CD20Bi), that combines rituximab targeting with non-major histocompatibility complex (non-MHC)-restricted cytotoxicity mediated by activated T cells (ATC).


METHODS
Activated T cells were obtained from anti-CD3 activated peripheral blood mononuclear cells (PBMC) of normal donors or the leukapheresis products of patients by culturing in the presence of interleukin-2 for 6-14 days. After ATC expansion, the cells were armed with CD20Bi. Killing activity was evaluated by 51Cr-release assay.


RESULTS
Arming ATC with as little as 5 ng CD20Bi/10(6) cells significantly increased cytotoxicity above unarmed ATC. CD20Bi-armed ATC (50 ng/10(6) cells) efficiently lysed CD20+ cell lines at E:T of 6.25-50, but not the nonhematologic, CD20- SK-BR-3 cell line. High levels of cytotoxicity mediated by CD20Bi-armed ATC (p < 0.05) could not be blocked by an 8000-fold excess of soluble rituximab. CD20Bi-armed ATC in the presence of complement killed ARH-77 cells, a rituximab-complement pathway-resistant multiple myeloma, significantly (p < 0.05) better than rituximab or unarmed ATC, suggesting that CD20Bi-armed ATC may be clinically effective for treatment of rituximab-resistant CD20+ hematologic malignancies.


CONCLUSIONS
Our findings demonstrate that CD20Bi-armed ATC enhance cytotoxicity against CD20+ B-cell lines and circumvent complement-mediated rituximab resistance, providing a strong rationale for this immune-based strategy for the treatment of rituximab-refractory CD20+ B-cell malignancies.
Gall
Jonathan M
JM
Cancer Immunotherapy Laboratory and Blood and Bone Marrow Transplant Programs, Adele R. Decof Cancer Center, Roger Williams Medical Center, Providence, RI 02908, USA.
Davol
Pamela A
PA
Grabert
Ryan C
RC
Deaver
Mark
M
Lum
Lawrence G
LG
D018033:Antibodies, Bispecific / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000276:immunology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects / Q000473:pathology*; D017252:CD3 Complex / Q000276:immunology*; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D003165:Complement System Proteins; D016207:Cytokines / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D000069283:Rituximab; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000970:Antineoplastic Agents; D017252:CD3 Complex; D016207:Cytokines; D000069283:Rituximab; D003165:Complement System Proteins