Optimization of culture conditions for activation and large-scale expansion of human T lymphocytes for bispecific antibody-directed cellular immunotherapy.
We investigated the optimal culture conditions (i.e., activation procedure, medium composition and type of culture vessel) for rapid in vitro expansion of large numbers (greater than 5 x 10(9) of blood T lymphocytes. These expanded lymphocytes can be targeted to be cytotoxic to ovarian carcinoma cells with a bispecific monoclonal antibody (BsAb) specific for CD3 and for the ovarian carcinoma-associated antigen MOv18. Both phytohemagglutinin (PHA) and monoclonal antibody (MAb) CD3 induced rapid T-cell proliferation, although the growth kinetics after PHA activation were slightly faster. A 50-fold increase in cell number was obtained after 14 and 16 days for PHA and CD3 MAb, respectively. The induction of BsAb-directed cytolysis was faster after CD3 MAb than after PHA activation of lymphocytes, but became similar around day 20. A mixture of media consisting of 78% RPMI 1640, 20% AIM-V and 2% human plasma (Mix-med) yielded better results than 100% AIM-V medium. Culture of lymphocytes in polyolefin bags, compared with tissue culture flasks, or cryopreservation did not affect lymphocyte yield and function. In most cultures the proportion of CD8+ lymphocytes increased, suggesting a growth advantage of CD8+ over CD4+ lymphocytes in this culture system. A protocol employing PHA activation, Mix-med and polyolefin bags has been used successfully to activate and expand blood lymphocytes for the first 5 patients entered into a phase-I/II clinical trial for the intraperitoneal treatment of ovarian carcinoma using CD3 x anti-MOv18 BsAb-directed T lymphocytes.
Lamers
C H
CH
Department of Immunology, Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.
van de Griend
R J
RJ
Braakman
E
E
Ronteltap
C P
CP
Bénard
J
J
Stoter
G
G
Gratama
J W
JW
Bolhuis
R L
RL
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology; D017252:CD3 Complex; D016827:CD8 Antigens / Q000032:analysis; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D003602:Cytotoxicity, Immunologic; D006801:Humans; D016130:Immunophenotyping; D007167:Immunotherapy / Q000379:methods; D007700:Kinetics; D008213:Lymphocyte Activation; D010835:Phytohemagglutinins; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D017252:CD3 Complex; D016827:CD8 Antigens; D003470:Culture Media; D010835:Phytohemagglutinins; D011948:Receptors, Antigen, T-Cell; C017589:ovarian tumor associated antigen