In Vitro Antitumoral Activity of Baculovirus-expressed Chimeric Recombinant Anti-CD4 Antibody 13B8.2 on T-cell Lymphomas

Abstract

A baculovirus-expressed chimeric recombinant IgG1 (rIgG1) antibody, with Cgamma1 and Ckappa human constant domains, was derived from the murine monoclonal antibody 13B8.2, which is specific for the CDR3-like loop of the CD4 molecule. The recombinant IgG1 antibody 13B8.2 was previously shown to inhibit HIV-1 replication and to abrogate the one-way mixed-lymphocyte reaction and block proliferation of CD3-stimulated peripheral blood CD4 lymphocytes from healthy donors. Before testing this recombinant anti-CD4 antibody in in vivo preclinical trials, in vitro mechanisms of action of rIgG1 13B8.2 were assessed using various CD4 T-cell lymphomas. The baculovirus-expressed rIgG1 13B8.2 antibody led to 14% to 40% proliferation inhibition of the lymphoblastic leukaemia-derived SUP-T1, the acute T lymphoma-derived CCRF-CEM and Jurkat, and the cutaneous T-Cell lymphoma-derived HUT-78 cell lines, but it did not affect the cell cycle nor induce cell apoptosis. rIgG1 antibody 13B8.2 bound the C1q fraction, leading to 9% to 17% complement-mediated lysis of the HUT-78, H9, Sup-T1, and the CCRF-CEM cell lines. No correlation was observed between cell sensitivity to rIgG1 13B8.2-triggered complement-dependent lysis and CD35-, CD46-, CD55-, and CD59-surface expression on T lymphoma cells. Using fluorescence-activated cell sorter analysis, the antibody was shown to bind to FcgammaRI/CD64-transfected IIA1.6, FcgammaRII/CD32-transfected CDw32L, and FcgammaRIII/CD16-transfected Jurkat CD16 cell lines. In correlation with these findings, rIgG1 13B8.2 induced 11% to 31% antibody-dependent cell-mediated cytotoxicity of the CCRF-CEM, SUP-T1, A2.01 CD4, and Jurkat cell lines. These convincing results on the activity of the recombinant chimeric anti-CD4 antibody 13B8.2 have led us to perform in vivo preclinical study in a murine xenograft model of CD4 lymphomas.

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

“…Recombinant IgG 1 Ab 13B8.2 (rIgG 1 13B8.2) has been proposed as a potential therapeutic agent for the treatment of CD4 ϩ malignant diseases because it induces complement-mediated lysis, Abdependent cell cytotoxicity, and growth arrest of T lymphoma cells (22). We demonstrated in this study that the baculovirus-expressed rIgG 1 13B8.2 Ab acts by causing the accumulation of the CD4 molecule inside the Brij 98-extracted rafts and by excluding the Zap70 kinase and downstream targets SLP-76, PLC␥1, and Vav-1 from the raft machinery, together with affecting Zap70 phosphorylation, which impairs further downstream activation events.…”

“…We evaluated a recombinant Ab (17,18) derived from the 13B8.2 murine anti-CD4 mAb, directed against the CDR3-like loop of CD4; this loop has not been fully exploited as a target by clinical Abs, mainly directed against other CD4 regions (11-13, 19, 20). This baculovirus-expressed recombinant Ab (rIgG 1 ) inhibits HIV replication at a post-gp120-binding step (17,18,21) and induces complement-mediated lysis, Ab-dependent cell cytotoxicity, and growth arrest of T lymphoma cells (22). The biological effects of rIgG 1 13B8.2 are partly due to signals that prevent NF-B nuclear translocation (23); ERK activation (24); and NF-AT, NF-B, and AP-1 binding to the IL-2 gene promoter (25).…”

The lipid-modulating effects of a CD4-specific recombinant antibody correlate with ZAP-70 segregation outside membrane rafts

“…Phosphatidylserine externalization in cells undergoing death receptor-mediated apoptosis seems to be Ca ++ -dependent [36]. Our finding that rIgG 1 13B8.2 lowers phosphatidylserine level in Jurkat T cells explains why treatment with rIgG 1 13B8.2 did not induce phosphatidylserine-dependent apoptosis in T cell lymphomas [19] and blocked CD3-induced Ca ++ increase and the subsequent signaling pathways [33]. Similarly, other CD4-specific antibodies have been shown to modulate phosphatidylserine level in vitro [37].…”

“…Analysis of key upstream events showed that modulation of ZAP-70 Tyr 292 and Tyr 319 phosphorylation occurred concomitantly with rIgG 1 13B8.2-induced ZAP-70 exclusion from the membrane rafts [16]. Such antibody-induced modulation of membrane raft signaling, which leads to inhibition both of NF-B nuclear translocation [17] and of binding to the IL2 gene promoter [18], partly explains the anti-proliferative effect of rIgG 1 13B8.2 in T cell lymphomas [19]; however the effects of rIgG 1 13B8.2 on lipid dynamics in membrane rafts remain unknown. Therefore, we decided to examine the effects of the anti-CD4 rIgG 1 13B8.2 antibody on the lipid composition of membrane rafts in a T lymphoma cell line.…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…There are few studies investigating CDC activity of IgG antibodies produced in insect cells. 23,26,27,100 In several cases, no CDC activation could be demonstrated, but this was true also for the parent antibody produced in mammalian cells. 23,26 In one study, insect cell produced antibodies could indeed induce C1q binding or CDC with human serum.…”