The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.

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We analyzed antigen-binding residues from the variable domains of anti-CD4 antibody 13B8.2 using the Spot method of parallel peptide synthesis. Sixteen amino acids, defined as Spot critical residues (SCR), were identified on the basis of a 50% decrease in CD4 binding to alanine analogs of reactive peptides. Recombinant Fab 13B8.2 mutants were constructed with alanine residues in place of each of the 16 SCR, expressed in the baculovirus cell system, and purified. CD measurements indicated that the mutated proteins were conformationally intact, with a ␤-sheet secondary structure similar to that of wild-type

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.