Anti-Human CD4 Recombinant Antibody (Zanolimumab)

A, zanolimumab does not fix complement factor C4 when bound to CD4+ T cells. Zanolimumab bound to cells does not induce complement activation as measured by deposition of C4. Representative of three independent experiments. The CD4+CD45RA+ T-cell population (left) and the CD4+CD45RO+ T-cell population (right) were measured using anti-CD45RA+-PE and CD45RO+-APC antibodies. The antibody OKT3, as positive control at the same concentrations, does induce C4 deposition. B, zanolimumab kills CD4+ T cells via ADCC. CD4+ T cells are efficiently killed by NK cell–dependent ADCC (left). Left, histograms of the CD4+ T cells. Columns, mean of four independent experiments; bars, SE. Right, histograms of the CD4+CD45RA+ and CD4+CD45RO+ T cells. Columns, mean of nine independent experiments; bars, SE. **, P < 0.005, Wilcoxon signed-rank test, CD4+CD45RO+ specific lysis by zanolimumab differs significantly from CD45+CD45RA+ specific lysis.

Rider, D. A., Havenith, C. E., de Ridder, R., Schuurman, J., Favre, C., Cooper, J. C., ... & Cambier, J. (2007). A human CD4 monoclonal antibody for the treatment of T-cell lymphoma combines inhibition of T-cell signaling by a dual mechanism with potent Fc-dependent effector activity. Cancer research, 67(20), 9945-9953.

A, dose-response plots of CD4 expression were carried out using purified blood CD4+ T cells incubated for 18 h with zanolimumab in the presence or absence of monocytes (Mo) incubated with or without IFNγ. B, dose-response plots of CD4 expression were carried out using CD4+ CEM-NKr cells incubated for 18 h with zanolimumab, F(ab′)2 fragments of zanolimumab, or an isotype control antibody (HuMab-KLH) in the presence or absence of THP-1 cells. The CD4 expression levels were measured using a noncompeting CD4 antibody, MT-477. Results are representative of four different experiments.

Rider, D. A., Havenith, C. E., de Ridder, R., Schuurman, J., Favre, C., Cooper, J. C., ... & Cambier, J. (2007). A human CD4 monoclonal antibody for the treatment of T-cell lymphoma combines inhibition of T-cell signaling by a dual mechanism with potent Fc-dependent effector activity. Cancer research, 67(20), 9945-9953.

A, dose-response plots of proliferation assays were carried out using purified CD4+ T cells stimulated with tetanus toxoid (TT; 10 μg/mL) at the concentrations shown. Points, mean of two independent experiments; bars, SE. B, dose-response plots of proliferation assays were carried out using purified CD4+ T cells stimulated with immobilized OKT3 mAb (0.1 μg/mL) coated onto latex beads, either with or without the addition of anti-CD28 (1.0 μg/mL) at the concentrations shown. Points, mean of four independent experiments; bars, SE. C, supernatants removed from proliferation assays after 48 h were assayed for IL-2 and IL-4. Points, mean of four independent experiments; bars, SE.

Rider, D. A., Havenith, C. E., de Ridder, R., Schuurman, J., Favre, C., Cooper, J. C., ... & Cambier, J. (2007). A human CD4 monoclonal antibody for the treatment of T-cell lymphoma combines inhibition of T-cell signaling by a dual mechanism with potent Fc-dependent effector activity. Cancer research, 67(20), 9945-9953.

Purified CD4+ T cells were incubated with (+) or without (−) zanolimumab for 10 min before incubation with mouse IgG1 (0 min) or OKT3-coated latex beads at 37°C for the times shown. Cells were lysed before samples were split into two and proteins were separated by 7% to 15% gradient SDS-PAGE. A, one membrane was then immunoblotted with the phosphotyrosine mAb 4G10 before stripping (top) and reprobing with LAT (middle) and TCRζ antibodies (bottom). B and C, the second membrane was probed with phospho-ZAP-70 (B) and phospho-Erk1/2 antibodies, phospho-p38, and phospho-AKT (C) and followed by stripping and reprobing with ZAP-70, Erk1/2, p38, and AKT, respectively, to assess loading. Results for the experiments are representative of four different experiments.

Rider, D. A., Havenith, C. E., de Ridder, R., Schuurman, J., Favre, C., Cooper, J. C., ... & Cambier, J. (2007). A human CD4 monoclonal antibody for the treatment of T-cell lymphoma combines inhibition of T-cell signaling by a dual mechanism with potent Fc-dependent effector activity. Cancer research, 67(20), 9945-9953.

A, CD4-associated p56lck tyrosine kinase activity is increased after CD4 ligation. Purified CD4+ T cells were incubated with zanolimumab, and CD4 was then immunoprecipitated from cell lysates, followed by in vitro kinase assays in the presence of α-casein before transfer to an immunoblotting membrane. CD4-p56lck autophosphorylation (top) and phosphorylated α-casein (middle top). Membranes were blotted for pY-394-p56lck and stripped and reprobed for both p56lck and CD4 as loading controls (middle, middle bottom, and bottom). B, Dok-1 phosphorylation is increased after CD4 ligation. Cells treated with zanolimumab were lysed and precleared with glutathione agarose beads coupled to an empty GST construct before Dok-1 was precipitated using a GST construct of the COOH-terminal SH2 domain of RasGAP (SH2C-RasGAP-GST). Samples were blotted with Dok-1 and GST antibody. C, SHIP-1 phosphorylation is increased after CD4 ligation. Cells were treated with either 10 μg/mL zanolimumab for 10 min before immunoblotting whole-cell lysates with a phosphospecific SHIP-1 antibody. D, phosphorylation of Dok-1 is dependent on the action of Src family kinases. CD4+ T cells were preincubated with the Src family inhibitors PP2 or damnacanthal (DAM) for 45 min before treatment with zanolimumab for 5 min and Dok-1 phosphorylation was then measured as in C. Results are representative of four different experiments.

Rider, D. A., Havenith, C. E., de Ridder, R., Schuurman, J., Favre, C., Cooper, J. C., ... & Cambier, J. (2007). A human CD4 monoclonal antibody for the treatment of T-cell lymphoma combines inhibition of T-cell signaling by a dual mechanism with potent Fc-dependent effector activity. Cancer research, 67(20), 9945-9953.