Anti-Human CD2 Recombinant Antibody (BTI-322) (CAT#: TAB-110CL)

Figure 1 Effect of BTI-322 on primary and secondary xenogeneic MLR.

(a) Human PBMCs (1 × 10⁶/ml) were cultured at a 1 : 1 ratio with irradiated SLAᵈᵈ porcine PBMCs, in the presence of 200 ng/ml BTI-322 or the humanized version MEDI-507, or control isotype-matched Ig, followed by measurement of [³H]-TdR incorporation (cpm) at days 3, 5, and 7. (b) Cells harvested after primary xenogeneic stimulation in the presence of these antibodies or controls were cultured for 3 days without stimulant, and then subjected to a secondary xenogeneic MLR, followed by measurement of [³H]-TdR incorporation (cpm) at days 3 and 5.

Xu, Y., Kolber‐Simonds, D., Hope, J. A., Bazin, H., Latinne, D., Monroy, R., ... & SCHUURMAN, H. J. (2004). The anti‐CD2 monoclonal antibody BTI‐322 generates unresponsiveness by activation‐associated T cell depletion. Clinical & Experimental Immunology, 138(3), 476-483.

Figure 2 Use of T cells as responder cells and effect of monocytes.

(a) PBMCs or nylon-wool-purified T cells (both 1 × 10⁶/ml) were cultured at a 1 : 1 ratio with irradiated SLAᵈᵈ porcine PBMCs, in the presence of 200 ng/ml BTI-322 or control Ig, followed by measurement of [³H]-TdR incorporation (cpm) at days 3, 5, and 7. (b) Purified responder T cells were supplemented with monocytes (adherent cells) at relative proportions [100% reflects the original proportion in the original PBMC preparation (10–20%)].

Xu, Y., Kolber‐Simonds, D., Hope, J. A., Bazin, H., Latinne, D., Monroy, R., ... & SCHUURMAN, H. J. (2004). The anti‐CD2 monoclonal antibody BTI‐322 generates unresponsiveness by activation‐associated T cell depletion. Clinical & Experimental Immunology, 138(3), 476-483.

Figure 3 Effect of BTI-322 on secondary MLR: SLAᶜᶜ, third-party SLAᵈᵈ and allogeneic restimulation after primary stimulation with SLAᵈᵈ cells.

(a) Cells (1 × 10⁶/ml) from primary 7-day xenogeneic MLR (1 : 1 responder : stimulator ratio) in the presence of 200 ng/ml BTI-322 or control Ig were cultured in secondary MLR with cells of the original SLA haplotype, third-party xenogeneic cells and allogeneic cells. Data shown are [³H]-TdR incorporation at various time-points during secondary culture, for cells cultured during the primary MLR. (b) The same experiment, using allogeneic stimulation in primary MLR. (c) The same experiment using suboptimal xenogeneic stimulation in primary MLR (1 : 0·125 responder : stimulator ratio).

Xu, Y., Kolber‐Simonds, D., Hope, J. A., Bazin, H., Latinne, D., Monroy, R., ... & SCHUURMAN, H. J. (2004). The anti‐CD2 monoclonal antibody BTI‐322 generates unresponsiveness by activation‐associated T cell depletion. Clinical & Experimental Immunology, 138(3), 476-483.

Figure 4 Effect of BTI-322 on primary and secondary stimulation with anti-TCR Vβ family-specific antibodies.

(a) PBMCs were incubated in primary culture with 10 µg/ml anti-TCR Vβ8 antibody, with or without BTI-322 or control Ig (100 ng/ml): [³H]-TdR incorporation was measured at day 7. (b) Cells after primary stimulation as in (a) were washed and cultured for 3 days without stimulator, and then subjected to secondary stimulation with the original anti-Vβ8 antibody or an irrelevant anti-Vβ13 antibody (10 µg/ml): [³H]-TdR incorporation was measured at day 3.

Xu, Y., Kolber‐Simonds, D., Hope, J. A., Bazin, H., Latinne, D., Monroy, R., ... & SCHUURMAN, H. J. (2004). The anti‐CD2 monoclonal antibody BTI‐322 generates unresponsiveness by activation‐associated T cell depletion. Clinical & Experimental Immunology, 138(3), 476-483.

Figure 5 Presence of CD3⁺Vβ8⁺ cells after stimulation of PBMCs with anti-TCR Vβ8 or anti-TCR Vβ13 antibody in the presence of BTI-322 or control Ig.

PBMCs (1 × 10⁶/ml) were stimulated for 7 days by control antibody (a,b), anti-Vβ8 antibody (c,d) or anti-Vβ13 antibody (e,f) at 100 ng/ml, in the presence of 100 ng/ml control antibody (a,c,e) or BTI-322 (b,d,f). Subsequently, flow cytometry was performed with a fluorescein isothiocyanate (FITC)-conjugated anti-CD3 antibody in combination with an anti-TCR Vβ8 antibody [indirect staining using a phycoerythrin (PE)-conjugated secondary antibody]. The position of the CD3⁺Vβ8⁺ double-positive population in the individual plots is indicated by circles, determined by flow cytometry on freshly isolated PBMCs from the same donor with appropriate isotype-matched control antibody and single-antibody staining. The CD3⁺Vβ8⁺ double-positive population comprises approximately 4·5% of the viable cells in (a,b,e and f); approximately 14% in (c); and approximately 1% in (d).

Xu, Y., Kolber‐Simonds, D., Hope, J. A., Bazin, H., Latinne, D., Monroy, R., ... & SCHUURMAN, H. J. (2004). The anti‐CD2 monoclonal antibody BTI‐322 generates unresponsiveness by activation‐associated T cell depletion. Clinical & Experimental Immunology, 138(3), 476-483.

Figure 6 Distribution of grades of GVHD during and after treatment with BTI-322.

Height of bar represents the number of patients with grade 0 (■), grade 1 (▧), grade 2 (░), or grade 3-4 (▩). Total number of patients represents evaluable survivors at each evaluation timepoint.

Przepiorka, D., Phillips, G. L., Ratanatharathorn, V., Cottler-Fox, M., Sehn, L. H., Antin, J. H., ... & McClain, J. B. (1998). A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease. Blood, 92(11), 4066-4071.

Figure 7 Absolute numbers of (A) CD2, CD16/56, and CD19/20 or (B) CD3, CD3/4, and CD3/8 lymphocytes in peripheral blood during and after treatment with BTI-322.

Przepiorka, D., Phillips, G. L., Ratanatharathorn, V., Cottler-Fox, M., Sehn, L. H., Antin, J. H., ... & McClain, J. B. (1998). A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease. Blood, 92(11), 4066-4071.

Figure 8 Serum concentrations (mean ± SE) of BTI-322 after infusion of the first (solid line) or fourth (dashed line) dose of drug.

Data are displayed for all 10 patients who had pharmacokinetic sampling.

Przepiorka, D., Phillips, G. L., Ratanatharathorn, V., Cottler-Fox, M., Sehn, L. H., Antin, J. H., ... & McClain, J. B. (1998). A phase II study of BTI-322, a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease. Blood, 92(11), 4066-4071.

Figure 9 BTI-322 prevents the infiltration of grafted skins with human T lymphocytes and subsequent dermal microvascular injuries.

Five weeks after human fetal skin grafting, mice were injected (A) with allogeneic PBL or B) with allogeneic PBL
BTI-322. Skin grafts were harvested 14 days after the PBL infusion. The degree of T-lymphocyte infiltration was scored using the five point scale (left panels) after immunostaining with an anti-human CD4 mAb (right, magnification 100). The data indicate the number of mice in each group (upper left corner) and the mean degree of infiltrationSD (upper right corner). Groups were compared using Student's t test. (C) Dermal microvascular injuries in skin grafts harvested from control mice (that did not receive human PBL) and from mice 14 days after PBL injection, with or without preventive therapy. The percentage of damaged human vessels (stained with an anti-human CD31) was determined using a square grid.

Snanoudj, R., Rouleau, M., Bidère, N., Carmona, S., Baron, C., Latinne, D., ... & Senik, A. (2004). A Role for CD2 Antibodies (BTI-322 and its Humanized Form) in the in vivo Elimination of Human T Lymphocytes Infiltrating an Allogeneic Human Skin Graft in SCID Mice: An Fcγ Receptor-Related Mechanism Involving Co-Injected Human NK Cells. Transplantation, 78(1), 50-58.

Figure 10 Effects of BTI-322, hu-BTI-322, and their F(ab’)2 fragments on human T-cell infiltration.

(A) Curative protocol: 14 days after PBL injection, skin grafted mice received two subcutaneous injections performed at a 3-day interval of vehicle or isotype-matched control antibody LoDNP 57, 10 μg of BTI-322 or hu-BTI-322, or 50 μg of the corresponding F(ab')2 fragments. Mice were killed 7 days after the first antibody injection. The degree of human lymphocyte infiltration was scored using the 5-point scale. Groups were compared using Student's t test. (B) Immunoblot analysis of whole and F(ab')2 fragments of BTI-322 and hu-BTI-322 revealed by horseradish peroxidase (HRP)- conjugated rabbit immunoglobulin (Ig)G specific for rat Ig and by a mouse HRP-conjugated IgG specific for human IgG, respectively. (C) Antigen-binding capacity of whole BTI-322, hu-BTI-322, and of their F(ab')2 fragments was evaluated by staining CD2+ Jurkat cells with 1 μg of these reagents. Secondary antibodies were fluorescein isothiocyanate-conjugated rabbit anti-rat IgG and rabbit anti-human kappa light chains.

Snanoudj, R., Rouleau, M., Bidère, N., Carmona, S., Baron, C., Latinne, D., ... & Senik, A. (2004). A Role for CD2 Antibodies (BTI-322 and its Humanized Form) in the in vivo Elimination of Human T Lymphocytes Infiltrating an Allogeneic Human Skin Graft in SCID Mice: An Fcγ Receptor-Related Mechanism Involving Co-Injected Human NK Cells. Transplantation, 78(1), 50-58.