Afuco™ Anti-Human CD2 ADCC Recombinant Antibody (BTI-322), ADCC Enhanced (CAT#: AFC-106CL)

Anti-CD2 ADCC Enhanced Antibody (BTI-322) is an ADCC enhanced antibody produced by our Afuco™ platform. BTI-322 is a monoclonal anti-CD2 antibody, for treatment of steroid-resistant acute graft-versus-host disease.

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Figure 1 cBTI-322 mAb is unable to form a mitogenic pair with another CD2 mAb but rather induces cell death.

Figure 1 cBTI-322 mAb is unable to form a mitogenic pair with another CD2 mAb but rather induces cell death.

A, 1 × 10⁵ nylon wool-purified T cells were stimulated for 4 days with 1 μg/ml of the indicated CD2 mAb.[³H]TdR incorporation corresponded to a 6-h pulse performed at the end of the culture period.B, 1 × 10⁵ T cells exposed for 6 days to the indicated CD2 mAb (1 μg/ml). Cell viability was then estimated by flow cytometry after staining with propidium iodide to detect dead cells (red fluorescence).

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 2 BTI-322 mAb alone has potent T cell death-inducing capacity.

Figure 2 BTI-322 mAb alone has potent T cell death-inducing capacity.

A, DNA synthesis of 5 × 10⁴ T cells exposed during 6 days to the indicated CD2 mAb (1 μg/ml) and to 100 U/ml IL-2. Values are means ± SD of triplicates cultures. B, 1.5 × 10⁵ T cells exposed in culture to 1 μg/ml CD2 mAb. Percentages of cells with fragmented DNA (<2N) were detected by flow cytometry after staining ethanol-permeabilized cells with propidium iodide.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 3 Relationship between T cell death and inhibition of CD3-induced T cell proliferation in BTI-322-treated T cells.

Figure 3 Relationship between T cell death and inhibition of CD3-induced T cell proliferation in BTI-322-treated T cells.

T cells (1 × 10⁵) were stimulated for 4 days with soluble OKT3 (250 ng/ml) and 100 U/ml IL-2 in the presence of 1 μg/ml CD2 mAb (D66 or T111), 500 ng/ml BTI-322, or 1 μg/ml Lo-Tact-1 mAb. A, T cell proliferation as assessed by [3H]TdR incorporation. B, percent cell death as estimated by trypan blue exclusion and cell morphology. C, Effect of graded concentrations of BTI-322 mAb; absolute numbers of viable cells and percent dead cells were estimated by trypan blue exclusion; the percentage of CD3+ cells was assessed by cytofluorometry.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 4 Phenotypic analysis of cells recovered from primary OKT3 + IL-2-stimulated cultures performed in the presence of BTI-322.

Figure 4 Phenotypic analysis of cells recovered from primary OKT3 + IL-2-stimulated cultures performed in the presence of BTI-322.

T cells were stimulated as in Figure 3 in the presence or absence of 10 ng/ml BTI-322, thoroughly washed, and recultivated for 6 days in the sole presence of 100 U/ml IL-2.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 5 BTI-322 alone is able to trigger the apoptotic death of activated T cells.

Figure 5 BTI-322 alone is able to trigger the apoptotic death of activated T cells.

A, T cells were stimulated for 4 days with 250 ng/ml OKT3 and 100 U/ml IL-2 and then exposed for 16 h to the indicated mAb at 1 μg/ml. Percentages of hypoploid cells were detected by flow cytometry after staining the DNA with propidium iodide (linear scales are represented). One experiment representative of four is shown. B, Ultrastructural changes associated with BTI-322-associated cell death.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 6 BTI-322-induced apoptosis occurs at low concentrations and is independent of the Fas-Fas-L system.

Figure 6 BTI-322-induced apoptosis occurs at low concentrations and is independent of the Fas-Fas-L system.

A, Nylon wool-purified T cells activated for 4 days with OKT3 + IL-2 and then subjected for 16 h to graded doses of BTI-322 in the presence of 10% monocytes.Effect of BTI-322 on T cell preparations treated twice with anti-CD14 mAb + complement,T cells exposed to "humanized" BTI-322, T cells exposed to F(ab′)2 fragments of BTI-322. T cells exposed to isotype-matched Lo-DNP-57 mAb.B, Anti-CD3-activated T cells exposed to 100 ng/ml BTI-322.C,Anti-CD3-activated T cells preincubated during 1 h with 10 μg/ml M3 or M33 anti-Fas mAb and then exposed for 16 h to 100 ng/ml BTI-322 or to 1/10 diluted yeast supernatant containing recombinant human Fas-L.In all these experiments, dead and apoptotic cells were enumerated by microscopic examination according to the criteria of trypan blue dye inclusion and cell morphology. Experiments are representative of four others (A) and of two others (B and C).

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 7 BTI-322 cannot inhibit T cell proliferation induced by mitogenic CD2 mAb or PHA-P.

Figure 7 BTI-322 cannot inhibit T cell proliferation induced by mitogenic CD2 mAb or PHA-P.

A, Resting T cells were incubated with GT2 and D66 mAb for 0.5 h in ice and then subsequently incubated with 1 μg/ml BTI-322 and FITC-labeled mAb (MARG26) specifically recognizing rat IgG. B, 1 × 105 T cells stimulated for 4 days with GT2 + D66 (1 μg/ml) in the presence or absence of 1 μg/ml BTI-322 and then stained with propidium iodide to show the percentages of dead cells. C, [3H]TdR incorporation of T lymphocytes stimulated for 4 days with 1 μg/ml GT2 + D66, or T cell numbers obtained after a 3-day stimulation with 2.5 μg/ml PHA or after a 4-day stimulation with anti-CD3 (250 ng/ml) and anti-CD28 mAb(2 μg/ml) plus IL-2. BTI-322 (1 μg/ml) or 1 μg/ml isotype-matched Lo-DNP-57 mAb (control cells) were added to the cultures.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 8 BTI-322 induces alloantigen-specific unresponsiveness.

Figure 8 BTI-322 induces alloantigen-specific unresponsiveness.

A, cells recovered from a 5-day MLC performed in the presence or absence of 100 ng/ml BTI-322.Expression of CD3 expression was assessed by double immunofluorescence staining. B, kinetic of the secondary alloproliferative responses of T cells recovered from a primary MLC performed in the presence of BTI-322 (100 ng/ml).

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 9 T cells cultured with BTI-322 in a primary MLC respond to soluble PPD and tetanus toxoid antigens in secondary cultures.

Figure 9 T cells cultured with BTI-322 in a primary MLC respond to soluble PPD and tetanus toxoid antigens in secondary cultures.

Cells recovered from a 6-day MLC performed in the presence or absence of 100 ng/ml BTI-322 were washed, rested for 2 days, and then stimulated for 6 days in secondary cultures with PPD (25 μg/ml) or with tetanus toxoid (1/250 dilution of the stock solution). The soluble Ags were presented by 10% autologous monocytes. Histograms represent the means±SD of triplicate values.

Dumont, C., Déas, O.,Mollereau,B.,Hebib,C., Giovino-Barry, V., Bernard, A. & Senik, A. (1998).Potent apoptotic signaling and subsequent unresponsiveness induced by a single CD2 mAb(BTI-322) in activated human peripheral T cells.The Journal of Immunology,160(8),3797-3804.

Figure 10 Distribution of grades of GVHD during and after treatment with BTI-322.

Figure 10 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 11 The absolute CD2+ cell count fell rapidly after infusion of BTI-322, remained low throughout the treatment period, and returned to pretreatment levels by study day 20.

Figure 11 The absolute CD2+ cell count fell rapidly after infusion of BTI-322, remained low throughout the treatment period, and returned to pretreatment levels by study day 20.

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 12 Serum concentrations of BTI-322 increased rapidly after infusion, and the concentration curves essentially decreased in parallel after the first and fourth doses.

Figure 12 Serum concentrations of BTI-322 increased rapidly after infusion, and the concentration curves essentially decreased in parallel after the first and fourth doses.

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

Figure 13 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 days3, 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 14 Use of T cells as responder cells and effect of monocytes.

Figure 14 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 15 Effect of BTI-322 on secondary MLR: SLAcc, third-party SLAdd and allogeneic restimulation after primary stimulation with SLAdd cells.

Figure 15 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 200ng/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 16 Effect of BTI-322 on primary and secondary stimulation with anti-TCR Vβ family-specific antibodies.

Figure 16 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 17 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.

Figure 17 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 18 Dermal microvessel injuries.

Figure 18 Dermal microvessel injuries.

Indicative of rejection, were visible in skin grafts. They consisted of parietal fibrin deposits, thickening of the vascular wall caused by endothelial cell proliferation (B),and expression of HLA-DR by endothelial cells (C).No HLA-DR staining was seen in the skin grafts of mice that did not receive PBL (not shown).Other signs of rejection were the dilation of the vascular lumen and the disruption of the endothelial cell layer (not shown). Such injuries were visible on day 10, when the human lymphocytes were just beginning to infiltrate the skin. They were more severe on day 21.In 9 of 10 mice, cells of the basal keratinocyte layer strongly expressed HLA-DR on their surface, provided they were infiltrated by human CD8+ T lymphocytes or simply in contact with them, which is another sign of rejection (C).

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 19 Prevention of Human Skin Allograft Rejection by Early Injection of the Anti-CD2 mAb BTI-322.

Figure 19 Prevention of Human Skin Allograft Rejection by Early Injection of the Anti-CD2 mAb BTI-322.

Early treatment with BTI-322 prevented human T-cell infiltration and subsequent vascular injury of the skin graft .The mean degree of lymphocyte infiltration was significantly lower in BTI-322-treated mice than in control mice: 0.2±0.6 versus 2.6±0.8, respectively (P<10−5).The percentage of damaged microvessels was reduced from 30.8±9.7% in control mice injected with the isotypic control antibody, LoDNP57 (n=10), to 7.6±10.3% in mice injected with BTI-322 (P<0.0005, n=8) C).

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.


Specifications

  • Host Species
  • Mouse
  • Derivation
  • Rat
  • Type
  • ADCC enhanced antibody
  • Species Reactivity
  • Human
  • Related Disease
  • Graft versus host disease

Product Property

  • Purity
  • >95% as judged by SDS-polyacrylamide gel electrophoresis
  • Storage
  • 4°C or -20°C, avoid repeated freezing and thawing.

Target

  • Alternative Names
  • CD2; CD2 molecule; T11; SRBC; LFA-2; T-cell surface antigen CD2; LFA-3 receptor; rosette receptor; erythrocyte receptor; lymphocyte-function antigen-2; T-cell surface antigen T11/Leu-5; CD2 antigen (p50), sheep red blood cell receptor

Product Notes

This is a product of Creative Biolabs' Hi-Affi™ recombinant antibody portfolio, which has several benefits including:

• Increased sensitivity
• Confirmed specificity
• High repeatability
• Excellent batch-to-batch consistency
• Sustainable supply
• Animal-free production

See more details about Hi-Affi™ recombinant antibody benefits.

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