Anti-Human CD4 Recombinant Antibody (Tregalizumab) (CAT#: TAB-260)

Figure 1 Tregalizumab is able to activate the suppressive capacity of regulatory T cells.

(A) Tregs were isolated and pre-incubated with plate-bound tregalizumab, OKT-3 mAb, or medium and transferred to a mixed lymphocyte reaction using allogeneic, CD3-depleted, irradiated PBMCs to activate the proliferation of Teffs. (B) The proliferation of allogenic stimulated Teffs with tregalizumab pretreated Tregs is shown compared with the proliferation without Tregs. (C) Tregalizumab engages the TCR signaling pathway. Incubation of Tregs with tregalizumab evokes signaling events in Tregs that mimic a signal through the T cell receptor complex without the need for direct CD3 or TCR stimulation. T cell receptor complex signaling represents a crucial event in the activation of Tregs since blockade of the corresponding signaling pathway resulted in the loss of suppressive activity of Tregs. Tregalizumab delivers a comparable signal as usually conferred by TCR binding in Tregs, resulting in activation/phosphorylation of the T cell receptor downstream signaling molecule ZAP-70 (ζ-chain-associated protein). This event leads to signals that finally trigger internalization of the tregalizumab–CD4 complex. As a consequence, CD4 expression levels on CD4+ T cells are downmodulated by tregalizumab in the physiological setting. This downmodulation causes a transient decrease of CD4 molecules on the cell surface, which is followed by a recovery of expression levels over time.

König, M., Rharbaoui, F., Aigner, S., Dälken, B., & Schüttrumpf, J. (2016). Tregalizumab–a monoclonal antibody to target regulatory T cells. Frontiers in immunology, 7, 11.

Figure 2 PK/PD of CD4 "Tregalizumab".

(A) PK/PD model was established to predict time course of CD4 downmodulation (% of baseline) after weekly dosing. (B) Mean CD4 downmodulation on CD4 T cells per dose group. CD4 modulation (±SEM) was measured by FACS analysis at weeks 0, 2, 4, 6, 8, 12, 16, and 24 just prior to the next dosing.

König, M., Rharbaoui, F., Aigner, S., Dälken, B., & Schüttrumpf, J. (2016). Tregalizumab–a monoclonal antibody to target regulatory T cells. Frontiers in immunology, 7, 11.

Figure 1 Anti-Human CD4 Recombinant Antibody (TAB-260) in SDS-PAGE

SDS-PAGE analysis of TAB-260 in non-reduced (Lane 1) and reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result, different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

The purity of TAB-260 was greater than 95% as determined by SDS-PAGE.

Figure 2 Anti-Human CD4 Recombinant Antibody (TAB-260) in SEC-HPLC

The purity of TAB-260 was greater than 95% as determined by SEC-HPLC.

Column: 3 µm, 7.8 x 300 nm
Mobile phase: 150 mM Sodium Phosphate Buffer, pH 7.0
Detection: UV 280 nm
Injection: 25 µl

Figure 3 Anti-Human CD4 Recombinant Antibody (TAB-260) in WB

Western blot analysis of TAB-260 was performed by loading Recombinant Human CD4 Protein onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with TAB-260 and HRP Goat Anti-Human IgG as a secondary antibody. Chemiluminescent detection was performed.
Lane 1: Reduced antigen (0.1 μg)
Lane 2: Reduced antigen (0.3 μg)
Lane 3: Reduced antigen (0.6 μg)

Figure 4 Anti-Human CD4 Recombinant Antibody (TAB-260) in DB

Dot Blot analysis of TAB-260 was performed by coating with Recombinant Human CD4 Protein.

TAB-260 incubation concentration: 2 μg/mL.
HRP-conjugated goat anti-Human IgG as a secondary antibody (1: 2000)

Figure 5 Anti-Human CD4 Recombinant Antibody (TAB-260) in ELISA

ELISA analysis of TAB-260 was performed by coating with Recombinant Human CD4 Protein. Then blocked with BSA, and incubated with Anti-Human CD4 Antibody (TAB-260). The HRP-conjugated goat anti-human IgG as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.