Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: The humanized CD4 specific monoclonal antibody (mAb) tregalizumab is currently being tested in phase II clinical trials in Rheumatoid Arthritis. In contrast to other anti-CD4 antibodies, tregalizumab is able to activate the suppressive properties of regulatory T cells (Tregs). Since tregalizumab was the first and only humanized anti-CD4 antibody described to be able to activate Tregs we asked for the underlying reason for this specific and unique functionality. To elucidate on the mode of action of this mAb, we focused on the signaling pathways engaged by tregalizumab.
Methods: Signaling events after cross-linking of tregalizumab, other anti-CD4 antibodies (RPA-T4, MT310, QS4120 or B-A1) or anti-CD3 (OKT-3) treatment were analyzed using intracellular staining of phosphorylated proteins (Lck, ZAP-70, LAT, SLP-76, PLC-gamma, MEK, Itk, ERK, PKC, MAPK and NF-kappaB). Furthermore, the ability of tregalizumab to induce suppressive properties in Tregs was evaluated using a mixed lymphocyte reaction system. Other CD4 antibodies were included as controls. Additionally, the binding mode of tregalizumab to soluble CD4 was resolved by co-crystallization and subsequent x-ray crystallography with a resolution of 2.9 angstrom.
Results: Upon binding of tregalizumab to the CD4 molecule on T cells and subsequent cross-linking, an intracellular signal was induced. As described for anti-CD3 or other anti‑CD4 antibodies, phosphorylation of the CD4 associated kinase Lck was observed. Nonetheless, significant signaling strength differences were observed between the different antibodies. Although tregalizumab induced the lowest phosphorylation signal on Lck, further downstream molecules were also activated. Tregalizumab mediated signaling additionally led to phosphorylation of ZAP-70, LAT, SLP-76, PLC-gamma and MEK, thus engaging several components of the T cell receptor pathway. However, tregalizumab induced no phosphorylation of Itk, ERK, PKC, MAPK and NF-kappaB as observed for anti-CD3 treatment or other anti-CD4 antibodies tested. Although inducing the weakest signal of all anti-CD4 antibodies, only tregalizumab was able to induce full functional activation of Tregs via CD4.
The new mode of action of tregalizumab may be explained by the special binding epitope. While all other tested anti-CD4 antibodies bound to domain 1 of CD4, the crystal structure of tregalizumab in complex with CD4 revealed binding to domain 2.
Conclusion: In summary, we hypothesize that binding to domain 2 of CD4 may be the underlying reason for inducing weak but unique signaling in CD4 T cells that is sufficient to activate the function of Tregs without activation of T effector cells. Thus, tregalizumab represents a unique and novel mode of action for treatment of autoimmune diseases with insufficient Treg activity. A phase IIb clinical trial is currently ongoing in Rheumatoid Arthritis in european countries to further evaluate clinical use of tregalizumab (Biotest Study 979).
Disclosure:
B. Helling,
Biotest AG,
3;
B. Daelken,
Biotest AG,
3;
H. Wallmeier,
Biotest AG,
5;
S. Aigner,
Biotest AG,
3;
C. Zuber,
Biotest AG,
3;
M. Koenig,
Biotest AG,
3;
A. Engling,
Biotest AG,
3;
F. Osterroth,
Biotest AG,
3;
N. Czeloth,
Biotest AG,
3;
C. Uherek,
Biotest AG,
3.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-therapeutic-antibody-tregalizumab-bt-061-induces-activation-of-regulatory-t-cells-by-engaging-a-unique-cd4-mediated-signaling-that-strongly-differs-from-signaling-events-induced-by-standard-anti/