Recombinant anti-CD3 monoclonal antibody(Shanghai Zhangjiang Biotechnology Co Ltd)

ScOKT3-gammaDeltaIgM VAEVD is a recombinant chimeric anti-CD3 antibody variant consisting of the light and heavy variable binding domains of the OKT3 monoclonal antibody and the CH3 and CH4 domains of a human IgM mutation linked by a human IgG3 hinge region. Due to the IgM Fc domains, scOKT3-gammaDeltaIgM VAEVD antibodies are able to form polymeric structures. Independent of their polymerization state, they possess in vitro CD3 modulating and immunosuppressive properties while inducing only minimal T cell activation compared to their monoclonal counterpart. To evaluate the in vivo efficacy of the antibodies, an adjuvant-induced chronic inflammation was established in human CD3 transgenic mice. Administration of four doses of 15 microg of isolated scOKT3-gammaDeltaIgM VAEVD monomers and pentamers significantly reduced diameters of inflamed ankle joints in a manner comparable to the monoclonal antibody OKT3. Additionally, the antibody treatment lead to a significant reduction of the cytokine levels (IL-2, TNF-alpha and INF-gamma) in the mice's sera. These results suggest that scOKT3-gammaDeltaIgM VAEVD antibodies may provide a useful alternative to the OKT3 mAb for clinical immunosuppressive treatment for auto-aggressive diseases or for organ-transplantation.

The isotype of anti-CD3 mAb has a dramatic effect on anti-CD3 induced T-cell activation, as was previously reported for switch variants (IgG2b to IgA) of a high-avidity IgG1 anti-CD3 mAb (CLB-T3/4.1). In order to study and compare the isotype dependency of T-cell activation with anti-CD3 mAb of various mouse and human subclasses, we now prepared recombinant anti-CD3 mAb. The variable region of the anti-CD3 Ig heavy chain was cloned, joined with genes for the heavy chain constant region and expressed in a cell line only secreting autologous mouse chi light chains. Thus we obtained cell lines that produced mouse (m) IgM, mIgG3 and chimaeric mouse/human (h) IgM, hlgG1, hlgG2, hlgG3, hlgG4, hlgE and hlgA2 anti-CD3. The matched set of mouse and mouse/human chimaeric anti-CD3 isotypes switch variants was then used to study activation of T cells in an accessory cell-dependent system. hlgG1, hlgG4, hlgE, mlgG2a and mlgE induced T-cell proliferation in PBMC of all donors tested, whereas PBMC from a subset of donors were unresponsive to stimulation with hlgG2, hlgG3, hlgA2, mlgG1 and mlgG2b anti-CD3 mAb. hlgM, mlgM and mlgA were only able to induce T-cell mitogenesis in combination with PMA. Our panel of anti-CD3 mAb variants may prove a powerful tool to study mouse and human isotype-dependent effector functions and their influence on T-cell activation requirements in detail.