Full text
| |
| Author(s): |
João Victor Batalha de Carvalho
Total Authors: 1
|
| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) |
| Defense date: | 2022-11-29 |
| Examining board members: |
Ana Maria Moro;
Denise Morais da Fonseca;
Keity Souza Santos;
Aryene Góes Trezena
|
| Advisor: | Ana Maria Moro |
| Abstract | |
CD3 is a protein chain set non-covalently associated with TCR. The CD3 complex is essential to T cell activation even to the correct TCR expression on the cell surface. TCR-CD3\'s chains are targets for immunotherapies that focus on the regulation of the T cell´s activity. Monoclonal antibodies (mAbs) anti-CD3 have the potential to treat autoimmune diseases (diabetes mellitus I, inflammatory bowel diseases), host-vs-graft disease prevention or reversion, and câncer immunotherapy (bispecific antibodies). Production of the murine anti-CD3 mAb by hybridoma has therapeutics limitations associated with its capacity to cause adverse reactions like T cell proliferation induction and pro-inflammatory cytokines secretion. To overcome those issues also associated with non-human molecules immunogenicity, some anti-CD3 mAbs were humanized like Otelixizumab, Visilizumab, Teplizumab. The experimental usage of humanized anti-CD3 mAbs has been associated with an increase of regulatory T lymphocytes (Treg) and immunoregulatory cytokines. To test the immunoregulatory potential of the humanized anti-CD3 from a murine hybridoma recloned at Butantan Institute, this study aims to evaluate in vitro the regulatory activity of clones of humanized anti-CD3 mAbs. The humanized molecule light and heavy chains genes were cloned into pCHO vector, then transfected into CHO-S. The clones were ranked by their specific productivity, cellular growth, and human CD3 binding capacity. For the binding assay by surface plasmonic resonance (SPR) we used an in-house recombinant antigen of the human CD3 gamma-epsilon. The recombinant antigen was made by DNA assembly technique and expressed in E. coli in inclusion bodies, then solubilized and in vitro refolded. The binding capacity of the humanized anti-CD3 was lower in comparison to the murine molecule, it indicates a low affinity of the humanized anti-CD3. From 280 clones expressing the humanized anti-CD3, some were selected for the in vitro functional assays with human PBMC. By flow cytometry, the proliferation rate induced by two clones was lower than that induced by the murine anti-CD3 treatment at two concentrations, 1 e 5 g/mL. The treatment with the humanized antibody increased the CD4+ subpopulation and decreased CD8+ cells in relation to the murine version. The Treg (CD4+ CD127-CD25highFoxp3+) subpopulation was increased when the humanized mAb was added and decreased with the murine antibody treatment. The secreted cytokine profile (48 h/192 h) due to the murine anti-CD3 treatment was highly inflammatory at 48 h and at 24 and 72 h in the intracellular assay. However, the two humanized clones showed a profile close to the non-treated group. The intracellular cytokines assay shows more granulocytes with humanized anti-CD3, and no IFN-γ e TNF-α were produced by those group of cells. These data contribute to the understanding of the humanized anti-CD3, although not conclusive. More studies are necessary for the possible constructions of the humanized anti-CD3 (monospecific, bispecific etc.). (AU) | |
| FAPESP's process: | 19/10724-2 - Recombinant monoclonal antibodies for therapeutic use |
| Grantee: | João Victor Batalha de Carvalho |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |