Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Abstract

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than OKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with OKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity.

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

“…The humanization process leading to the FvFc anti-CD3 antibody versions has suggested the immunoregulatory profile required for tolerance induction [11], thus making the complete IgG humanized anti-CD3 an attractive candidate for additional studies. Although the modular design of immunoglobulins is very compatible with protein engineering [15], constructions other than the ones found in nature have not yet been approved by the regulatory agencies.…”

“…The vector pMIRES harboring the humanized FvFc contained an IRES sequence element allowing a dicistronic expression with the Neo gene [11]. Recombinant DNA was transfected into CHO cells (ATCC 9096) with Lipofectamine 2000 (Invitrogen, USA).…”

“…In this work, two versions of humanized anti-CD3 were obtained by the transfecting CHO cells with vectors containing either FvFc (lacking CH 1 and C j domains) or IgG 1 (containing the complete heavy and light chain genes) of humanized anti-CD3. Both constructs, FvFc and IgG, were previously assembled by our partners [10,11]. Initial studies with the product from FvFc transiently transfected cells [11] demonstrated a favorable immunoregulatory profile, suggesting that the antibody making the humanized construct is a promising candidate for the treatment of transplant or auto-immune disease patients in need of achieving an operational tolerance status.…”

Produção de novos anticorpos Anti-CD20 na forma de FvFc em células de mamíferos

“…Este promotor tem como principal característica promover altos níveis de transcrição do gene da proteína recombinante (Mariati et.al., 2010). Para tanto ele possui o íntron A com uma deleção de 600 pares de base (Quilici et al, 2013 (Caldas et al, 2000), e anti-CD3 (Silva et al, 2009a) e deste modo, foi proposta a humanização e produção de anticorpos anti-CD20 totalmente humanos em células de mamíferos que foram gerados pela tecnologia de V domain shuffling e selecionados por phage display pela aluna de doutorado Barbara Maciel Sidou Pimentel (dados não publicados).…”

“…Embora, a obtenção de clones estáveis seja a mais adequada para atingir altos níveis de produção, ela requer maior tempo e custos mais elevados associados com a geração de uma linha de células de mamíferos estavelmente transfectadas de alta produção; por custos de produção decidiu-se trabalhar com a população mista de células CHO-K1 e HEK-293 produtoras dos FvFcs recombinantes, segundo (Silva et al, 2009b),esta estratégia é possível embora não seja a mais eficaz. Diante desses resultados, optou-se por manter a cultura de células nessas condições com o intuito de gerar um acúmulo de sobrenadante suficiente para preparação das proteínas para as próximas etapas do trabalho.…”