Knowledge based improvement of an anti-human CD3 humanized antibody
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1
EMBRAPA, Brazil
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2
Brasilia University, Brazil
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3
National Institute of Science and Technology for Immunology Investigation/INCTiii, Brazil
Molecular development of humanized antibodies has been effective in improving clinical use of biopharmaceuticals. To minimize cytokine release syndrome, which is a side effect caused by ortholog antibody use, our group developed two constructions of humanized anti-CD3 antibodies consisting in an IgG1 human construction containing conserved CDRs from a mouse anti-human CD3 antibody (OKT3). These recombinant constructions were able to interact with human CD3 besides of its lower capacity to compete with OKT3, suggesting loss of affinity induced by humanization process (Silva et al, 2009). Humanized molecules also induced lower production of IFN gamma, higher of IL-10 and had less mitogenic effect than OKT3, being promising for its substitution in patient treatment (Silva et al, 2009). In order to improve performance of our humanized constructions we performed site-directed mutagenesis based on the crystal structure of CD3-OKT3 antibody complex which identified key binding residues that were selected and modified by assisted PCR mutagenesis. Basically, aminoacids at positions 31 (S/R; VHS31R) and 74 (T/K; VHT74K) of the heavy chain and positions 45 (L/R; VLL45R) and 70 (F/Y; VLF70Y) of the light chain were changed in RVL molecule, a construction used by used by Silva et al (2009) and as one of the controls of our experiments. Two other constructions, TVL, which has a mutation at the CDR domain, and murine FvFc, which conseved human IgG1 and murine anti-CD3 CDR intacts, were also used at the assays in order to evaluate the site-directed mutagenesis. All of seven different humanized anti-CD3 antibodies were produced in Chinese hamster ovary cells (CHO) as FvFc fragments. Flow cytometer analyses demonstrate that RVL, TVL and VHS31R and VHT74K constructions displayed a higher capacity of interact to CD3 at T cell surface (Figure 1). Other constructions were also capable to bind CD3, but at lower frequency. Proliferation assays indicated a lower mitogenic response in result of PBMC treatment with RVL, TVL and murine FvFc constructs tested although more tests have to be run. The results indicated that these site-direction mutations affect the binding affinity of these molecules, specially aminoacids outside of the CDR region that does not interact with CD3 antigen. More tests will be conducted to evaluate affinity binding constants and biological effects of all constructions.
Figure 1: Humanized antibody constructions binds to CD3 on surface of TCD4 cells. Humanized mutant antibodies obtained by site-directed mutagenesis (0,3ug/ml) were incubated with 300.000 leukocytes isolated from human blood collected by venipuncture. After 30 minutes of incubation cells were washed and incubated with anti-IgG1 human-FITC and anti-CD4PE antibodies for flow cytometry analyses. Dot plot reveals the percentage of TCD3+TCD4+ cells indicating different affinities among mutant constructions. (A) Mouse anti-CD3 human (OKT3 clone) showing the gate used for all analysis, (B) Humanized anti-CD3 RVL construction, (C)TVL, (D) Murine FvFc, (E) VHS31R, (F) VHT74K, (G) VLL45R, (H) VLF70Y.
Acknowledgements
Financial support: MCT/CNPq
References
Silva, H., Vieira, P., Costa, P., Pimentel, B., Moro, A., Kalil, J., Maranhão, A., Coelho, V., Brigido, M. (2009). Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells. Immunol Lett. 15, 129-136.
Keywords:
anti-CD3,
site-direct mutagenesis,
humanized antibody,
protein expression and purification,
CHO Cells
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Immune receptors and signaling
Citation:
Carvalho
WA,
De Paula
J,
Gulis
G,
Bezerra
M,
Simi
K,
Balbino
A,
Bento
F,
Rodrigues
R,
Maranhão
A and
Brigido
M
(2013). Knowledge based improvement of an anti-human CD3 humanized antibody.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00581
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Received:
14 May 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Dr. Wanessa A Carvalho, EMBRAPA, Juiz de Fora, Brazil, wanessa.carvalho@embrapa.br