Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.
The TCR-CD3 complex consists of the clonotypic disulfide-linked TCRalphabeta or TCRdeltagamma heterodimers, and the invariant CD3delta, epsilon, gamma and zeta chains. We generated plasmid constructs expressing the extracellular domains of the CD3delta, epsilon or gamma subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3delta, epsilon or gamma subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3epsilon-Ig with either the CD3delta-Ig (CD3epsilondelta-Ig) or the CD3gamma-Ig (CD3epsilongamma-Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3epsilondelta-Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3epsilondelta-Ig and anti-CD3 mAb ranged from 10(-8) to 10(-9) M. Based on these results, a single-chain (sc) construct encoding the CD3delta chain linked to the CD3epsilon chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3deltaepsilon-scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3epsilondelta-Ig at a higher affinity than CD3epsilongamma-Ig, suggesting potential structural differences between the CD3epsilondelta and CD3epsilongamma subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR-CD3 complex, and to identify molecules that can interfere with TCR-CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.
Law
Che-Leung
CL
Xcyte Therapies, Inc., Seattle, WA 98104, USA.
Hayden-Ledbetter
Martha
M
Buckwalter
Sonya
S
McNeill
Lisa
L
Nguyen
Hieu
H
Habecker
Phil
P
Thorne
Barbara A
BA
Dua
Raj
R
Ledbetter
Jeffrey A
JA
D000915:Antibody Affinity; D017252:CD3 Complex / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry*; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D008957:Models, Genetic; D016853:Muromonab-CD3 / Q000276:immunology; D011487:Protein Conformation; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017252:CD3 Complex; D018984:Epitopes, T-Lymphocyte; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D016853:Muromonab-CD3; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011993:Recombinant Fusion Proteins