International Immunology, Vol. 14, No. 4, pp. 389-400,
April 2002
© 2002 Japanese Society for Immunology
Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR–CD3 complex
1 Xcyte Therapies, Inc., Seattle, WA 98104, USA 2 Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA 3 Present address: Seattle Genetics, Inc., Bothell, WA 98021, USA 4 Present address: Targeted Genetics Corp., Seattle, WA 98101, USA
Correspondence to: J. A. Ledbetter, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA; E-mail: jledbetter{at}pnri.org
Transmitting editor: E. A. Clark
The TCR–CD3 complex consists of the clonotypic disulfide-linked TCR
ß or TCR
heterodimers, and the invariant CD3, 
, and 
chains. We generated plasmid constructs expressing the extracellular domains of the CD3, or subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3, or subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3–Ig with either the CD3–Ig (CD3–Ig) or the CD3–Ig (CD3–Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3–Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3–Ig and anti-CD3 mAb ranged from 10–8 to 10–9 M. Based on these results, a single-chain (sc) construct encoding the CD3 chain linked to the CD3 chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3–scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3–Ig at a higher affinity than CD3–Ig, suggesting potential structural differences between the CD3 and CD3 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.
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