Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses.

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse-human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')2 fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')2 fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.

Kinetics of T-cell receptor binding to peptide/I-Ek complexes: correlation of the dissociation rate with T-cell responsiveness.

Recognition by T-cell antigen receptors (TCRs) of processed peptides bound to major histocompatibility complex (MHC) molecules is required for the initiation of most T-lymphocyte responses. Despite the availability of soluble forms of TCRs and MHC heterodimers, this interaction has proven difficult to study directly due to the very low affinity. We report here on the kinetics of TCR binding to peptide/MHC complexes in a cell-free system using surface plasmon resonance. The apparent association rates for the interactions of related peptide/MHC complexes to one such TCR are relatively slow (900-3000 M-1.s-1) and dissociation rates are very fast (0.3-0.06 s-1) with t1/2 of 2-12 s at 25 degrees C. The calculated affinity of the engineered soluble molecules compares well with previously reported competition data for native TCRs or competition data reported here for native peptide/MHC complexes, indicating that these soluble heterodimers bind in the same manner as the original molecules expressed on cells. We also find that the peptide variants which give weaker T-cell stimulatory responses have similar affinities but distinctly faster dissociation rates compared with the original peptide (when loaded onto the MHC molecule) and that this later property may be responsible for their lower activity. This has implications for both downstream signaling events and models of TCR-peptide antagonists.

Biochemical evidence for a homophilic interaction of the alpha 3 beta 1 integrin.

The purpose of this study was to determine if the alpha 3 beta 1 integrin could interact in a homophilic manner. Several earlier reports have shown that certain integrin adhesion receptors, namely alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 localize to intercellular adhesion structures and, therefore, may participate in cell-cell interactions (Carter, W. G., Wayner, E. A., Bouchard, T. S., and Kaur, P. (1990) J. Cell Biol. 110, 1387-1404; Kaufmann, R., Frosch, D., Westphal, C., Weber, L., and Klein, C. E. (1989) J. Cell Biol. 109, 1807-1815; Hynes, R. O. (1987) Cell 48, 549-554; Symington, B. E., Takada, Y., and Carter, W. G. (1993) J. Cell Biol. 120, 523-535). We present data herein suggesting that the integrin alpha 3 beta 1 may interact homophilically in such cell-cell adhesion structures which contain this specific receptor or, alternatively, in receptor aggregates found in focal adhesions. The alpha 3 beta 1 receptor was purified by affinity chromatography on either human laminin or peptide GD-6-Sepharose and subsequently used as a substrate in cell adhesion assays. The immobilized alpha 3 beta 1 supported the adhesion of cells containing alpha 3 beta 1, and this attachment was specifically inhibited by monoclonal antibodies to both beta 1 and alpha 3 subunits. In addition, an affinity matrix containing purified alpha 3 beta 1 showed specific binding of only alpha 3 beta 1 from detergent extracts of cell surface proteins and such binding was cation-dependent. Finally, using biosensor technology involving the principle of surface plasmon resonance (BIAcore, Pharmacia Biosensor), alpha 3 beta 1, when bound to a carboxymethyl dextran-modified gold surface, was found to bind only other soluble alpha 3 beta 1 receptors and did not bind other purified integrins, including alpha 5 beta 1 and alpha v beta 3. These data strongly suggest that alpha 3 beta 1 likely interacts in a homophilic manner under our experimental conditions.