The I Binding Specificity of Human V H4-34 (V H4-21) Encoded Antibodies is Determined by Both V HFramework Region 1 and Complementarity Determining Region 3

Abstract

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the V H4-34 (V H4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds V H4-34 encoded antibodies and serves as a marker for the V H4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of V HFR1, heavy (H) chain complementarity determining region 3 (CDR H3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other V Hfamilies, CDR H3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the V H4-34 gene segment and the CDR H3 are essential for the interaction between CA and the I antigen, with the CDR H3 being fundamental in determining the fine specificity of antigen binding (I versusi). Mutants with substantially altered CDR H1 and CDR H2 regions bind I as long as the FR1 is V H4-34 encoded and the CDR H3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for V H4-34 FR1 explains the almost exclusive use of the V H4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDR H3 dictates the fine specificity and strength of binding.