Characterization of human blood group scFv antibodies derived from a V gene phage‐display library

Abstract

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.

Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

“…This is important since the failure to detect the binding of another anti-D scFv to intact erythrocytes was attributed to the lower than desired affinity of the fragment for the D antigen (Hughes-Jones et al, 1994).…”

“…Isolation of a scFv fragment with anti-D reactivity was first reported by Marks et al (1993). This anti-D scFv was subsequently shown to be capable of agglutinating D positive erythrocytes with the aid of a cross-linking antibody directed against the c-myc peptide tag of the scFv (Hughes-Jones et al, 1994). Soluble Fabs, generated as fusion proteins, were also shown to be capable of binding and agglutinating D positive red blood cells in the presence of a cross-linking second antibody (Siegel and Siberstein, 1994;Dziegiel et al, 1995).…”

Selection and characterization of cell binding and internalizing phage antibodies

“…In the first such example, a panel of single chain Fv (scFv) antibodies were generated whose specificity could be deciphered using panels of erythrocytes with known antigen expression profiles. scFv binding and specifically agglutinating erythrocytes expressing the blood group B antigen (500,000 sites/cell), Rh(D) and Rh(E) (20,000 sites/cell) and the Kpb antigen (5,000 sites/cell) were isolated [30, 31]. The Rh(D) and Rh(E) antigens are multispanning membrane proteins with 10 and 12 transmembrane domains respectively and have hypothesized channel or pore transporter activity.…”

Introduction to Antibody Engineering and Phage Display

“…Naïve libraries provide a readily available resource of antibody fragments with the artificial VH and VL pairings obtained giving rise to antibody fragments with binding activities against many different antigens [32, 33, 34, 35]. Immune libraries are more likely to contain high affinity antibody fragments to the antigen against which the individual was immunised.…”

“…We have used red blood cells to select scFvs against Rh antigens from naïve and synthetic libraries [33, 34, 39]. Whole blood flow cytometry has been used to isolate cell–population–specific phage antibodies.…”