Characterization of human anti-high molecular weight-melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library.

The human high molecular weight-melanoma-associated antigen (HMW-MAA) meets the criteria to be used as an immunogen for immunotherapy of malignant melanoma, because it is expressed by a large percentage of melanoma lesions with limited heterogeneity and has a restricted distribution in normal tissues. The high immunogenicity of the HMW-MAA in BALB/c mice has resulted in the development of a large number of anti-HMW-MAA monoclonal antibodies (mAbs). In contrast, no human anti-HMW-MAA mAbs have been described. Because the latter may serve as useful probes to characterize the antigenic profile of the HMW-MAA, human anti-HMW-MAA single-chain fragments of the variable region (scFvs) were isolated by panning synthetic scFv library 1 on purified HMW-MAA. Colony hybridization studies and nucleotide sequence analysis revealed that scFv 19, 44, 56, and 61 belong to the V(H)3 gene family and use the DP-38 germ-line gene segment but have a diverse third complementarity-determining region. The human scFvs share some characteristics with mouse anti-HMW-MAA mAb but also display some distinct features. Like mouse mAbs, human scFvs recognize determinants of HMW-MAA with a heterogeneous cellular and molecular distribution in human melanoma cells. Furthermore, like some mouse mAbs, human scFvs react with rat neural cells expressing the chondroitin sulfate proteoglycan NG2, which shows 81% homology to the HMW-MAA. However, at variance with mouse mAbs, the human scFvs show poor reactivity with guinea pig melanoma cells. Lastly, human scFv 61 stains both benign and malignant lesions of melanocytic origin, although with a lower frequency than mouse mAbs. Analysis of the clinical significance of the differential expression of the scFv 61-defined determinant in melanoma lesions will be facilitated by its reactivity with formalin-fixed melanoma lesions. In contrast to mouse mAb, scFv 61 immunoprecipitates the >450-kDa chondroitin sulfate proteoglycan component of the HMW-MAA, but not its 250-kDa subunit from melanoma cells. Thus, contrary to the current view about the structure of HMW-MAA, our results demonstrate that the two components are not associated. The described scFv antibodies, which represent the first example of human anti-HMW-MAA antibodies, have provided novel information about the structure of this antigen. Future studies will assess the impact of these in vitro-assembled antibody fragments on the identification of antigenic determinants of the HMW-MAA that can be recognized by the human immune system.