As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.In the human scalp there is a very complex balance among many microorganisms. One of these organisms, Malassezia furfur, is an anthropophilic fungus that belongs to the physiological skin flora. Increased turnover of M. furfur has been implicated in the formation of dandruff (6,11,38,42) and other skin diseases, such as psoriasis (1). Significantly, more M. furfur was found on the scalps of people with dandruff than on the scalps of people without dandruff (34,39). Reductions in the numbers of M. furfur cells on the scalps of dandruff sufferers resulted in a decrease in the severity of the dandruff (32).To date, the treatment and/or prevention of dandruff has involved the use of chemical antifungal compounds in shampoos (23), compounds such as ketoconazole (33), selenium sulfide (6), cyclopyrox olamine, piroctone olamine, zinc pyrithione, and sulfur-containing substances (38).Here we describe a novel approach for preventing the formation of dandruff by inhibition of M. furfur with antibodies. For successful use of antibodies in consumer goods they must meet certain requirements regarding cost of production, specificity, affinity, and especially stability under application conditions.Camelid heavy-chain antibodies have been shown to have great potential in many biotechnological applications (9, 13, 25, 43) because of their unique characteristics involving production, folding, and stability (12, 30). They lack light chains, and therefore the variable domain of the heavy chain (VHH) is the single binding domain (14). The simple, one-domain structure of these VHHs give them unique characteristics, but they have properties with regard to specificity and affinity that are similar to the properties of conventional antibodies (41).Furthermore, the extralong protruding third binding loop (CDR3) of VHHs is considered an advantage for efficient inhibition of enzymes and...

We report for the ¢rst time the a⁄nity maturation of Fab antibody fragments using £uorescent-activated cell sorting (FACS) of yeast-displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies speci¢c for di¡erent protein targets has been demonstrated by £ow cytometry and immuno£uorescence microscopy. We have a⁄nity matured a Fab antibody speci¢c for the tetravalent antigen streptavidin using FACS of yeastdisplayed repertoires diversi¢ed by error-prone polymerase chain reaction. A panel of variants with up to 10.7-fold improvement in a⁄nity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC-CDR1 (H34R) and LC-CDR3 (Y96H or Y96F) and gave a 10.7-fold and 6.3-fold a⁄nity improvement over the starting antibody, respectively. The ability to e⁄ciently a⁄nity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an e⁄cient method for this purpose.