Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

Abstract

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...

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

“…Camelid VHH is famous for its high thermodynamic stability (25)(26)(27). Unique applications are proposed to utilize this high stability of VHH, such as detection of caffeine in hot beverages (28) and prevention of dandruff by VHH mixed with shampoo containing a high concentration of surfactant (29). Stabilization by the engineered disulfide bond reinforces the utility of VHHs.…”

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

“…The VHH domain can be easily cloned and expressed to high levels in bacteria and yeast (26,27). This notion, together with advantageous characteristics in terms of stability and solubility (20,64,81), has led to successful development of camelid VHH in a number of applications against a range of biological targets (2,13,14,19,21,57,58,69,83,84), including neutralization of rotavirus (28,60). We hypothesized that the small size of VHH in combination with their protruding CDR3 loops and their preference for cleft recognition may allow them to recognize conserved motifs on gp120 that are occluded from conventional antibodies.…”

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

“…These VHH are the smallest naturally occurring intact soluble antigen-binding units with a molecular weight of about B15 kDa. 16 VHH are stable at high temperatures, 19,20 can bind antigen in high salt concentrations, 21,22 cripple intracellular viral replication, 23 block enzymatic activity, 24 are able to pass the blood-brain barrier, 25 and can be used for various immunological applications like classical antibodies. We have selected a nonimmune VHH phage display library against six different recombinant HIF-1a protein fragments.…”