Integrin and Antibody Engineering for Therapeutics

Integrin Engineering

As part of the innate immune response, activated phagocytes are recruited at the sites of inflammation to fight infection. The specific trafficking of these immune cells from the bloodstream heavily relies on cell surface adhesion molecules. For instance, the rolling and firm adhesion of leukocyte from the bloodstream require interactions of its surface molecules sialyl-Lewisx and integrin αLβ2 (lymphocyte function-associated antigen-1, LFA-1) against E-selectin and intercellular adhesion molecule-1 (ICAM-1) respectively on the vascular endothelial surface. Especially the importance of integrins in leukocyte functions has been illustrated by the lack of or abnormally sustained binding which are associated with leukocyte adhesion deficiency and various autoimmune-related diseases.

Integrins are noncovalently associated heterodimeric (α and β subunits) cell surface molecules showing immense diversity of 24 known pairs between 18 α and 8 β subunits. The adhesiveness of integrins is regulated through various cell signaling pathways termed inside-out signaling. Upon activation, integrins undergo a dramatic switchblade-like conformational change accompanied with opening of its subunit legs at the C-termini as seen in Figure 1. The conformational shift in turn remodels the ligand-binding site, reshaping the molecule ready for interaction with its ligands at unprecedented high-affinity states. Allosteric studies of one of the integrin binding domains, the inserted domain (I-domain) present in a subset of α subunits, has revealed that the molecule can bind to its physiologic ligand up to 200,000 times higher affinity than the inactive state.

Because of its value as a therapeutic target and the unique regulation of activation in integrins, we are interested in developing therapeutic antibodies that are specific only to the active conformations. Therapeutic antibodies that can specifically antagonize active integrins will have less side effects and longer half-lives. To be able to harness the binding domains in its active states, we employ two different protein engineering methods termed directed evolution and rational design. As for directed evolution, random mutagenesis PCR on the gene of interest, in vivo homologous recombination, and display of protein on yeast surface enable us to generate enough diversity and bridge between genotype and phenotype. Selective pressure against known antibodies or ligands directs the evolution of the diverse population towards the phenotypic properties of interest, that is allosterically activated ligand binding integrin domains. We are currently studying α4β1 (very late antigen-4, VLA-4) which lacks I-domain in the α subunit. Headpiece and head domains of the integrin are designed as a single chain on yeast surface, shown in Figure 2, to employ directed evolution.

Engineered ligand binding domains of the integrin VLA-4 will prove useful in many applications. The high affinity conformation of the engineered molecule will represent the active VLA-4 in leukocytes, providing the framework for the development of function-blocking antibodies. The comparison between the inactive or wild-type and active or high affinity VLA-4 ligand binding domains will render structural and mechanistic basis of the currently available antagonists. The expression of the head or headpiece VLA-4 integrin may also provide opportunities for crystallization.

Protein-protein Interactions

Protein-protein interactions are the fundamental events in cell signaling, and are the major therapeutic targets in many diseases. Various useful techniques exist for the study of protein-protein interactions such as the yeast two-hybrid system and fluorescence resonance energy transfer (FRET). However, it still remains difficult to apply the existing systems to protein engineering for the development of therapeutics and the study of protein allostery. Recently, we have developed the yeast surface two-hybrid (YS2H) system to study protein interactions in the secretory pathway in yeast, where a pair of proteins fold and associate in the process of transport from endoplasmic reticulum, Golgi, and to the cell surface. YS2H system express two proteins within the same cell using bidirectional GAL1/10 promoters as seen in Figure 3, one as a fusion to agglutinin for cell surface display and the other as a releasable form.

YS2H is an efficient platform for directed evolution and direct readout of protein-protein interaction to probe protein allostery. The wild-type of the ligand binding domain of αLβ2 integrin, I-domain, and the activation-specific ligand-mimetic scFv AL-57 are functionally expressed as surface displayed and releasable form respectively on YS2H system. Our findings demonstrate that directed evolution through random mutagenesis on αL I-domain enable efficient sorting of allosterically activated mutants such as the previously reported mutation F265S. Moreover, the selection and sorting was done by the C-terminal Myc tag of scFv AL-57 as schematically presented in Figure 4, circumventing the need of production and purification of the antibody and enabling effective screening with relatively more accessible antibodies such as 9E10 anti-Myc antibody. Current application of YS2H involves engineering scFv AL-57 to react cross-species to both human and murine αL I-domain and affinity maturation of alpaca light chain only antibody VHH-C3 against botulinum neurotoxin serotype B.

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