New classes of therapeutic proteins under development

T cells play a central role in cell-mediated immunity; however, the molecular mechanism underlying TCR recognition was not well understood until the late 1980s. Unlike antibodies that can recognize pathogens or their toxins directly, T cells recognize only short peptides derived from pathogens in complex with MHC molecules on the surface of antigen presenting cells (APCs) through their TCRs. The nature of the interaction between TCRs and peptide-MHC (pMHC) complexes determines the function of the induced cellular immune responses. Therefore, both TCRs and MHC molecules can be potentially used in protein therapeutics (see the section titled "Targets of Protein Therapeutics and Modes of Action," in this chapter).

Major Histocompatibility Complex (MHC) Proteins

Malfunction of the MHC system, consisting of class I (MHCI) and class II (MHCII) MHC proteins, has been implicated in many diseases, such as malaria, rheumatoid arthritis, type-I diabetes, and graft rejection. This has spurred great interest in developing MHC-based immunotherapeutics and immunodiagnostics methods. The development of pMHC tetramer, a multimeric form of peptide-MHC complexes, has revolutionized the field of T cell research. It enables direct detection and identification of antigen-specific T cells, modulation of T cell responses in vivo to treat graft rejection and autoimmune diseases, and detailed monitoring of cellular immune responses induced by immunotherapy, which is critical for a better understanding of tumor immunology and improved immune-based therapies. However, there are some limitations of pMHC tetramers that stem from their difficult recombinant production and the low affinity of the pMHC monomer. Therefore, it is highly desirable to engineer MHC molecules with improved solubility or higher TCR-binding affinity.

Phage Display

Phage display of MHCs is challenging, because MHC molecules are large, heterodi-meric membrane proteins with high glycosylation and multiple disulfide bonds. In addition, in the absence of their transmembrane domains, the a and p polypeptide chains are unable to assemble properly and tend to aggregate. To date, there are only three successful examples of MHCI phage display, with the first one reported in 2000 (Le Doussal et al. 2000; Vest Hansen et al. 2001; Kurokawa et al. 2002). Although the displayed pMHC complexes were correctly folded and capable of binding specific antigenic peptides, no significant interaction with relevant T cells was detected. Therefore, it is necessary to further optimize and develop novel design of the pMHC phage display system for efficient and stable T cell recognition.

Cell surface Display

Compared to phage display, yeast surface display is more effective for the display of MHC proteins and has been used to express both MHCI (Brophy et al. 2003; Jones et al. 2006) and MHCII (Starwalt et al. 2003; Esteban and Zhao 2004; Boder et al. 2005; Wen et al. 2008) proteins. Functional display of a mutant single-chain murine

MHCI protein was evidenced not only by recognition of conformation-specific antibodies but also by direct binding of a specific TCR that has been engineered to have high affinity (see the section titled "T Cell Receptors (TCRs)," in this chapter) (Brophy et al. 2003). More significantly, yeast cells displaying pMHC complexes upregulated the surface expression level of an early activation marker on naive T cells isolated from mice. Although the authors did not rule out the possibility of T cell autostimulation, this study clearly suggested that yeast display could be used for directed evolution of pMHC complexes. Indeed, the same group later successfully isolated stabilized mutants of a single-chain murine MHCII protein (which is known to be unstable and difficult to work with) from either a focused library created by site-directed mutagenesis or a library created by random mutagenesis (Starwalt et al. 2003). In a similar study, mutants of a single-chain human MHCII protein without a covalently attached peptide were stably displayed on yeast cell surface (Esteban and Zhao 2004). These MHCII proteins exhibited specific and fast peptide-binding kinetics, as well as high thermostability. Interestingly, although the single-chain gene construct did not include any peptide, the peptide-binding groove of the displayed MHCII mutants was not empty, but occupied presumably by yeast endogenous peptides, indicating the importance of binding peptides in stabilizing MHCII expression. By incorporating an MHCII-binding peptide in the single-chain construct, the wild-type pMHC complex was functionally displayed on yeast cell surface without introduction of any mutations and capable of activating immobilized hybridoma T cells (Wen et al. 2008). More importantly, the authors demonstrated that yeast display could be used in combination with expression cloning to identify T cell epitopes from a pathogen-derived peptide library.

Recently, baculovirus-infected insect cells have also been used for MHC display (Crawford et al. 2004; Wang et al. 2005; Crawford et al. 2006). As higher-order eukaryotic cells, insect cells have fewer problems with MHC expression compared to yeast. So far, baculovirus display of MHC has been used only to identify T cell epitopes/mimotopes from peptide libraries. Nevertheless, the ability of insect cell-displayed pMHC complexes to directly activate relevant T cells (Crawford et al. 2004) makes this system a promising platform for pMHC engineering.

T Cell Receptors (TCRs)

TCRs are a pivotal element in almost every aspect of T lymphocytes, including their development, proliferation, differentiation, activity, and specificity. Therefore, researchers are exploring the potential of soluble TCRs to be used as immuno-therapeutic or immunodiagnostic reagents to specifically target the pMHC complex (Miles et al. 2006), just as antibodies are used to neutralize or opsonize their antigens. However, there are several obstacles impeding therapeutic applications of TCRs, including difficult recombinant production, instability, and low pMHC binding affinity. Therefore, efforts have been devoted to the engineering and design of soluble, stable, and high-affinity TCRs.

Protein Fusions and Rational Design

Until recently, there was no generally applicable method of producing soluble TCRs (Molloy et al. 2005). Many of the initial strategies, such as removing exposed hydrophobic residues, or fusion to antibody constant regions or thioredoxin (Andrews et al. 1996; Shusta et al. 1999), worked for a very limited number of TCRs. Later, Jun/Fos leucine zipper domains were introduced as fusions to the C-termini of the a/p TCR extracellular domains, respectively (Willcox et al. 1999). The incorporation of leucine zipper domains significantly stabilized the TCR, while maintaining its ligand specificity; however, it raised the potential of immunogenicity. Another generally applicable method involved introducing a non-native interchain disulfide bond, predicted by molecular modeling based on the TCR crystal structure, in the TCR invariant region (Boulter et al. 2003). The resulting disulfide-stabilized TCR (dsTCR) was highly stable, and the sequence/structural change was minimal compared to wildtype TCR, reducing its possibility of being immunogenic. More importantly, the dsTCR construct enabled phage display (Figure 7.3A), which represented a powerful directed evolution platform for TCR engineering (Li et al. 2005). By using the dsTCR format, ten different human class I- and class Il-restricted TCRs were successfully displayed on phage particles (Li et al. 2005).

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