The ability of cross-reactive antibodies to bind multiple related or unrelated targets derived from different species provides not only superior therapeutic efficacy but also a better assessment of treatment toxicity, thereby facilitating the transition from preclinical models to human clinical studies. This chapter provides some guidelines for the directed evolution of cross-reactive antibodies using yeast surface display technology. Cross-reactive antibodies are initially isolated from a naïve library by combining highly avid magnetic bead separations followed by multiple cycles of flow cytometry sorting. Once initial cross-reactive clones are identified, sequential rounds of mutagenesis and two-pressure selection strategies are applied to engineer cross-reactive antibodies with improved affinity and yet retained or superior cross-reactivity.

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique\'s utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme\'s specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.