Generating Diverse CD80 Mutant Library

Yeast surface display libraries containing the randomly mutated IgV domain of human CD80 (residues 35-141) were generated by error-prone PCR as described^{21 (link)}. Multiple selection strategies were employed to generate a diverse set of candidates. All intermediate selection outputs were assayed for CD28, CTLA-4, and PD-L1 binding by flow cytometry. The pool of mutants from which the lead candidate domain (H2393) was isolated and evolved as follows. A randomly mutated library (2.7 × 10⁸ clones, avg 4.6 mutations/clone) was screened with 2 parallel flow sorting progressions: (A) 3 nM CTLA-4-Fc⁺ (produced at Alpine Immune Sciences) followed by two successive 300 nM CD28-Fc⁻ (R&D Systems) sorts and (B) two successive 300 nM CD28-Fc⁻ sorts followed by a 3 nM CTLA-4-Fc⁺ sort. Yeast plasmid DNA isolated from the A and B terminal sorts was pooled and used as template to build a subsequent error-prone PCR library. This second-round library was screened via the following sort progression: 50 nM PD-L1-Fc⁺ (R&D Systems) followed by 20 mM CTLA-4-Fc⁺, followed by 10 nM PD-L1-Fc⁺ followed by 25 nM CD28-Fc. Each sort step was followed by outgrowth in YPD media (Himedia) at 30 °C and expression induction in media derived from Benatuil et al. (2010), with the substitution of dextrose/galactose with dextrose only at 22 °C before progressing to the next sort.