Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders

Abstract

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K_d = 6-9 nM).

Keywords: Gp2 proteins enhance; PD-L1 binders; combinatorial libraries; sitewise amino acid constraint.

Figure 1. Gp2 protein scaffold structure and second generation library designs

(a) Cartoon ribbon structure and amino acid sequence of the Gp2 scaffold (PDB ID: 2WNM). The paratope region (red) is allowed to remain wild-type length or extend by one or two amino acids. A region near paratope loop two that previously displayed higher than expected mutation rates is shown in green. (b) Combinatorial library design: codon or amino acid diversity for each site in each library. cdr+ represents base antibody CDR diversity and cdr− represents the closest match to that diversity while removing cysteine (and thus arginine, glycine, and tryptophan due to degenerate codon limitations, except when glycine is added on a separate codon (cdr− +G)). Single amino acid abbreviations are used for diversity at constrained sites, at approximately equal frequency except where denoted by underline (higher) or double underline (highest). Full details in Supplemental Tables 1 and 2.

Figure 2. Potential of disulfide bonded cysteines in first generation Gp2

Observed frequencies of cysteines in evolved binders at indicated positions were multiplied to calculate predicted frequency (i.e. pairwise frequency if independent). The predicted frequency was compared to the actual pairwise frequency and the absolute difference and calculated mutual information are shown. High mutual information indicates synergistic interaction between the particular pair of cysteines. The sites with the top three values for difference and mutual information are shown in cartoon structure (PDB ID: 2WNM).

Figure 3. Sitewise analysis of Gp2 domain

(a) Sitewise enrichment or depletion of each amino acid from initial naïve library to evolved binding sequences isolated from the first generation Gp2 library. 3 × 10⁴ binding sequences against multiple epitopes on eight targets were aligned, analyzed for sitewise amino acid frequencies, and compared to the unsorted naïve library. Sites with more than a 5% change are labeled. (b) Sitewise amino acid frequencies in natural homologs to Gp2. Thirty-two Gp2 homologs were identified, aligned, and analyzed. Amino acids with above 5% prevalence are labeled. (c) Computationally estimated change in folding energy. The (de)stabilization (ΔΔG_folding in kcal/mol) upon mutation of the indicated site to the indicated amino acid was computed in FoldX for at least 40 random paratopes in 4 wild-type structures. Positive values represent less stable folds. (d) Sitewise solvent accessible surface area of wildtype Gp2. Values calculated from the GetArea webserver (2WNM) or the Accessible Surface Area tool from Center for Information Biology at Ochanomizu University (2WNM, 2LMC, 4LK0, 4LLG) for 4 protein data bank files were averaged. Area is normalized to surface area for each amino acid in the unfolded state and compared to the median accessibility (0.31): area(site i) – area(median). Diversified paratope is highlighted in red. The ₃₀EWQ₃₂ region, which is diversified in a sublibrary, is highlighted in green.

Figure 4. Conserved cysteine pairs impact Gp2 library efficacy differently depending on location

Cysteines were conserved at sites 7 and 12 (Intra 1), sites 36 and 39 (Intra 2) and sites 8 and 36b (Inter). Sequence frequency change from each library before (white) and after (grey) evolution were compared to the cysteine-free library (CDR−).

Figure 5. Allowance of C, G, R, and W aids Gp2 library efficacy in loop 1 and hinders efficacy in loop 2

Libraries either had high diversity at each paratope site based on the antibody CDR (base) or had multiple sites constrained to lower diversity (constrained). The evaluation of a cysteine-free library (and limitations of degenerate nucleotides) resulted in libraries that contained (+) or lacked (−) the amino acids C, G, R, and W at certain sites. Change in sequence frequencies from before (white) and after (grey) evolution were compared between libraries.

Figure 6. A particular sitewise amino acid constraint hinders Gp2 binder discovery

Enrichment in sequence frequency from the combined naïve library to evolved binders is shown for libraries where nine paratope sites were constrained (red) based on first generation binder and natural homolog sequences, computational stability, and solvent accessibility, or where these sites were left as full diversity CDR (white with black lines). The CDR diversity was either cysteine-free (CDR−) or contained all amino acids (CDR+).

Figure 7. Extending the loop two paratope region is detrimental to Gp2 binder discovery

A three amino acid region (₃₀EWQ₃₂) near loop 2 that showed high mutation rates in the first generation library was diversified to six amino acids at each site, including wild type. The other sites in this extended paratope library were diversified identical to the cysteine-free, sitewise constrained library. The change in sequence frequency before (white) and after (grey) evolution was compared to the non-extended paratope, cysteine-free, sitewise constrained library.

Figure 8. PD-L1 binding Gp2 protein characterization

(a) The sequences of PD-L1 binding Gp2 domains recovered from library sorting in which blue lettering indicates the diversified paratope region. Clones C and G were unable to be produced in the soluble fraction. (b) The produced clones were used to label CHO-hPD-L1 cells at a medium (150 nM, gray) and high (3 μM, black concentration. Binding was detected using a fluorescently tagged anti-His₆ antibody with flow cytometry. Binding is visible at 3 μM but nearly undetectable above background at 150 nM. Theoretical affinity curves were fit using data from strong PD-L1 binding Gp2 clones to determine the maximum fraction bound. Proteins were also used to label untransfected CHO-K1 cells as a negative control and showed much lower signal than on transfected cells (white).

Figure 9. Deep sequencing of affinity matured PD-L1 binding Gp2 domains

Gp2 domains that detectably bound 50 nM PD-L1 during yeast flow cytometry were grouped based on uniqueness and their frequency was quad-root damped. Observed percentages of each amino acid at each site are shown. Amino acids allowed in the initial library at that site are outlined. The parental amino acid has thick outlines.

Figure 10. Characterization of strongest binding evolved and parental PD-L1 binding Gp2

A) Clones E1 and E4 were produced at measurable levels in the soluble fraction using E. coli. Their binding affinity (K_D) was determined with n=3 trials (90% confidence interval indicated); N.E. indicates not expressed. (B) Purified Gp2-PDL1_E4 was added at the indicated concentrations to CHO cells (transfected for PD-L1 expression: filled circles; untransfected: empty triangle). Binding was detected by fluorophore tagged anti-His₆ antibody via flow cytometry. The best-fit estimate of the K_d (6 nM) and the 90% confidence interval (3 – 13 nM) are indicated by solid and dashed lines, respectively. (C) CHO cells transfected for PD-L1 expression were treated, at 4 °C, with 300 nM Gp2-PDL1_E4 in the absence or presence of initial treatment with 67 nM atezolizumab. Binding was detected by fluorophore tagged anti-His₆ antibody via flow cytometry.