Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes

Abstract

We have combined phage display and array screening for the rapid isolation of pairs of interacting polypeptides. Our strategy, named SAC (selection by avidity capture), is based on the avidity effect, the formation of highly stable complexes formed by multivalent interactions; in our case, between a receptor (multivalently displayed on phage) and a ligand (coexpressed as a multimeric fusion protein). Capture of the long-lived interaction complex allows the isolation of phage bearing cognate interaction pairs, as we demonstrate for a range of interactions, including Ab-antigen pairs and the rapamycin-dependent interaction of FKBP-12 and FRAP. Cognate phage are enriched by SAC up to 1000-fold and interacting pairs can be identified by array screening. Application of SAC to Ab-antigen interactions as a model system yielded over 140 specific Abs to a single antigen and 92 Abs to three different fetal human brain antigens in a single round of SAC each. Our results suggest that SAC should prove useful for the identification and study of receptor-ligand interactions in particular among extracellular proteins, as well as for the rapid generation of specific Abs to multiple antigens.

Figure 1

General scheme of SAC. (1) A receptor polypeptide fused to g3p and a ligand polypeptide (fused to a multimerizing domain, e.g., GST) are coexpressed in the same cell and exported to the periplasm, where (2) they associate to form a multivalent high-avidity complex that is incorporated into nascent phage particles (3). Phages bearing a cognate interaction complex are captured on a solid support by way of the GST domain. Because both plasmids contain an f1 packaging origin, both are packaged into phage particles (4). Selected phage are plated for double antibiotic resistance and arrayed. Receptor and ligand proteins are coexpressed and cognate pairs detected (e.g., by capture of the GST-ligand fusion protein with an anti-GST IgG and detection of cognate receptor binding with an antireceptor-horseradish peroxidase conjugate).

Figure 2

Effect of multivalency. The cognate receptor–ligand pair (scFv anti-BSA 13CG2 × GST-protein L B1 domain) is rescued with two different helper phages. ELISA signal is plotted against phage titer. Phage are diluted stepwise by a factor of 2, starting from 10¹² colony-forming units (cfu) per ml (R408, filled circles) and 10¹¹ cfu/ml (R408Δg3p, open squares). Phages rescued with R408 are predominantly monovalent, leading to inefficient avidity complex formation. Phages rescued with R408Δg3p are multivalent and avidity complexes are readily formed.

Figure 3

Interaction matrix ELISA of avidity complexes on phage. Receptors and K_D of cognate interactions row A, Fab 9E10 [anti-c-myc, K_D = 80 nM (21)]; row B, FKBT-12 [K_{D(FKBP-rapamycin, FRAP)} = 2 nM (24)]; row C, FRAP (see B); row D, scFv M12 [anti-M, K_D = 21 nM (13)]; row E, scFv T14 [anti-T, K_D = 5 μM (this work)]; row F, scFv D4 [anti-D (not determined)]; row G, c-Abl SH3, [we tested binding to two proline-rich target peptides, p41, K_D(p41) = 1.5 μM, and 3BP1, K_D(3BP1) = 35 μM (39)]; row H, scFv Mab32 [anti-huTNFα, K_D = 26 nM (14)] are combined with ligands column 1, GST-c-myc; column 2, GST-FRAP; column 3, GST-FKBP-12; column 4, GST-M; column 5, GST-T; column 6, GST-D; column 7, GST-41p and GST-3BP1 peptide (shaded); column 8, anti-hen egg lysozyme (HEL) FvD1.3-huTNFα fusion protein (22); column 9, anti-HEL diabody HyHEL10/5-c-myc (40); column 10: GST-protein L B1 domain K_D(huVk1) = 130 nM (27)]. SAC phage were rescued and all combinations assayed by ELISA [with either anti-GST IgG (columns 1–7 and 10) or HEL (columns 8 and 9) for capture of phage bearing avidity complexes].

Figure 4

Stability of avidity complexes on phage. Dependence of avidity complex formation between FKBP-12 on phage and GST-FRAP on the extraneous mediator rapamycin and avidity complex stability is assayed by ELISA. Phage are rescued in the presence (+) or absence (−) of rapamycin, or are precipitated before the addition of rapamycin (PEG/+) and captured with anti-GST IgG. Avidity complex formation strictly depends on rapamycin. Once formed, FKBP-12 × GST-FRAP avidity complexes on phage are stable to repeated precipitation with PEG.

Figure 5

Results of SAC selection. Array screen of 3,072 double-spotted clones before and after one round of SAC selection. Clones were arrayed in a 4 × 4 pattern (see expanded image). (A and B) Screening for cognate binding pairs before (A) and after (B) selection. Cognate interaction pairs are captured with anti-GST IgG and detected with protein L-horseradish peroxidase. Selected clones are circled. Positive controls GST-M × anti-M scFv M12 and GST-T × anti-T scFv T4 are boxed. (C) Direct screen for antigen M-specific binders after selection.