Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

Abstract

The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer's disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer's and related neurodegenerative diseases.

Keywords: Alzheimer; antibody; drug development; inhibitor; self-assembly.

Fig. 1.

Schematic outline of the phage display selection strategy. (Step 1) Monomer-binding members of the libraries were captured by Aβ42 monomers on magnetic beads that were removed using a magnet (negative selection, A). Ca. 10¹³ virions (16 pmol) were added to 100 pmol of monomers (blue line) on beads, thus providing capacity to bind all phages displaying scFvs with high affinity for monomers. (Step 2) The solutions with unbound phages were added to magnetic beads with Aβ42 fibrils to capture phages with fibril-binding scFvs (positive selection, B), followed by extensive washing to discard nonbinding members. Fibril-bound phages were eluted with acid, neutralized, and used to infect E. coli to produce an enriched scFv phage display library. Step 3 = step 1, and step 4 = step 2, except that the enriched libraries from step 2 were used. Step 5 = step 1, and step 6 = step 2, except that the enriched libraries from step 4 were used. (Step 7) Fibril-bound phages were eluted with acid, neutralized, and used to infect E. coli, spread on LB agar plates and single clones picked for production of phages with displayed scFvs. The medium after removal of E. coli cells by centrifugation was used in SPR experiments for ranking their affinity for Aβ42 fibrils immobilized on sensor chips (C). (Step 8) The strongest binding candidates were used to infect E. coli to produce isolated scFvs for more detailed SPR analysis and Aβ42 aggregation kinetics (D).

Fig. 2.

Simulated kinetic profiles in the presence of inhibitors of discrete microscopic steps in protein aggregation reactions. (Top) Microscopic processes included in the reaction scheme. The AmyloFit interface (28) was used to simulate the changes in the time evolution of the fibril mass upon selective reduction of (A) the rate constants of primary nucleation (k_n), (B) the rate constants of secondary nucleation (k₂), and (C) the rate constants of elongation (k₊). In each case, the remaining two rate constants were fixed at the values obtained for Aβ42 in 20 mM sodium phosphate buffer with 0.2 mM EDTA, pH 8.0, 37 °C. The reference (black) curves in each panel were calculated using the rate constants (20) measured for Aβ42 alone in the absence of inhibitor under the same conditions: k_n = 3 × 10⁻¹ M^-1·s⁻¹, k₂ = 1 × 10⁴ M⁻¹·s⁻¹, k₊ = 3 × 10⁶ M⁻¹·s⁻¹. All calculations assumed that the reaction was initiated at time 0 from a solution containing 3 µM Aβ42 monomer. The color codes and numbers refer to the fold-reduction of the selected microscopic rate constant (e.g., 10 indicates that the rate constant is reduced 10-fold). The rate of formation of nuclei by primary and secondary processes was calculated for the cases of selective reduction of (D) k_n, (E) k₂, and (F) k₊.

Fig. 3.

Kinetic analyses of the effects of scFvs that were found to inhibit selectively secondary nucleation. The aggregation kinetics starting from 3 µM Aβ42 monomer in the absence (gray) and presence of I48 (A), I2 (B), J44 (C), and J57 (D) at concentrations ranging from 0.3 to 3.0 µM with color code shown as an Inset in B. The fitted curves shown here were generated by the AmyloFit interface (28) keeping k₊ and k_n the same for all scFv concentrations, whereas the rate constant k₂ was allowed to take specific values at the different scFv concentrations. (E and F) The relative change in k₂ obtained from the fits in A–D, with linear and logarithmic y axis, respectively. The solid lines show fitted curves using an equation for competitive binding of the scFvs and the Aβ42 monomers for the fibril surface as described in SI Appendix. (G and H) Cryo-TEM images of Aβ42 fibrils formed in the absence (G) and presence (H) of scFv J57.

Fig. 4.

Kinetic analyses to discard scFvs that affect other processes in addition to secondary nucleation. The aggregation kinetics of 3 µM solutions of Aβ42 monomers in the absence (black) and presence (colors) of scFvs I68 (A), J7 (B), and J46 (C) and two control scFvs selected randomly from the I and J libraries (D and E) at 0.3–3.0 µM. The fitted curves in A–C were generated by fixing k_n and k₊, allowing k₂ to have different values at each scFv concentration. Note that the data for these three scFvs cannot be fitted on this assumption nor on the assumption of a selective reduction of k_n, making it likely that they also inhibit elongation.

Fig. 5.

Seeded aggregation kinetics. Shown are the aggregation kinetics of solutions of 3 µM Aβ42 monomer in the absence (E) or presence of 2 µM scFv I2 (A), I48 (B), J44 (C), and J57 (D) supplemented with no (black), 30 nM (cyan), 90 nM (green), 300 nM (yellow), or 900 nM (red) seeds at time 0. The initial slopes in the presence of 30% (900 nM) seed fibrils (F) were fitted using straight lines. (G) The obtained elongation rate constant, k₊, relative to the case of no seed is shown as bars, and the rate constant for secondary nucleation, k₂, relative to the case of no seed is shown as bars in lighter colors.

Fig. 6.

The scFv structure and amino acid compositions. (A) Space-filling model of an IgG (molecular mass,150 kDa; PDB ID code 1IGY) with the two heavy chains in blue, the two light chains in red, and the residues in the CDR regions in green. (B and C) An scFv (molecular mass, 27 kDa; PDB ID code 5DFW) including the variable domain of one heavy chain (V_H) and one light chain (V_L) . In C, the scFv is rotated to get a better view of the CDRs, and the 18 residues that are varied in CDR2 and CDR3 of V_H and V_L in the Tomlinson libraries are shown in darker green. (D and E) Result of DNA sequencing of the four scFvs that inhibit secondary nucleation (I2, I48, J44, and J57). For each library (I and J), the observed average frequency of each amino acid in the 18 diversified positions in the selected clones (green bars) is compared with the average frequency in the libraries (gray bars). Green asterisks indicate enrichment after selection.