Rapid discovery and optimization of therapeutic antibodies against emerging infectious diseases

Abstract

Using a comprehensive set of discovery and optimization tools, antibodies were produced with the ability to neutralize SARS coronavirus (SARS-CoV) infection in Vero E6 cells and in animal models. These anti-SARS antibodies were discovered using a novel DNA display method, which can identify new antibodies within days. Once neutralizing antibodies were identified, a comprehensive and effective means of converting the mouse sequences to human frameworks was accomplished using HuFR (human framework reassembly) technology. The best variant (61G4) from this screen showed a 3.5-4-fold improvement in neutralization of SARS-CoV infection in vitro. Finally, using a complete site-saturation mutagenesis methodology focused on the CDR (complementarity determining regions), a single point mutation (51E7) was identified that improved the 80% plaque reduction neutralization of the virus by greater than 8-fold. These discovery and evolution strategies can be applied to any emerging pathogen or toxin where a causative agent is known.

Fig. 1

Schematic of DNA display. A Fab library is constructed using a single vector containing a Fab light chain and the heavy chain cloned in-frame with a zinc finger DNA binding protein. The vector also contains the DNA binding site for the zinc finger. The library is transformed into a Rosettagami E coli host and the Fab-zinc finger fusion protein is produced. It binds to the appropriate encoding plasmid, the cells are lysed, and the library screened against an antigen bound to magnetic Dynal beads. Beads containing bound Fab are magnetically separated from unbound Fab and plasmids containing functional Fabs are eluted from the beads.

Fig. 2

Representative ELISA data of SARS-CoV-reactive Fabs isolated by DNA display. (A) Zinc finger-Fab fusion proteins analyzed in an ELISA using the spike protein as a capture reagent on 48 wells of a 96-well Maxisorp plates. Bovine serum albumin coated on the remaining 48 wells was used to determine specificity of binding. (B) Relative specific activity is the functional activity from Fig. 2A normalized to the amount of fusion protein determined using an ELISA measuring relative expression levels.

Fig. 3

Binding of the spike protein–antibody mixture to Vero E6 cells. Vero E6 cells were analyzed by flow cytometry using a bandpass filter of 580/30 with collection of 10 000 cells. (A) Cells incubated with streptavidin–phycoerythrin only, (B) cells incubated with 4 nM spike protein and a bacterial lysate from cells expressing an irrelevant antibody, (C) same as (B) but with an anti-spike antibody that does not block binding, (D–E) same as (B) but with three unique anti-spike antibodies that block binding of spike to its receptor. All antibodies were added at a 12 nM concentration with the exception of (D) which was at 8 nM. Listed % represents the % of cells following in the defined gate.

Fig. 4

Functional activity of human framework reassembly (HuFR™) antibody variants. (A) The top 10 antibody variants from the heavy chain library as determined by functional spike ELISA normalized to the relative expression of antibody variant. The specific activity for each antibody was further normalized to the wild-type (WT) control antibody (i.e. WT: chimeric antibody, 4049Fab14). (B) The top 10 antibody variants from the light chain library determined as described in (A). Numbers within bars indicate the corresponding heavy chains. (C) Purified antibody candidates were tested in the plaque reduction neutralization test (PRNT). The number of plaques resulting in 50 and 80% neutralization is noted. Statistical analysis at the approximate WT antibody concentration for 80% neutralization (0.78 µg/ml) indicates better neutralization (i.e. fewer plaques) for 61G4 (P < 0.04). Duplicates of each variant were assayed in the ELISA and PRNT experiments.

Fig. 5

Functional activity of GSSM™ and reassembled combination antibody variants. (A) The top 10 antibody variants from the GSSM™ as determined by functional spike ELISA normalized to the relative expression of the antibody variant. The specific activity for each antibody was normalized to the wild-type (WT) control antibody (WT: chimeric antibody, 4049Fab14). (B) Purified antibody candidates from (A) tested in the plaque reduction neutralization test (PRNT). The number of plaques resulting in 50 and 80% neutralization is noted. Statistical analysis at the approximate WT antibody concentration for 80% neutralization (0.78 µg/ml) indicates better neutralization (i.e. fewer plaques) for 51E7 and 52G3 (P < 0.02 and P < 0.03, respectively). (C) Top 10 antibody variants from the combination library (containing the best GSSM™ mutants placed in the best framework backbones) determined as described in (A). (D) Purified antibody candidates from (C) tested in the PRNT. Data was not collected for one of the 10 variants. Statistical analysis at the WT antibody concentration for 80% neutralization (1.56 µg/ml) indicates better neutralization (i.e. fewer plaques) for several antibodies (i.e. P < 0.02 for 2978/15, 2992/15, 2978/10, 2702/10; P < 0.03 for 2703/10; P < 0.04 for 2703/15 and P < 0.05 for 2699/10). Duplicates of each variant were assayed in the ELISA and PRNT experiments.