A single donor is sufficient to produce a highly functional in vitro antibody library

Abstract

Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 10⁹ human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.

Fig. 1. Process flow for B-cell purification, V-region amplification, and cloning into pDAN5 phage display vector.

a Schematics of B-cell purification of a single donor repertoire from LeukoPak. b FACS characterization of the CD19⁺ paramagnetically isolated cells for CD37⁺ (left) and CD20⁺ (right) staining. c Rescue of V domain diversity from donor and creation of the scFv phage antibody library: specific constant domain primers are used for the RT-reaction, followed by V domain amplification with IGKV/IGLV and IGHV-specific forward and reverse primers (PCR1). A second PCR introduces vector and linker overlaps (PCR2) that are exploited for the assembly of the scFv genes (PCR3). The genes are cloned into the pDAN5 phagemid vector by restriction enzyme digestion and transformed into bacteria. Upon superinfection with M13K07 helper phage, the phage is produced (primary library) and used to infect the recombinase positive bacteria (Cre+) at a multiplicity of infection of 1:200. The presence of incompatible lox sites between IGKV/IGLV and IGHV domains and downstream g3p (in black and white) allows recombination to occur between different VH and VL genes carried by different phagemid vectors. The process yields a phage population (secondary library) that has greater recombinatorial diversity than the primary library. A final phage production step, at a multiplicity of infection ≤1 restores the essential phenotype/genotype coupling (tertiary library/final library).

Fig. 2. Heavy and light chain diversity accumulation curves.

a Species accumulation curves for HCDR3 at varying hamming distance criteria reveal continued accumulation at all edit string distance cutoffs (Hamming 0–3) and using accumulation cutoffs (N ≥ 2). b Species accumulation curves of light chain CDR3 at varying hamming distance criteria reveal saturation of diversity around 7–8 × 10⁵ for the combined heavy and light chain for hamming distances 1–3 and for abundance cutoffs of N ≥ 2.

Fig. 3. Relative cluster of primer sets corresponding to germline sequences for insight into off-target.

a Unsupervised hierarchical clustering of 18 bp region at 5′ portion of the V-region shows three predominant clusters that include many cross-family members. b Analysis of the off-target gene-family amplification for IGHV1a as an example of the primer specificity for the IGHV1 family. c Analysis of the off-target gene-family amplification for IGHV2a as an example of the primer specificity for the IGHV2 family. d Analysis of the off-target gene-family amplification of IGHV3a to represent the primer specificity for the IGHV3 family. e Analysis of the off-target amplification for IGHV4. f Analysis of the off-target gene-family amplification of IGHV5a to represent the primer specificity for IGHV5. g Analysis of the off-target amplification for IGHV6a.

Fig. 4. Primer pair clonal dominance, overlap and relative diversity, and V-region distribution of heavy and light chain in the final library.

a Cross-family comparison of clonotype dominance by individual primer sets. b Relative overlap of the HCDR3 among IGHV1–5 primer sets. c HCDR3 clonal dominance, d Cumulative clonal rank of HCDR3 assessing % representation by different copies of individual clones (e.g., 1 singletons, 2 duplicates). e LCDR3 cumulative clonal rank of the (IGKV or IGLV) LCDR3. f LCDR3 clonal dominance of the IGKV or IGLV library. Scaffold representation and Tiller’s developability criteria analysis for the f IGHV domains, g IGKV domains, and h IGLV domains.

Fig. 5. Biophysical characteristics in the final library show a broad distribution of binding features.

a Circos graph of V/J coupling in the heavy chain. b Circos graph of V/J coupling within combined IGKV/IGLV library. c HCDR3 length distribution for different IGHV:IGHJ combinations. d Calculation of hydrophobicity index using GRAVY for different IGHV:IGHJ combinations.

Fig. 6. Phage and yeast selection against four therapeutically relevant targets shows highly developable candidates and biased biophysical profiles.

a Limited cross-reactivity of yeast scFv across the different panel of antigens at 200 nM. b Yeast scFv-display selection against a wide range 2–200 nM of antigen, shows minimal background (0 nM) with a noticeable population obtained from each selection campaign. c, d Pairwise Levenshtein distances distribution of the merged HCDR1–3 and HCDR3 amino acids. e, f HCDR3 and merged HCDR1–3 amino acid net charge tabulated using pH = 7 on Lehninger pK scale, showing the clear disparity of binders. g, h Gravy hydrophobicity index analysis of merged HCDR1–3 and HCDR3 amino acids. i HCDR3 length distribution across different antigens. j–m Analysis of the selected scaffold and their developability profile.

Fig. 7. Affinities and expression levels of clones selected against human CD73.

a expression level and affinity of human-specific IgG clones b expression level and affinity of IgG antibodies selected against CD73 recognizing both the human and the murine proteins.