Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

Abstract

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro,the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.

Figure 1.

LYNDAL antibody-pIII fusion protein expression. Induced periplasmic preparations of individual clones from 40 sublibraries were analyzed by (A) dot blot and, (B) western blot. (A) Dot blot analysis using the anti-c-myc-tag antibody 9E10 showed that 261/320 clones (82%) expressed detectable amounts of antibody-pIII fusion proteins (framed boxes). Negative control (NC) growth medium; positive control (PC) purified scFv (5 µg in 1:2 dilutions). (B) Immunoblotting with monoclonal anti-pIII antibody of 30 random clones with detectable protein expression in dot blot revealed for all clones with exception of clones 10, 14 and 22 (90%) protein bands migrating at an apparent molecular weight of approximately 80 kDa corresponding to scFv-pIII fusion proteins. Size of molecular weight markers are indicated (kDa).

Figure 2.

Phylogenetic analysis of LYNDAL sequences. Germline sequences of 280 randomly sequenced clones containing verified scFv genes were determined. Functional sequences from the VBASE database were employed for drawing three unrooted phylogenetic trees for the VH (A), VL-kappa (B) and VL-lambda (C) subset by means of the Phylogeny.fr web tool, followed by grouping the sequences to corresponding antibody families (black cycles). All functional sequences that were identified within the analyzed sample are marked green (77%), and non-represented germline sequences are labeled red. The nucleotide distance scales are indicated with a value of 30% distance for VH and VL-kappa, and 20% distance for VL-lambda.

Figure 3.

Diversity of LYNDAL antibody sequences. Sequence analysis of 280 randomly chosen clones from all sublibraries against the VBASE database identified all antibody V gene families (A) and most of the functional VH (88%), VL-kappa (63%) and VL-lambda (74%) germline sequences (B). Comparison of LYNDAL functional germline gene usage against the three independent human antibody databases VBASE, VBASE2, and IMGT (C). Error bars are shown as standard deviations (SD) of mean values.

Figure 4.

Enrichment of gB-specific LYNDAL antibodies. LYNDAL of seven donors with high anti-HSV titers (No. 1, 3 - 8) were combined for panning against target gB-1. Polyclonal phage ELISA (A) confirmed enrichment of target-specific scFv phage antibodies during selection rounds 1 to 3. The error bars represent standard deviations of duplicates. Screening of individual clones by monoclonal phage ELISA revealed an increase of specific binders between round 2 (B) and 3 (C). Phage antibodies were detected using an anti-phage peroxidase conjugate in combination with colorimetric substrate TMB.

Figure 5.

Sequence analysis of gB-specific LYNDAL antibodies. Germline sequences of the 34 selected scFvs were determined and phylogenic relationships analyzed by drawing phenograms employing the Phylogenic.fr web tool. The germline sequences as well as corresponding antibody families are shown for each clone.

Figure 6.

Analysis of somatic mutations within enriched LYNDAL antibodies. Variable genes of the 34 gB-specific scFvs were aligned to the closest respective germline sequences and the number of nucleotide (A) and amino acid (B) mutations was determined. Mean values and corresponding standard deviation are shown for VH, VL, and combined VH/VL genes. The distribution of amino acid mutations was analyzed separately for the VL and VH domains (C). The number of mutations in the framework regions 1 - 3 and complementarity determining regions 1 and 2 were determined and normalized to the length of each corresponding region. Results are presented as mean mutation frequency, i.e., the average probability of observing an amino acid mutation within the investigated segment when compared with its corresponding germline sequence. Error bars represent standard deviations of the mean values.

Figure 7.

Binding analysis of gB-specific antibodies from LYNDAL. Specificity of scFvs for binding to cell surface glycoprotein B of HSV-1 and HSV-2 infected Vero cells was analyzed by flow cytometry.

Figure 8.

Equilibrium-binding curves for antibody 28. Binding activities of monovalent scFv and bivalent (scFv)₂ was measured on either (A) HSV-1 or (B) HSV-2 infected Vero cells by flow cytometry. Error bars represent standard deviations of the mean values. MFI_max, maximum median fluorescence intensity.

Figure 9.

In vitro neutralization of HSV by LYNDAL-selected antibody. Efficacy of clone 28 for neutralizing HSV-1 strain F (A) or HSV-2 strain G (B) was studied by plaque reduction neutralization tests (PRNT) using Vero cells. Neutralization capacity was assessed with serial dilutions of either monovalent scFv or bivalent (scFv)₂ and concentrations were determined that neutralized 50% of viruses (PRNT₅₀). The neutralizing humanized monoclonal antibody, IgG hu2c, was used as a control and comparator. Experiments were performed in duplicate. Error bars represent standard deviations of the mean values.