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.

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune Phage Display Libraries

“…However, immobilization of antigens/virions on plastic can alter conformation of coated proteins leading to antibodies recognizing epitopes that are not found on intact virions. Alternatively, binding affinity of antibody fragments can also be estimated by flow cytometry using antigen overexpressing cell lines [30], e.g., by using Vero cell being infected with HSV [18,28].…”

“…Although the isolation of peripheral blood mononuclear cells (PBMC) from whole blood (the main source for plasmablasts) is often described for retrieving antiviral antibodies, B-cell sources such as spleen, lymph nodes (many memory B-cells), or bone marrow (the main source of plasma cells) might be considered for library construction. Good protocols for the cloning of combinatorial scFv phage display libraries including the primer sets for PCR amplification of antibody genes can be found elsewhere [17,[20][21][22] and are out of the scope of this chapter.…”

“…This strategy has been successfully used for generating neutralizing human antibodies against HSV as a proof-of-principle [18]. The following protocol will systematically describe the procedure of generating broadly neutralizing antiviral antibodies from isolated human B-cells by a phage display technique.…”

“…Hence, the preparation of combinatorial immune libraries from immunized or infection cured donors can deliver a broader range of highly functional antibodies, even from smaller libraries (i.e., potent antiviral antibodies with broadly neutralizing efficacies). For instance, the selection of combinatorial immune repertoires (>10 8 independent clones) from HSV seropositive donors resulted in a broad panel of various high-affinity binders with partly HSV-neutralizing properties [17,18].…”

“…the VH repertoire into the VL phagemid [17,18,[20][21][22]. In the three-step cloning strategy, two separate VH and VL libraries are prepared before exercising one repertoire and including it into the phagemid containing the other repertoire [23].…”