**1. Introduction**

Antibodies are powerful tools for the prophylaxis and treatment of viral infections. The use of antibodies against severe, life-threatening infections began in the 1890s when Robert Koch

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

demonstrated that administration of sheep antiserum against diphtheria toxin to a girl dying from diphtheria infection led to her rapid recovery and survival [1]. On that basis, numerous attempts to treat potentially deadly viral diseases such as influenza, severe acute respiratory syndrome (SARS), or Ebola by administrating sera from survivors have successfully been undertaken [2]. Further improvements led to the development of polyclonal hyperimmunoglobulin G (IgG) preparations, consisting of purified antibodies from seropositive donors [3]. Several hyperimmunoglobulins against human cytomegalovirus (CMV), hepatitis B virus (HBV), rabies, and other viral infections are available on the market. Although polyclonal preparations provide strong antiviral activity, the major disadvantage of such hyperimmunoglobulin preparations is that they include a high amount of nonspecific antibodies and only a low proportion of neutralizing antibodies. The development of hybridoma technology by Köhler & Milstein in 1975 revolutionized science and medicine and led to the isolation of numerous monoclonal antibodies [4]. Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of therapeutics has grown significantly [5]. In 2016, 5 of the top 20 pharmaceuticals were therapeutic antibody drugs [6]. The vast majority of monoclonal antibodies is approved for the treatment of cancers, multiple sclerosis, or rheumatoid arthritis [7]. However, numerous potent human or humanized antiviral antibodies against H5N1 influenza virus, human immunodeficiency virus (HIV), herpes simplex virus (HSV), human cytomegalovirus (CMV), hepatitis C virus (HCV), Ebola virus, severe acute respiratory syndrome (SARS) virus, and other viral infections are in preclinical development, clinical studies, or even approved for antiviral treatment [2, 7–12]. Antibodies mostly neutralize free viruses by targeting the initial stages of virus infection described as the binding of free virions to permissive target cells followed by entry and replication [2]. Neutralizing antibodies not only provide new tools for prophylaxis and therapy of viral diseases, but also identify conserved epitopes that may be used to design new vaccines capable of conferring broader protection [11]. However, enveloped viruses such as HIV-1, HSV-1/2, CMV, and measles can also move between adjacent cells without diffusing through the extracellular environment (cell-to-cell spread). This mechanism facilitates rapid viral dissemination, promotes immune evasion from the host's immune response, and enhances the progression of disease [13]. We recently described a murine monoclonal antibody (mAb) capable of inhibiting the cell-to-cell spread of HSV. This antibody proved to be highly effective in the prevention of drug-resistant HSV infections in highly immunodeficient NOD/SCID mice indicating the enormous potential of antibody blocking mechanisms crucial for the virus spread [12]. Due to these unique features, this antibody was humanized for clinical applications and is now being tested in phase I and II trials. Besides the generation of antiviral antibodies by humanization approaches, neutralizing antibodies can also be isolated from humans cured of viral infections, such as SARS or Ebola [9, 14]. Various methods have been developed for the isolation of antibodies from humans including B-cell immortalization and single-cell expression cloning [15]. Among these approaches, high-throughput screening of phage-displayed antibody libraries has become one of the leading technologies for generating human therapeutic antibodies (**Figure 1**) [16]. Nowadays, very large phage display libraries from naive (IgM) B-cell repertoires (>1010 independent clones) are widely used for the selection of human antibodies against a broad panel of targets including human self-antigens to identify high-affinity binders. However, these antibodies are not affinity-matured by the human immune system. 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).

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune...

from HSV seropositive donors resulted in a broad panel of various high-affinity binders with

Although various other *in vitro* selection platforms have been developed during the last decades (e.g., ribosome display and yeast display), phage display using filamentous phages is predominantly used for library generation since it is robust, inexpensive, and allows the automation of the selection and screening process. Due to the limitation in the production of fulllength IgG antibodies in *Escherichia coli*, only smaller antibody fragments (e.g., scFvs or Fabs) can be efficiently expressed within the *E*. *coli* periplasm as functional proteins. Several systems

**Figure 1.** Isolation of human neutralizing antiviral antibodies (nAbs) by phage display technique. Lymphocytes comprising B-cells from humans harboring neutralizing antibodies with unique features, e.g., Ebola disease survivors are isolated from blood, spleen, lymph nodes, or bone marrow by standard techniques (e.g., PBMCs by Ficoll density gradient centrifugation). Lymphocytes RNA is prepared and transcribed into single-stranded cDNA that is used as the source for PCR amplification of the variable heavy (VH) and light chain (VL) genes. Variable genes are randomly cloned into phagemid vectors as scFv antibody fragments prior to electroporation of phagemids into *E*. *coli* bacteria to produce combinatorial immune libraries. Library glycerol stocks are then used for the generation of a bacterial culture that is superinfected with a helper phage to produce phages presenting different scFvs on their surface. Specific binding scFvphages are enriched over several selection rounds by stringent washing and elution using antigen/virions immobilized on immunotubes. After screening for monoclonal binders on ELISA plates, the best specific binders are directly produced as monovalent scFvs in bacteria cultures or cloned into appropriate expression vectors for the production of Fab, or various bivalent antibody formats prior to functional analysis (e.g., virus neutralization capacity and affinity).

independent clones)

77

http://dx.doi.org/10.5772/intechopen.70139

For instance, the selection of combinatorial immune repertoires (>108

partly HSV-neutralizing properties [17, 18].

immune system. 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 (>108 independent clones) from HSV seropositive donors resulted in a broad panel of various high-affinity binders with partly HSV-neutralizing properties [17, 18].

demonstrated that administration of sheep antiserum against diphtheria toxin to a girl dying from diphtheria infection led to her rapid recovery and survival [1]. On that basis, numerous attempts to treat potentially deadly viral diseases such as influenza, severe acute respiratory syndrome (SARS), or Ebola by administrating sera from survivors have successfully been undertaken [2]. Further improvements led to the development of polyclonal hyperimmunoglobulin G (IgG) preparations, consisting of purified antibodies from seropositive donors [3]. Several hyperimmunoglobulins against human cytomegalovirus (CMV), hepatitis B virus (HBV), rabies, and other viral infections are available on the market. Although polyclonal preparations provide strong antiviral activity, the major disadvantage of such hyperimmunoglobulin preparations is that they include a high amount of nonspecific antibodies and only a low proportion of neutralizing antibodies. The development of hybridoma technology by Köhler & Milstein in 1975 revolutionized science and medicine and led to the isolation of numerous monoclonal antibodies [4]. Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of therapeutics has grown significantly [5]. In 2016, 5 of the top 20 pharmaceuticals were therapeutic antibody drugs [6]. The vast majority of monoclonal antibodies is approved for the treatment of cancers, multiple sclerosis, or rheumatoid arthritis [7]. However, numerous potent human or humanized antiviral antibodies against H5N1 influenza virus, human immunodeficiency virus (HIV), herpes simplex virus (HSV), human cytomegalovirus (CMV), hepatitis C virus (HCV), Ebola virus, severe acute respiratory syndrome (SARS) virus, and other viral infections are in preclinical development, clinical studies, or even approved for antiviral treatment [2, 7–12]. Antibodies mostly neutralize free viruses by targeting the initial stages of virus infection described as the binding of free virions to permissive target cells followed by entry and replication [2]. Neutralizing antibodies not only provide new tools for prophylaxis and therapy of viral diseases, but also identify conserved epitopes that may be used to design new vaccines capable of conferring broader protection [11]. However, enveloped viruses such as HIV-1, HSV-1/2, CMV, and measles can also move between adjacent cells without diffusing through the extracellular environment (cell-to-cell spread). This mechanism facilitates rapid viral dissemination, promotes immune evasion from the host's immune response, and enhances the progression of disease [13]. We recently described a murine monoclonal antibody (mAb) capable of inhibiting the cell-to-cell spread of HSV. This antibody proved to be highly effective in the prevention of drug-resistant HSV infections in highly immunodeficient NOD/SCID mice indicating the enormous potential of antibody blocking mechanisms crucial for the virus spread [12]. Due to these unique features, this antibody was humanized for clinical applications and is now being tested in phase I and II trials. Besides the generation of antiviral antibodies by humanization approaches, neutralizing antibodies can also be isolated from humans cured of viral infections, such as SARS or Ebola [9, 14]. Various methods have been developed for the isolation of antibodies from humans including B-cell immortalization and single-cell expression cloning [15]. Among these approaches, high-throughput screening of phage-displayed antibody libraries has become one of the leading technologies for generating human therapeutic antibodies (**Figure 1**) [16]. Nowadays, very large phage display libraries from naive (IgM) B-cell repertoires (>1010 independent clones) are widely used for the selection of human antibodies against a broad panel of targets including human self-antigens to identify high-affinity binders. However, these antibodies are not affinity-matured by the human

76 Antibody Engineering

Although various other *in vitro* selection platforms have been developed during the last decades (e.g., ribosome display and yeast display), phage display using filamentous phages is predominantly used for library generation since it is robust, inexpensive, and allows the automation of the selection and screening process. Due to the limitation in the production of fulllength IgG antibodies in *Escherichia coli*, only smaller antibody fragments (e.g., scFvs or Fabs) can be efficiently expressed within the *E*. *coli* periplasm as functional proteins. Several systems

**Figure 1.** Isolation of human neutralizing antiviral antibodies (nAbs) by phage display technique. Lymphocytes comprising B-cells from humans harboring neutralizing antibodies with unique features, e.g., Ebola disease survivors are isolated from blood, spleen, lymph nodes, or bone marrow by standard techniques (e.g., PBMCs by Ficoll density gradient centrifugation). Lymphocytes RNA is prepared and transcribed into single-stranded cDNA that is used as the source for PCR amplification of the variable heavy (VH) and light chain (VL) genes. Variable genes are randomly cloned into phagemid vectors as scFv antibody fragments prior to electroporation of phagemids into *E*. *coli* bacteria to produce combinatorial immune libraries. Library glycerol stocks are then used for the generation of a bacterial culture that is superinfected with a helper phage to produce phages presenting different scFvs on their surface. Specific binding scFvphages are enriched over several selection rounds by stringent washing and elution using antigen/virions immobilized on immunotubes. After screening for monoclonal binders on ELISA plates, the best specific binders are directly produced as monovalent scFvs in bacteria cultures or cloned into appropriate expression vectors for the production of Fab, or various bivalent antibody formats prior to functional analysis (e.g., virus neutralization capacity and affinity).

the VH repertoire into the VL phagemid [17, 18, 20–22]. 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]. After electroporation of the phagemids into electrocompetent *E*. *coli* bacteria, the antibody libraries are grown on selection plates and stored as frozen bacterial glycerol stocks. Prior to PCR amplification of the variable antibody genes, B-cells from respective donors need to be isolated. In the case of viral infection and depending on when the infection occurred, the isolation of the short-lived plasmablast pool during the early antibody response or the long-lived memory B-cell and plasma cell pool may be preferred. 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, 20–22] and are out of the scope

Detailed Protocols for the Selection of Antiviral Human Antibodies from Combinatorial Immune...

http://dx.doi.org/10.5772/intechopen.70139

79

Here, we present a methodology for the recovering of potential therapeutic antibodies with unique antiviral properties from human B-cell repertoires (for workflow see **Figure 2**). 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

Before starting antibody phage display, please be aware that phages are highly stable and decontamination of workspace and consumables is hard to achieve. It is best to do phage work in a special lab keeping equipment/material separated from the common bacterial workspace, especially when antibody library construction is performed. If not possible, phage work should be carried out in at least a separate workspace including a separate hood, shaker, and centrifuge. Inactivation of phage solutions can be done by incubation with diluted bleach (caution, always wear personal protection during handling) and/or sterilizing workspaces with UV light. For decontamination of tubes and Erlenmeyer flasks, bleach can be added to water-filled tubes and incubated overnight before washing, rinsing, and autoclaving. In common, single-use material is preferred for phage work. Collect phage-contaminated solutions in glass flasks and inactivate by adding bleach before dumping. Only use polypropylene (PP) tubes since phages might stick to other kinds of plastics. To prevent contamination to

Presented protocols are intended for the selection and screening of antibody libraries based on the scFv antibody format being cloned in phagemid vectors as pIII fusion with an intrinsic amber stop codon and under the *lac* promoter (inducible by IPTG, repressible by glucose). Many current antibody phage display libraries are constructed in phagemid vectors with listed features (e.g., most derivatives of pHEN, pComb3X, pHAL, and pCANTAB), although

of this chapter.

a phage display technique.

**2. Preliminary notes**

pipettes, always use barrier tips.

**Figure 2.** Workflow of the selection and screening procedure. For the selection of antigen-specific scFv-phages, logphase library cultures are packed by superinfection with helper phages (for the preparation of helper phages, see Protocol I) that provide all proteins necessary for phage propagation (see Protocol A). After IPTG induction, expressed scFv-pIII fusions are inserted within the produced phages leading to the presentation of scFv antibody fragments on the phage surface. After determination of phage titer (see Protocol B), specific binding scFv-phages are enriched over several selection rounds by stringent washing and elution on recombinant antigen/virions that have been immobilized onto immunotubes (see Protocol C). Successful enrichment of specific binding scFv-phages can be analyzed by polyclonal phage ELISA (ppELISA) (see Protocol D) prior to screening of monoclonal antibodies as scFv-phages by monoclonal phage ELISA (mpELISA) (see Protocol E-I) or as soluble scFvs (see Protocol E-II). After the identification of bacterial colonies encoding for full-length scFvs by colony PCR and sequencing (see Protocol F), soluble scFvs can be produced in the periplasm of bacteria (see Protocol G) or variable antibody genes can be cloned into mammalian expression vectors to produce Fab or various bivalent antibody fragments. Finally, antibody fragments can be analyzed for their neutralizing activity in functional assays like the plaque reduction neutralization test (PRNT) (see Protocol H).

for displaying antibody fragments on the phage surface have been developed over time using different vectors and phage coat proteins for display. The most common type (3 + 3 system) is based on phagemid vectors where the antibody gene fragments are cloned as fusions with the pIII phage gene. Cloning of the antibody gene repertoires can be done by using different strategies in one, two, or three independent steps where the variable light and heavy chain genes are PCR-amplified and randomly combined into reliable phagemid vectors. In the-one step cloning strategy, the VH and VL genes are separately amplified with an overlapping, additional linker sequence and combined by assembly PCR [19]. In the two-step cloning strategy, mostly the VL gene repertoire is cloned first into the phagemid followed by insertion of the VH repertoire into the VL phagemid [17, 18, 20–22]. 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]. After electroporation of the phagemids into electrocompetent *E*. *coli* bacteria, the antibody libraries are grown on selection plates and stored as frozen bacterial glycerol stocks. Prior to PCR amplification of the variable antibody genes, B-cells from respective donors need to be isolated. In the case of viral infection and depending on when the infection occurred, the isolation of the short-lived plasmablast pool during the early antibody response or the long-lived memory B-cell and plasma cell pool may be preferred. 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, 20–22] and are out of the scope of this chapter.

Here, we present a methodology for the recovering of potential therapeutic antibodies with unique antiviral properties from human B-cell repertoires (for workflow see **Figure 2**). 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.
