**2. Antibody library screening**

A critical step in the establishment of antigen-specific monoclonal antibody fragment clones is the screening of the recombinant-antibody libraries [6, 7]. Methods for screening the antibody libraries can be largely divided into two strategies [24]: the display and repertoire cloning strategies (**Figure 1**).

integrated [28]; bacterial surface display [29]; and mammalian cell surface display [30, 31] for human antibody discovery. In these display systems, panning is applied for screening [13]. The antigen is immobilized on the surface of a microtiter plate, and the scFv library can be screened with phage display and with ribosome/mRNA display. Weakly bound clones are removed by washing, retaining the specific clones bound to the antigen (**Figure 1A**). This panning method is characterized by repeated selection, proliferation, and the enrichment of positive clones for enabling the processing of large libraries. For yeast, bacterial and mammalian cell surface display FACS with the cells displaying the recombinant antibody fragments

**Figure 1.** Strategies for antibody library screening. (A) Display strategy: scheme of the phage display panning process. (B) Repertoire cloning strategy: scheme of the cloning and assay process. (C) Detection of antigen-specific antibody

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In contrast, in repertoire cloning strategies, the antibody library is transformed into *E. coli*; the scFvs are expressed and secreted from a single clone, and scFvs are screened by ELISA (**Figure 1B**). Clones are selected based on assays, using scFv characteristics such as the affinity; thereby, this method offers advantages such as low false-positive rates and the ability to reliably identify clones with a high affinity. However, an assay must be performed for each individual *E. coli* clone, and only the positive clones are selected. There is no enrichment process in the screening method, and only limited libraries can be used for antibody selection.

Particularly, antibody repertoires from immunized animals with a clone number of approxi-

are suitable for the repertoire cloning but not naïve and synthetic antibody

using labeled antigen is applied.

fragments released from a bacterial colony by a colony assay.

mately 106

**2.2. Repertoire cloning strategies for antibody library screening**

#### **2.1. Display strategies for antibody library screening**

In the display strategy [25], the antibody fragment and its gene, i.e., the antigen recognition function and information, are joined, and antibody fragments with an affinity against the antigen are screened. Phage display systems in which an scFv joined to the filamentous phage coat protein, g3p, is displayed on the phage are extensively used [15, 16]. Other display systems include yeast display systems in which scFvs are displayed on the surface of yeast [26]; mRNA display [27]; ribosome display in which a ribosome, mRNA, and an scFv are

from the lymph tissue of immunized animals and linked together for creating a single-chain variable fragment (scFv) library, and Fab libraries are constructed too. In general, the antibody fragments used for screening are the scFvs. Currently, entirely synthetic libraries [8–11] and naïve libraries [12] are being used. These antibody gene libraries are incorporated into a phagemid or plasmid and expressed in phage or *Escherichia coli* (*E. coli*). Further, panning [13] or colony assays [14] are performed to isolate scFvs possessing affinity to the antigen, thereby establishing monoclonal antibodies. This step, the screening of antibody libraries, is critical for establishing monoclonal antibody fragments with a high affinity and specificity against the antigen. One of the most extensively used methods is the phage display method [15, 16]. The display of the antibody repertoires on the surface of bacteriophages and their selection through panning enables the isolation of monoclonal antibodies [17]. Phage display is also widely used for affinity maturation [18, 19], in which mutations are introduced into the variable domains of an antibody gene mainly into CDRs to produce antibodies with a higher affinity as the original clone [20]. In addition, cell surface panning techniques [1, 21, 22] are being developed to establish antibodies recognizing membrane proteins on living cells that are difficult to produce using the conventional methods. Technologies that enable liquid panning rather than immobilizing the antigens to a solid phase have also been proposed for phage display to establish antibodies that recognize protein conformation [23]. Screening with a colony assay induces the actual expression of the scFvs themselves and involves a direct confirmation of the antigen-antibody binding, lending it the advantage of a low falsepositive rate. In addition, the method can be easily used to screen libraries in the order of magnitude larger than those that can be screened with the hybridoma technology. However, this method poses several problems: it requires extensive and complex manipulation of assay steps, the expression of antibody fragments could be at times nonexistent or very low, and the extensive manipulation during the assay can lead to contamination and death of the *E. coli* cells, potentially preventing gene retrieval. Although this technique is not complete and not widely applied, further development and improvement can render it highly beneficial.

A critical step in the establishment of antigen-specific monoclonal antibody fragment clones is the screening of the recombinant-antibody libraries [6, 7]. Methods for screening the antibody libraries can be largely divided into two strategies [24]: the display and repertoire cloning

In the display strategy [25], the antibody fragment and its gene, i.e., the antigen recognition function and information, are joined, and antibody fragments with an affinity against the antigen are screened. Phage display systems in which an scFv joined to the filamentous phage coat protein, g3p, is displayed on the phage are extensively used [15, 16]. Other display systems include yeast display systems in which scFvs are displayed on the surface of yeast [26]; mRNA display [27]; ribosome display in which a ribosome, mRNA, and an scFv are

**2. Antibody library screening**

**2.1. Display strategies for antibody library screening**

strategies (**Figure 1**).

104 Antibody Engineering

**Figure 1.** Strategies for antibody library screening. (A) Display strategy: scheme of the phage display panning process. (B) Repertoire cloning strategy: scheme of the cloning and assay process. (C) Detection of antigen-specific antibody fragments released from a bacterial colony by a colony assay.

integrated [28]; bacterial surface display [29]; and mammalian cell surface display [30, 31] for human antibody discovery. In these display systems, panning is applied for screening [13]. The antigen is immobilized on the surface of a microtiter plate, and the scFv library can be screened with phage display and with ribosome/mRNA display. Weakly bound clones are removed by washing, retaining the specific clones bound to the antigen (**Figure 1A**). This panning method is characterized by repeated selection, proliferation, and the enrichment of positive clones for enabling the processing of large libraries. For yeast, bacterial and mammalian cell surface display FACS with the cells displaying the recombinant antibody fragments using labeled antigen is applied.

#### **2.2. Repertoire cloning strategies for antibody library screening**

In contrast, in repertoire cloning strategies, the antibody library is transformed into *E. coli*; the scFvs are expressed and secreted from a single clone, and scFvs are screened by ELISA (**Figure 1B**). Clones are selected based on assays, using scFv characteristics such as the affinity; thereby, this method offers advantages such as low false-positive rates and the ability to reliably identify clones with a high affinity. However, an assay must be performed for each individual *E. coli* clone, and only the positive clones are selected. There is no enrichment process in the screening method, and only limited libraries can be used for antibody selection.

Particularly, antibody repertoires from immunized animals with a clone number of approximately 106 are suitable for the repertoire cloning but not naïve and synthetic antibody repertoires with high clone numbers (10<sup>9</sup> –1012 clones). During the assay, a clone from an *E. coli* library is cultured, and its expression is induced. The reactivity of the expressed scFv against the antigen is measured. Clones exhibiting high reactivity are selected as the positive clones. In this method, only a few thousands of clones can be examined simultaneously, even if a multi-well microtiter plate is used. Although this number is higher than the clones obtained by the hybridoma technique, positive clones cannot be efficiently obtained, when the positive ratio is low.

However, as several antibody fragments are themselves toxic to *E. coli*, these clones will be lost during panning, even if they possess a high affinity. Conversely, repeated panning may result in the relatively preferential propagation of clones with reduced *E. coli* toxicity, even if the clones do not possess a high binding capacity. Toxicity to *E. coli* can increase the background, resulting in several false-positive clones being obtained. This situation renders panning extremely difficult; it is not easy to establish single positive clones only through several rounds of panning [14]. Although the phage display is a powerful tool for establishing mono-

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As an alternative antibody-screening tool, the colony assay can be used which is sometimes superior to the phage display method [42]. The advantage of this method is that the antibodyantigen binding can be directly observed during the screening process, reducing the selection of false-negative clones [24]. Thus, the colony assay presents notable advantages over the

In the colony assay (**Figure 2**), antibody libraries are expressed in *E. coli* for the selection of clones with a favorable affinity to the antigen. An scFv library is transformed into *E. coli* cells, and afterward transformed *E. coli* cells are plated on appropriate agar plates. After growing of the colonies, they are lifted onto a filter. Further, an expression-inducing reagent such as isopropyl-β-D-thiogalactopyranoside (IPTG) is applied, inducing the expression and secretion of scFvs from the *E. coli* cells (**Figure 2A**). scFvs with the desired affinity will diffuse out and bind the antigen coated on the membrane beneath the colonies. However, scFvs without affinity will not bind the antigen (**Figure 2B**), and the unbound scFvs are washed away. Then, the bound scFvs with an affinity against the antigen are detected using an enzymatic method. The His-tags attached to the scFvs are detected with anti-His antibodies (**Figure 2C**). Positive

clones are identified as the colonies corresponding to positive signals (**Figure 2D**).

tightly controlled IPTG concentration for expression of the scFvs.

Dreher et al. [43, 44] improved the colony assay by developing the filter-sandwich colonyscreening assay (hereafter, the filter-sandwich assay) for selecting positive clones; *E. coli* colonies are grown directly on a hydrophilic filter, which is then transferred to an antigen-coated membrane soaked with IPTG solution and placed on an agar plate containing IPTG to induce antibody fragment production. The antibody fragments produced by the colonies diffuse out and bind to the antigen on the membrane. The presence of antibody fragments bound to the membrane is then detected, and the spot is superimposed on the colony. This method circumvents the difficult technique of lifting the colony [14, 36]. In addition, the filter-sandwich assay was further optimized. The procedure can now be performed by a single step [45] under

clonal antibodies, it is used less frequently than expected [39].

**3. Colony assay for antibody library screening**

phage display and biopanning method.

**3.1. Principle of the colony assay**

**3.2. Filter-sandwich assay**

Colony assay in which periplasmic expression and *E. coli* colony formation lifted onto filters are used provides a method for handling large libraries (**Figure 1C**). In colony assay, the clones do not need to be picked up individually before screening; all the colonies on the plate can be assayed simultaneously. Thus, numerous clones can be assayed from a single plate. Antibody fragments released by bacteria were detected by a phage plaque assay in earlier experiments [32, 33]. Libraries of the antibody fragments were expressed in *E. coli* using phage λ vectors [34, 35]. Then, the active fragments secreted from the viable *E. coli* colonies were detected by colony-lift immunoassay [36]. With the colony assay, considerably larger libraries can be dealt with because the number of colonies screened can be easily increased.

#### **2.3. Screening with a phage display**

In a phage display system, panning is used to isolate phages that display the antibody fragments exhibiting affinity to the antigen (**Figure 1A**). Positive clones are established by selecting only the phages that displays antibody fragments (primarily scFvs) fused to the g3p coat proteins on the surface of the filamentous phage, which have affinity to the antigen. This method has the advantage of processing large libraries (~1011) [3, 37]. The antigen is immobilized, and the recombinant phage bound to the antigen is left intact; weakly bound recombinant phages are washed away. The remaining recombinant phages, which possess a binding capacity are detached from the antigen by acid treatment and infected into *E. coli*. Further, *E. coli* cells are cultured to propagate positive clones. The *E. coli* clones expressing the phagemids are then infected with a helper phage, and the phages displaying scFvs with binding capacities are collected. Panning is performed repeatedly for the selected group of phages. The repeated selection and propagation of positive clones enrich clones with antibodies comprising binding capacities to the antigen. Then, single clones are isolated at the final step with high binding capacities [38]. This method renders it possible to handle large libraries.

One limitation of this method is that the high background during panning selection often results in false-positive clones. A specific antigen-binding activity is typically not the only driving force exploited during the panning process [39, 40]. Multiple rounds of panning have been documented to frequently cause a strong bias for antibodies directed against immunodominant epitopes and abundant proteins [41], resulting in the loss of the library's diversity and of valuable antibody clones. Several factors influence the selection of the antigen-specific clones and produce undesired effects; these factors include a high efficiency of expression and folding despite poor antigen-binding activity, the nonspecific hydrophobic binding properties of the phage particle itself, and a superior compatibility with the host cells, not related to the antibody fragment affinity.

However, as several antibody fragments are themselves toxic to *E. coli*, these clones will be lost during panning, even if they possess a high affinity. Conversely, repeated panning may result in the relatively preferential propagation of clones with reduced *E. coli* toxicity, even if the clones do not possess a high binding capacity. Toxicity to *E. coli* can increase the background, resulting in several false-positive clones being obtained. This situation renders panning extremely difficult; it is not easy to establish single positive clones only through several rounds of panning [14]. Although the phage display is a powerful tool for establishing monoclonal antibodies, it is used less frequently than expected [39].
