Cocktail immunization also provides the opportunity to augment an antibody response by providing helper epitopes (see **Figure 6**). Indeed, it is possible to induce a specific antibody response even if the target protein differs from the endogenous ortholog by only a single amino acid substitution, as exemplified by the classic Thy1 (CD90) alloantigens (47). Inbred strains of mice express either Thy1.1 or Thy1.2. Immunization of a Thy1.1+ mouse with cells expressing Thy1.2 induces a potent Thy1.2-specific antibody response, but only if the cells used for immunization co-express other disparate alloantigens that provide helper determinants (48). As explained in the previous section, antibody responses are driven by antigen-specific Th. This T cell help depends on both a peptide that can be presented by the particular MHCII molecules expressed by the immunized animal and a corresponding specific TCR that has escaped thymic selection. In order to obtain T cell help, the B cell needs to display MHCII-bound peptides on the cell surface. The MHCII presented peptides need not be derived from the membrane protein itself. They can also be derived from proteins encoded by a co-delivered cDNA expression vector during cocktail immunization, provided that the co-delivered protein is taken up and processed by the B cell displaying a specific antibody directed against the membrane protein of interest. In other words, suitable peptides must be delivered to the B cell together with the membrane protein of interest. It is unlikely that naive B cells endocytose intact EC or Langerhans cells. More likely, B cells bind and endocytose membrane-bound vesicles derived from these cells. If one or more distinct membrane proteins are contained in such vesicles, they can serve as a source for MHCII bound peptides.

Similarly, peptides binding to MHCI molecules typically drive cytotoxic T cell responses against the cells transfected with the cDNA expression construct during immunization (49, 50). Such a cytotoxic T cell response may indirectly support an antibody response by enhancing the release of vesicles containing the membrane protein from transfected cells, which can then be transported to the draining lymph nodes, thereby increasing the chances for encounter with and uptake by an antigen-specific B cell. As in case of MHCII-binding peptides, MHCI-binding peptides presented to cytotoxic T cells need not be derived from the target membrane protein of interest. With cDNA cocktail immunizations such peptides can be provided by a co-transfected protein, e.g., an antibiotic resistance marker under control of a mammalian promoter. Mixtures of cDNA expression vectors against two different membrane proteins of interest may therefore increase the chances of providing suitable peptides for activation of helper and cytotoxic T cells.

### CLONING OF THE VHH REPERTOIRE AND IDENTIFICATION OF SPECIFIC BINDERS

**Figure 7** schematically outlines the procedure for cloning and sequencing of specific Nbs from immunized camelids. RNA extracted from blood lymphocytes (**Figure 7A**) is transcribed into cDNA and the Nb-coding region is then PCR amplified using appropriate primer pairs (**Figure 7B**). The-PCR-amplified VHH repertoire is then subjected to next-generation sequencing (**Figure 7C**) and cloning into pro- and or eukaryotic expression vectors (**Figures 7D,E**, respectively).

The fact that camelid heavy-chain antibodies recognize their target antigen *via* a single immunoglobulin domain provides a huge technical advantage for cloning of specific binders in comparison to conventional antibodies that recognize their target antigen via a pair of variable domains derived from two distinct protein chains. Cloning of the binding module from heavy-chain antibodies does not face the pairing problem of conventional antibodies. In other words, the VHH-domain can be PCR amplified and cloned directly into a mammalian expression vector so as to fuse the Nb to the hinge and Fc domains of a conventional mouse, human, or rabbit antibody (**Figure 7E**). It is then feasible to express such Nb-based heavy-chain antibodies in transiently transfected HEK cells, e.g., in a 96-well format and to screen the supernatants for production of specific binders. A second important advantage over conventional antibodies is that the entire ~350 bp coding sequence of each Nb is readily determined by a single sequence run. It can, thus, easily be determined whether identified binders are derived from the same or from distinct Nb families.

transfected with the membrane protein of interest. Bound phage are eluted and panning can be repeated, e.g., on another cell type transfected with the protein of interest. Panning can be performed in the presence of available antibodies or Nbs in order to select for Nbs binding to a distinct epitope. (E) During cloning into the mammalian vector, the Nb is genetically fused to the hinge and Fc domains of a conventional rabbit, mouse, or human IgG. Individual clones are transfected into HEK cells. Heavy-chain antibodies secreted into the HEK cell supernatant are then screened for binding to cells transfected with the membrane protein of interest by ELISA or immunofluorescence microscopy using appropriate enzyme- or fluorochrome-conjugated secondary antibodies.

Next-generation sequencing of the PCR-amplified VHH repertoire (**Figure 7C**) provides information about expanded clones and about the degree of somatic hypermutation in expanded clones (12, 51, 52). If the samples are prepared from an appropriate source, e.g., a lymph node biopsy at the peak of B-cell clonal expansion or a sample of blood at the peak of migration of expanded clones from the lymph node to the bone marrow, most of the expanded clones are detected by NGS contain specific binders against the target protein of interest. Therefore, the results of next-generation sequencing can be used as a guide to specifically clone and express members of the expanded clones by simply ordering the coding sequence for each Nb as a synthetic DNA fragment and cloning this into a suitable mammalian heavy-chain antibody expression vector (**Figures 7C–E**).

A library of phage displayed Nbs can be used to select specific binders by panning of the library on immobilized antigen (**Figure 7D**). For membrane proteins, selection can be performed on cells transiently or permanently transfected with the protein of interest. In order to avoid selection of Nbs binding to irrelevant target proteins displayed on the surface of the cells used for selection, additional rounds of selection can be performed while changing the cell background. This can be achieved by using distinct cell types transfected with the same membrane protein of interest, e.g., human HEK cells in the first round and hamster CHO cells in the second round. Moreover, negative selections can be performed to remove phage binding to other cell surface proteins, using cells not expressing the protein of interest, e.g., untransfected HEK or CHO cells.

A convenient strategy for identifying binders using CHO cells transiently transfected with the same plasmid that was used for immunization is illustrated in **Figure 8**. We typically co-transfect CHO cells with expression vectors encoding the membrane protein of interest and a green fluorescent protein (GFP) fused to a nuclear localization signal (**Figure 8**). 24–48 h after transfection, the 10–20% of co-transfected cells are distinguished from untransfected cells by their green fluorescent nucleus. Nb-based rabbit IgG heavy-chain antibodies that bind specifically to cotransfected cells can readily be identified using a fluorochromeconjugated anti-rabbit IgG secondary antibody.

# ASSESSING THE SPECIFICITY, AFFINITY, AND RELATIVE BINDING EPITOPES OF TARGET-SPECIFIC Nbs

The immunofluorescence assay described in **Figure 8** also provides a convenient means to assess the specificity of the selected Nbs, i.e., by comparing the reactivity of the Nbs with CHO cells transfected with related membrane proteins. This provides information on the cross reactivity of the selected Nbs with orthologs of the membrane protein of interest from other species (e.g., human, mouse, rat) or with paralogs of the same species. The same assay can also be used to assess the relative affinity of the selected Nbs, i.e., by performing titration analyses.

Similarly, cross-blocking assays can provide information about the relative binding epitopes of different Nbs directed against the same membrane protein. To this end, cells are incubated sequentially with a large excess of one Nb before addition of the second Nb. The two Nbs must be distinguishable, e.g., *via* linkage to a moiety that permits independent detection of at least the second Nb. There are several options to achieve this, e.g., conjugation of the purified Nb-based heavy-chain antibody to biotin or a fluorochrome. Alternatively, the Nbs can be fused to two different IgG isotypes, e.g., rabbit IgG and human IgG1 allowing detection by isotype-specific secondary antibodies. The same procedure can be used to asses whether the selected Nbs bind to distinct or overlapping epitopes with commercially available monoclonal antibodies against the same target.

## GENETIC IMMUNIZATION TO RAISE Nbs AGAINST SECRETORY AND INTRACELLULAR PROTEINS

The strategy described here for raising Nbs against membrane proteins in native conformation can, in principle, be adapted also for raising Nbs against secretory or intracellular proteins in native conformation. Most secretory proteins can readily be produced as recombinant proteins and can thus also be used directly for classic adjuvant-assisted protein immunizations. For panning

Figure 8 | Identification of specific binders using CHO cells transiently co-transfected with expression vectors for the membrane protein of interest and nuclear GFP. CHO cells were transiently co-transfected with expression vectors for GFP fused to a nuclear localization sequence and the cDNA expression vector used for immunization, in this case encoding the homotrimeric ligand-gated P2X7 ion channel. Cells plated onto 96-well plates were analyzed 24 h after transfection by immunofluorescence microscopy. Green nuclei clearly distinguish transfected CHO cells from untransfectd CHO cells (blue nuclei stained with the DNA-binding dye Hoechst 33342). Cells were screened for cell surface binding of specific antibodies and bound antibodies were detected with a phycoerythrin (PE)-conjugated secondary antibody. (A) Hoechst dye, (B) GFP, (C) PE, and (D) merged images. Reprinted from Cellular Immunology 236, Sahil Adriouch, Gudrun Dubberke, Philip Diessenbacher, François Rassendren, Michel Seman, Friedrich Haag, Friedrich Koch-Nolte, Probing the expression and function of the P2X7 purinoceptor with antibodies raised by genetic immunization, 72–77, ©2005, with permission from Elsevier.

of phage libraries on secretory proteins, the target protein can be coated onto the walls of a standard 96-well ELISA plate or after biotinylation—be captured on streptavidin-coated beads. Screening for specific binding can conveniently be performed on transiently transfected cells. In this case, signals can be enhanced by treatment of cells with Brefeldin A for 4–6 h prior to analysis in order to trap secretory proteins in the ER and/or Golgi apparatus. Moreover, cells need to be fixed and permeabilized in order for Nbs or Nb-based heavy-chain antibodies to access the Golgi and ER compartments.

cDNA immunization can effectively induce specific antibody responses also against intracellular proteins, e.g., GFP or neomycin phosphotransferase (our own unpublished observations), even though these intracellular proteins are not directly accessible to the BCR on the B cell plasma membrane. Presumably, cytolysis mediated by specific cytotoxic T cells results in the release of intracellular proteins and their transport to the B cell compartment in draining lymph nodes *via* the lymphatics. We have found that antibody responses against intracellular proteins can be enhanced by forced expression of the entire proteins or distinct domains thereof on the cell surface. To this end, the open reading frame for the entire intracellular protein or for a structurally independent subdomain of the protein is genetically fused upstream to an ORF encoding a signal peptide and downstream to an ORF encoding a GPI-anchor signal sequence. In many cases, this results in forced display of the protein or protein domain on the cell surface. However, not all intracellular proteins or protein domains fold properly in the oxidative environment of the ER. Moreover, if the protein (domain) contains an internal glycosylation site (e.g., N X S/T), it can be aberrantly glycosylated in the ER. Furthermore, unpaired cysteine residues can engage in disulfide bond formation in the ER. These problems can often be circumvented by site-directed mutagenesis, e.g., by conservative substitutions such as asn to gln, or cys to ser. It may be convenient to attach an epitope tag—e.g., a FLAG tag to the N-terminus of the protein (domain), so that successful cell surface expression can be monitored by flow cytometry or immunofluorescence microscopy with a tag-specific antibody. If successful, immunization and selection of Nbs then follow the procedures described above for membrane proteins.

# ADVANTAGES AND LIMITATIONS OF BALLISTIC cDNA IMMUNIZATION

Key advantages of the cDNA immunization approach are the high purity of the immunogen and the presentation of the target protein in its native conformation. Moreover, this approach offers the opportunity to co-immunize with other proteins of interest, e.g., target ortholog(s) of other species, a foreign protein(s) as a source of MHC-binding peptides, and inflammation-promoting cytokines. A limitation of cDNA immunization vs. adjuvant-assisted protein or cellular immunizations is the prolonged induction of the immune response resulting from the time required for transcription, translation, and posttranslational modifications. Moreover, lymph node swelling and serum titer typically are lower with cDNA than with protein immunizations. Consequently, it is more difficult to judge the optimal timing of lymphocyte sampling. Another limitation is the high cost of the gene gun. A recent report describes the successful selection of ChemR23 specific Nbs (a GPCR) upon immunization of llamas with the dermojet (AKRA DERMOJET), a needle-less injection device (34). Nbs elicited by cDNA immunization do not differ in terms of affinity, specificity, or other fundamental properties from Nbs elicited by alternative approaches, such as liposomes or virus-like particles bearing membrane proteins.

# CONCLUSION AND PERSPECTIVES

Nanobodies and Nb-based heavy-chain antibodies have already proved valuable experimental tools in infection, immunity, oncology, and numerous other settings. The first Nb expected to be licensed for clinical applications next year (caplacizumab) is directed against a soluble protein (von Willebrand factor). This will likely pave the way for a wave of new Nb-based reagents to enter the clinic. Membrane proteins expressed by immune cells and tumor cells in particular represent potential therapeutic Nb targets in immunology and oncology. Conventional immunization strategies with purified protein and/or transfected cells are hampered by the poor solubility of many membrane proteins outside of a lipid environment and by the multitude of other antigens in transfected cells. The cDNA immunization strategy described here provides a powerful tool for the development of Nbs directed against membrane proteins.

# AUTHOR CONTRIBUTIONS

FH and FK-N conceived the project. All authors established experimental procedures. TE, SM, JW, and FK-N wrote the manuscript. All authors reviewed and approved the manuscript.

### ACKNOWLEDGMENTS

We thank Bianca Morgenrot and Sarah Hewald, UKE central animal facility, for excellent technical help with llama immunizations.

### FUNDING

This study was supported by grants No. 310/4, No. 310/5, No. 310/6, No. 310/7, No. 310/8, and SFB1192-B5 from the Deutsche Forschungsgemeinschaft to FH and FK-N.

# REFERENCES

**Conflict of Interest Statement:** FH and FK-N receive a share of antibody sales *via* MediGate GmbH, a wholly owned subsidiary of the University Medical Center Hamburg-Eppendorf. TE, SM, JW, and FK-N are co-inventors on patent applications on nanobody transgenic mice and/or CD38- or P2X7-specific nanobodies.

The reviewer GH and handling editor declared their shared affiliation.

*Copyright © 2018 Eden, Menzel, Wesolowski, Bergmann, Nissen, Dubberke, Seyfried, Albrecht, Haag and Koch-Nolte. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from *In Vitro* Display Libraries

*Kevin A. Henry1 , Dae Young Kim1 , Hiba Kandalaft1 , Michael J. Lowden1 , Qingling Yang1 , Joseph D. Schrag2 , Greg Hussack1 , C. Roger MacKenzie1,3 and Jamshid Tanha1,3,4\**

*1Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada, 2Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, QC, Canada, 3School of Environmental Sciences, University of Guelph, Guelph, ON, Canada, 4Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada*

#### *Edited by:*

*Abdul Qader Abbady, Atomic Energy Commission of Syria, Syria*

#### *Reviewed by:*

*Serge Muyldermans, Vrije Universiteit Brussel, Belgium Oscar R. Burrone, International Centre for Genetic Engineering and Biotechnology, Italy*

#### *\*Correspondence:*

*Jamshid Tanha<EMAIL_ADDRESS>
*This is NRC-HHT publication number: 53364.*

#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology Received: 30 September 2017 Accepted: 27 November 2017 Published: 12 December 2017*

#### *Citation:*

*Henry KA, Kim DY, Kandalaft H, Lowden MJ, Yang Q, Schrag JD, Hussack G, MacKenzie CR and Tanha J (2017) Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries. Front. Immunol. 8:1759. doi: 10.3389/fimmu.2017.01759*

Human autonomous VH/VL single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from *in vitro* display libraries constructed *via* synthetic randomization of rearranged VH/VL domains. Here, we describe the design and characterization of three novel human VH/VL sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential VH/VL sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) *in vitro* randomization of the CDRs of three VH/VL sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three VH/VL libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 VHs and 7 VLs in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2–3 µM), but had highly variable expression yields (range: 0.1–19 mg/L). Despite our efforts to identify the most stable VH/VL scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing VH/VL sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some VH/VL sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence clearly dramatically impacts the stability of human autonomous VH/VL immunoglobulin domain folds, and sequence-stability tradeoffs must be taken into account during the design of such libraries.

Keywords: single-domain antibody, synthetic antibody, human VH/VL, phage display, protein engineering

# INTRODUCTION

The concept of an autonomous single immunoglobulin variable domain (single-domain antibodies or sdAbs) as the smallest representation of an antigen-binding-competent antibody was first described by Ward et al. in the mouse (1). With the discovery of naturally occurring heavy chain-only antibodies in *Camelidae* (2) and in cartilaginous sharks (3) several years later (the single variable domains of which can recognize antigen autonomously), it became clear that sdAbs represented not only a theoretical possibility but a viable immunological solution to the problem of antigen recognition. Although the human humoral immune system produces only conventional antibodies with paired heavy and light chains and not sdAbs, the question of whether human sdAbs (autonomous variable heavy- or light-chain domains, VHs or VLs) could be isolated and/or molecularly engineered *in vitro* was brought to light.

The identification, engineering and biophysical characterization of a handful of non-antigen-specific human VH/VL sdAbs has been extensively reported and discussed (4). The first efforts to produce human VH/VL sdAbs with novel antigen-binding specificities used "camelized" scaffolds that incorporated the solubilizing framework region (FR) substitutions found in camelid sdAbs (5–9). Although this approach yielded antigen-specific sdAbs with excellent solubility and biophysical properties, it relied on undesirable sequence deviation from the human IGHV germline. Later, rare fully human rearranged VH and VL variable domains were discovered that were autonomously stable and monomeric and large phage display libraries were constructed by randomizing their complementarity-determining regions (CDRs), although it was clear from the mid-2000s that certain CDR sequences (potentially low in hydrophobic content and rich in negative charge) were better compatible with solubility and stability of these molecules (9–11). There are now many examples of fully human antibodies (primarily VHs) isolated from such libraries against a variety of targets, including α-amylase (12), β-galactosidase (13, 14), *Candida albicans* MP65 and SAP-2 (15), carbonic anhydrase (12), CD154 (16), CD28 (17), CD40 (18, 19), CD40L (20), *Clostridium difficile* toxin B (21), EGFR (22), glypican-2 (23), glypican-3 (24), human serum albumin (HSA) (25–27), lysozyme (28–30), maltose-binding protein (31), MDM4 (32), mesothelin (33), TNF-α (34), TNFR1 (35), and VEGF (22). These fully human VH/VL sdAbs exhibit a variety of antigen-binding modes and functional activities and several have entered clinical development, where they have been generally well-tolerated albeit unexpectedly immunogenic (36, 37).

Here, we report the design, construction and characterization of three novel phage-displayed, synthetically randomized human VH/VL sdAb libraries. We attempted to circumvent the unfavorable biophysical properties of many human VH/VL sdAbs by (i) selecting ultra-stable VH/VL sdAbs tolerant to CDR modification as library scaffolds, (ii) maximizing randomized sequence diversity in CDRs using trinucleotide mutagenesis, and (iii) spiking the library with negatively charged residues to encourage solubility. Similarly to the experiences of others, we were able to isolate monomeric, high-affinity VH/VL sdAbs from the libraries against some antigens but not against others. The stochastic process of selecting binders from human VH/VL sdAb libraries is likely a consequence of fundamental tradeoffs between CDR sequence and human VH/VL sdAb stability and aggregation resistance.

#### MATERIALS AND METHODS

#### Identification of Human Autonomous VH/VL sdAb Scaffolds

The human autonomous VH and VL sdAb scaffolds used in this study (**Table 1**; Figure S1 in Supplementary Material) were

*a Full-length amino acid sequences are listed in Figure S1 in Supplementary Material. bIMGT numbering.*

isolated as previously described by To et al. (38) and Kim et al. (39). Disulfide-stabilized versions of each VH/VL sdAb (bearing an intradomain disulfide linkage formed between Cys residues at IMGT positions 54 and 78) were produced by overlap extension PCR as described in Kim et al. (40).

# CDR Shuffling

All three CDRs of each VH/VL scaffold were simultaneously exchanged for those listed in **Table 2** (20 VH/VL scaffolds × 12 CDR sets = 240 CDR-shuffled variants). DNA constructs encoding the CDR-shuffled variants were synthesized commercially (GeneArt/ Life Technologies, Regensberg, Germany) and subcloned into the pSJF2H bacterial expression vector (12).

# Design and Construction of Synthetic Human VH/VL sdAb Phage Display Libraries

Three phage-displayed sdAb libraries were constructed by *in vitro* randomization of the sdAb scaffolds VH428, VHB82SS and VL383SS. Briefly, nondegenerate oligonucleotides spanning each sdAb were chemically synthesized using the phosphoramidite method (GeneArt/Life Technologies) and purified by HPLC. CDRs were randomized *via* incorporation of defined mixtures of trinucleotide phosphoramidite building blocks (41) during oligonucleotide synthesis. Oligonucleotides were assembled without amplification by Klenow fragment extension, gel purified using a GeneJET™ gel extraction kit (Thermo-Fisher, Waltham, MA, USA) according to the manufacturer's instructions, and resuspended in a total volume of 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Non-amplified library DNA was quantified by real-time PCR using Fast SYBR™ Green master mix (Thermo-Fisher), and a StepOnePlus™ real-time PCR system (Thermo-Fisher), then approximately 232–1,069 ng (4.3 × 1011 to 1.5 × 1012 molecules) of DNA was PCR-amplified with Phusion® high-fidelity DNA polymerase (Thermo-Fisher) using M13F and M13R primers (sequences in Table S1 in Supplementary Material), gel purified using a GeneJET™ gel extraction kit, then digested and ligated between the *Nco*I and *Not*I sites of the phagemid vector pMED1 (42). *Escherichia coli* TG1 cells were transformed with the ligation products by electroporation, yielding final library sizes between 1.0 × 1010 and 2.3 × 1010 independent transformants, ≥95% of which carried VH/VL sdAb inserts as shown by colony PCR.

# Antibodies, Proteins and Reagents

Recombinant human CEACAM6 (residues 145–232) and mouse GITR (residues 20–153) ectodomains were produced by transient transfection of HEK293-6E cells as previously described (43). HSA was from Sigma-Aldrich (St. Louis, MO, USA; Cat. No. A9511).

Table 2 | Amino acid sequences of complementarity-determining region (CDR) sets introduced into human VH/VL single-domain antibody (sdAb) scaffolds for biophysical stability assessment.

*a IMGT positions 24–40 in the acceptor sdAb were replaced with the indicated sequence. bIMGT positions 56–69 in the acceptor sdAb were replaced with the indicated sequence. c IMGT positions 105–117 in the acceptor sdAb were replaced with the indicated sequence. dIMGT positions 35–40 in the acceptor sdAb were replaced with the indicated sequence. e IMGT positions 55–66 in the acceptor sdAb were replaced with the indicated sequence. f IMGT positions 105–117 in the acceptor sdAb were replaced with the indicated sequence.*

Recombinant human CTLA4 ectodomain (residues 1–162) was from Sino Biological (Beijing, China; Cat. No. 11159-H08H). *E. coli* 0157:H7 intimin (residues 658–934) fused C-terminally to maltose-binding protein (MBP-intimin) was from GenScript (Piscataway, NJ, USA). Horseradish peroxidase-conjugated antibodies used in ELISA (mouse anti-M13, Cat. No. 27942101; mouse anti-c-Myc, Cat. No.<PHONE_NUMBER>1; rabbit anti-6 × His, Cat. No. A190-114P) were from GE Healthcare (Piscataway, NJ, USA), Bethyl Laboratories (Montgomery, TX, USA) and Roche Diagnostics (Basel, Switzerland), respectively. M13K07 helper phage was from New England BioLabs (Ipswich, MA, USA) and M13K07ΔpIII hyper phage was from Progen Biotechnik (Heidelberg, Germany).

# Isolation of Antigen-Specific Human VH/VL sdAbs by Panning

Phage particles displaying monovalent VH/VL sdAbs were prepared by rescue of the three synthetic human VH/VL sdAb phagemid libraries with M13K07 helper phage as previously described (42). Briefly, 2 × YT broth (4 L) containing 1% (w/v) glucose and 100 µg/mL ampicillin was inoculated with 1 mL phagemid-bearing *E. coli* TG1 cells (7.3 × 1010 to 1.6 × 1011) and grown at 37°C with 250 rpm shaking to an OD600 of 0.5. Phagemid-bearing cells were superinfected with M13K07 helper phage at a multiplicity of infection of 20:1 at 37°C for 30 min with no shaking, then pelleted and resuspended in 6 L of 2 × YT broth containing 100 µg/mL ampicillin and 50 µg/mL kanamycin. The next day, phage particles were purified from bacterial supernatants using two rounds of polyethylene glycol precipitation; neither heat treatment nor filtration steps were used to remove residual bacterial cells. For panning, 5 µg of each protein in 35 µL phosphate-buffered saline (PBS), pH 7.4, was adsorbed overnight at 4°C in wells of NUNC MaxiSorp™ 96-well microtiter plates (Thermo-Fisher). The next day, wells were blocked with 200 µL PBS containing either 2% (w/v) bovine serum albumin (BSA) or 2% (w/v) skim milk for 1 h at 37°C, then rinsed 3 × with PBS. Approximately 5 × 1011 infective library phage particles (5 × 1012 virions) were added to wells in 100 µL of PBS containing 1% BSA or skim milk and 0.1% (v/v) Tween-20 for 2 h at room temperature. The blocking protein (BSA and skim milk) was switched in alternate rounds of panning, except for panning on HSA in which only skim milk was used. Wells were washed 5 × with PBS containing 0.1% Tween-20, 2 × with PBS and then bound phage were eluted for 10 min with 50 µL of 100 mM triethylamine, neutralized with 50 µL 1 M Tris-HCl, pH 7.5, and used to reinfect exponentially growing *E. coli* TG1 cells. The cultures were superinfected with M13K07 helper phage. The next day, amplified phage were purified by polyethylene glycol precipitation from 10 mL overnight cultures and used in subsequent panning rounds. After four or five rounds of selection, antigen-specific VH- or VL-displaying phage clones were identified by their binding in polyclonal and monoclonal phage ELISA as previously described (42).

# Selection for Monomeric and Expressible Human VH/VL sdAbs by Panning

Phage particles displaying monovalent VH/VL sdAbs were prepared by rescue of phagemid libraries with M13K07 helper phage as described above. Phage particles displaying multivalent VH/ VL sdAbs on all copies of pIII were prepared as described above, except that: (i) smaller volumes (100 mL) of 2 × YT broth were inoculated with 1 mL phagemid-bearing *E. coli* TG1 cells, (ii) phagemid libraries were rescued with M13K07ΔpIII hyper phage at a multiplicity of infection of 10:1, and (iii) the final overnight culture volume was 150 mL of 2 × YT broth.

For panning, 5 µg of either protein A (for the VHB82SS library; Thermo-Fisher) or protein L (for the VL383SS library; Thermo-Fisher) in 50 µL PBS, pH 7.4, was adsorbed overnight at 4°C in wells of NUNC MaxiSorp™ 96-well microtiter plates. The next day, wells were blocked with 300 µL PBS containing either 2% BSA or skim milk for 1 h at 37°C, then rinsed 1 × with PBS. Approximately 1011 infective library phage particles (1012 virions) were added to wells in 100 µL of PBS containing 1% skim milk and 0.1% Tween-20 for 2 h at room temperature. Wells were washed 5 × with PBS containing 0.1% Tween-20, 2 × with PBS and then bound phage were eluted for 10 min with 50 µL of 100 mM triethylamine, neutralized with 50 µL 1 M Tris-HCl, pH 7.5, and used to reinfect exponentially growing *E. coli* TG1 cells. The cultures were superinfected with either M13K07 helper phage or M13K07ΔpIII hyper phage. The next day, amplified phage were purified by polyethylene glycol precipitation from 10 mL overnight cultures and used in subsequent panning rounds. After three rounds of selection, pools of sdAb-phage (either phage bound by and eluted from protein A/L, or phage amplified from overnight cultures) were interrogated using next-generation DNA sequencing (NGS) as described below.

### Soluble VH/VL Protein Expression

Monomeric VH/VL sdAbs bearing C-terminal 6 × His and c-Myc tags were expressed from overnight cultures of *E. coli* TG1 cells grown in 250 mL to 1 L of 2 × YT broth under IPTG (isopropyl β-D-1-thiogalactopyranoside) induction, then extracted from periplasmic space by osmotic sucrose shock and purified by immobilized metal affinity chromatography as previously described (42). For small-scale expression screening, 5 mL overnight cultures were grown as above, lysed using FastBreak™ reagent (Promega, Madison, WI, USA) and sdAbs purified using PureProteome™ nickel magnetic beads (EMD Millipore, Billerica, MA, USA). Expression yields from 5 mL cultures were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA) as per manufacturer's instructions with a VHH sdAb of known concentration as the protein standard. Titration ELISAs using soluble VH/VL sdAbs were performed as previously described (42, 44, 45), using either anti-6 × His or anti-c-Myc secondary antibodies to detect binding.

# Size Exclusion Chromatography (SEC) and SEC with Multiangle Light Scattering (MALS)

Size exclusion chromatography analyses of monomeric VH/VL sdAbs were conducted using a Superdex™ 75 GL column (GE Healthcare) connected to an ÄKTA FPLC protein purification system (GE Healthcare) as previously described (42). UPLC-SEC-MALS analyses of VH/VL sdAbs were conducted essentially

#### Figure 1 | Continued

Biophysical stability assessment of complementarity-determining region (CDR)-shuffled human VH/VL single-domain antibody (sdAb) variants. (A,B) Expression yields of CDR-shuffled VL (A) and VH (B) sdAbs from 5 mL overnight cultures, as determined by Bradford assay. Two outliers (VL382 + CDR set 9, expression yield 467.7 µg/mL and VH428SS + CDR set 2, 356.4 µg/mL) are indicated by green triangles. (C,D) Aggregation tendencies of CDR-shuffled VL (C) and VH (D) sdAbs, as determined by SEC-MALS. (E,F) Melting temperatures (*Tm*s) of CDR-shuffled VL (E) and VH (F) sdAbs, as determined by thermal shift assay. (G,H) Turbidity upon thermal denaturation of CDR-shuffled VL (G) and VH (H) sdAbs, as measured by spectrophotometry at 360 nm wavelength. Dashed lines represent the range of turbidity measurements for unheated VH/VL sdAbs. The aggregation tendencies, *Tm*s and turbidities of CDR-shuffled variants of VH429 were not characterized due to inadequate expression yields. Horizontal lines represent mean (C,D,G,H) or median (A,B,E,F) values and wild-type VH/VL sdAbs with unmodified CDRs are shown in red. Open and solid circles represent VH/VL sdAbs with, or without, a stabilizing exogenous disulfide linkage. The number of data points in each panel reflects whether the experiment was conducted in singlicate (C,E) or duplicate (A,B,D,F–H) and for panels (C–H), whether the CDR-shuffled variant expressed sufficiently for analysis.

as previously described (39) using an Acquity BEH-125 column (Waters, Milford, MA) connected to an Acquity UPLC H-Class Bio system (Waters) with miniDAWN™ MALS detector and Optilab® UT-rEX™ refractometer (Wyatt Technology, Santa Barbara, CA, USA). VH/VL sdAbs (10–20 µg) were injected at 30°C in a mobile phase consisting of calcium- and magnesiumfree DPBS (GE Healthcare) at a flow rate of 0.4 mL/min. Weighted average molecular mass (*M*MALS) was calculated using a protein concentration determined using A280 from the PDA detector with extinction coefficients calculated from amino acid sequences. Data were processed using ASTRA 6.1 software (Wyatt).

#### Surface Plasmon Resonance (SPR)

All monomeric human VH/VL sdAbs were SEC-purified and buffer-exchanged into HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20, pH 7.4) prior to SPR analyses. All SPR analyses were performed on a Biacore™ 3000 instrument (GE Healthcare) at a temperature of 25°C. Briefly, proteins (CEACAM6, 370 resonance units (RUs); HSA, 1614 RUs; GITR, 796 RUs; MBP-intimin, 1536 RUs) were immobilized *via* amine coupling on research-grade CM5 sensor chips (GE Healthcare) in 10 mM acetate buffer, pH 4.0, according to the manufacturer's instructions. VH/VL sdAbs were injected at concentrations ranging from 1 nM to 10 µM, at flow rates of 20–50 µL/min and with contact time between 120 s and 300 s, then allowed to dissociate for 7–10 min. All surfaces were regenerated using 10 mM glycine, pH 1.5. Ethanolamine-blocked flow cells served as reference surfaces. The data were fitted to a 1:1 interaction model and binding affinities (*K*Ds) and/or kinetic parameters were determined either by steady-state analysis or by multicycle kinetic analysis using BIAevaluation 4.1 software (GE Healthcare).

Surface plasmon resonance analyses of two VL sdAbs (VLSS-2 and VLSS-5) against MBP-intimin were conducted as described above, except that 234 RUs of MBP-intimin were immobilized on a research-grade C1 sensor chip (GE Healthcare) *via* amine coupling in 10 mM acetate buffer, pH 4.5, according to the manufacturer's instructions. These two VL sdAbs were injected in HBS-EP buffer containing either 150 or 500 mM NaCl with an extended contact time (600 s).

#### Protein Turbidity Assays

VH/VL sdAb samples (0.2 mL) in 1.5 mL microcentrifuge tubes were heated to 85°C in a heating block for 10 min, then allowed to cool to room temperature for 30 min. Absorbance at 360 nm was measured pre- and post-heat treatment in a quartz NanoQuant Plate™ (Tecan, Männedorf, Switzerland) using an Infinite® M200 PRO microplate reader (Tecan). VH/VL sdAb concentrations were adjusted to 0.25 mg/mL in PBS prior to analysis.

#### Thermal Shift Assays

VH/VL sdAb samples (45 µL) in 96-well thin-wall optical plates (Bio-Rad) were mixed with 5 µL SYPRO® Orange (diluted 1:100 from 5,000 × stock; Life Technologies, Carlsbad, CA, USA) and sealed with optical quality sealing tape (Bio-Rad). Using an iQ™ 5 real-time PCR system (Bio-Rad), a temperature ramp of 1°C/min was applied and thermal unfolding was monitored by measuring fluorescence at 0.5°C intervals as previously described (46, 47). The wavelengths for excitation and emission were 490 and 575 nm, respectively. Melting temperatures (*T*ms) were calculated as the temperature at which the maximum rate of change in fluorescent signal (d(RFU)/d*t*) was achieved. VH/VL sdAb concentrations were adjusted to 1 mg/mL in PBS prior to analysis.

#### Circular Dichroism

*T*ms were also determined by circular dichroism as previously described (39, 40). Ellipticity of VH/VL sdAbs (100 µg/mL) was measured at wavelengths between 205 and 210 nm in 100 mM sodium phosphate buffer, pH 7.4. Ellipticity measurements were normalized to a percentage scale and then *T*ms were determined by plotting percent folded versus temperature and fitting the data to a Boltzmann distribution.

### Next-Generation DNA Sequencing

VH/VL sdAb libraries were interrogated using an Illumina MiSeq instrument as described previously (44, 45, 48). Amplicons for NGS were prepared using FR1- and FR4-specific barcoded primers (sequences in Table S1 in Supplementary Material) using as template either phagemid DNA (~10 ng) or phage virions (~106 particles).

#### Statistical Analyses

Descriptive statistics (mean, median) were used to describe datasets as described in figure legends. No inferential statistical tests were used.

### RESULTS

Antigen-specific human autonomous VH/VL sdAbs do not exist in nature, and are most commonly isolated from synthetically randomized *in vitro* display libraries. These molecules are notoriously unstable and aggregation prone (4), which probably negatively

#### Figure 2 | Continued

Design and construction of the VL383SS, VH428, and VHB82SS synthetic human VH/VL single-domain antibody (sdAb) libraries. (A) Design of the human VH/VL sdAb libraries. The parental VH/VL sdAb sequence is shown at top with IMGT numbering. Intradomain disulfide linkage-forming cysteine residues at positions 54 and 78 are shown in magenta. Randomization positions are highlighted in either red (no length polymorphism) or green (length polymorphism). Arabic numerals indicate the codon mixture (X1, X2, X3, X4, or X5) incorporated at each randomization position. (B) Expected amino acid frequencies at VH/VL sdAb library randomization positions. (C) Observed amino acid frequencies at VH/VL sdAb library randomization positions, measured from phagemid DNA isolated from *Escherichia coli* TG1 cells. Crossed-out cells indicate a frequency of <0.1%. (D) CDR3 length distributions of the VH/VL sdAb libraries. (E) Degree of randomized sequence diversity (Levenshtein distance, or number of amino acid changes with respect to the parental scaffold) in the VH/VL sdAb libraries. Analyses shown in (C–E) are representative of 7.7 × 104 to 7.9 × 105 sequences per library.

Table 3 | Attrition rates of human VH/VL single-domain antibodies (sdAbs) through isolation and characterization.

*a Double entries reflect two independent isolation attempts.*

impacts the selection of antigen-specific binders from synthetic VH/VL sdAb libraries. In an effort to mitigate these factors, we attempted to identify ultra-stable VH/VL sdAb scaffolds upon which we could construct highly diverse, stability-enhanced phage-displayed VH/VL sdAb libraries designed to yield soluble and thermostable antigen-specific human sdAbs.

### Biophysical Stability Assessment of Wild-Type Human VH/VL sdAb Scaffolds

The *T*ms and aggregation tendencies of 22 potential VH sdAb scaffolds and 18 potential VL sdAb scaffolds were determined by circular dichroism and SEC (Tables S2 and S3 in Supplementary Material). Five VH and five VL sdAb scaffolds as well as their disulfide-stabilized equivalents (**Table 1**) were selected as the most promising candidates for library construction based on: (i) primarily monomeric folding, (ii) high thermostability with reversible thermal unfolding, and (iii) reasonable expression yields (>1 mg/L) from overnight *E. coli* TG1 cultures. As reported previously (39, 40), incorporation of a disulfide linkage spanning IMGT positions 54–78 increased the *T*m of each scaffold by ~5–20°C but also had unpredictable effects on expression yields.

### Tolerance of Human VH/VL sdAb Scaffolds to CDR Modification

As a preliminary investigation into which of the 20 VH/VL sdAb scaffolds might best tolerate library randomization, we grafted 12 sets of exogenous CDRs (**Table 2**) into each scaffold and assessed the resulting molecules' expression level (Bradford assay), aggregation tendency (SEC-MALS), *T*m (thermal shift assay), and ability to refold after thermal denaturation (turbidity assay). For VL sdAb scaffolds, the 12 sets of exogenous CDRs were derived from non-antigen-specific human VL sdAbs isolated from previously constructed libraries with expression yields ≥10 mg/L and for VH sdAb scaffolds, the 12 sets of exogenous CDRs were selected from either human VH sdAbs of unknown antigen specificity isolated from previously constructed libraries or camelid VHH sdAbs with expression yields ≥3 mg/L.

The VL sdAb scaffolds yielding the best-expressing CDRshuffled variants were VL383SS, VL325SS and VL335SS (**Figure 1A**). CDR-shuffled variants of VL383SS and VL325SS were primarily monomeric by SEC-MALS, while CDR-shuffled variants of VL335SS showed some tendency to aggregate (**Figure 1C**). All three of these VL sdAb scaffolds, especially VL325SS, yielded CDR-shuffled variants with high *Tm*s (**Figure 1E**); however, many CDR-shuffled variants of VL325SS showed significant turbidity upon heat denaturation (**Figure 1G**), reflecting their inability to refold as soluble monomers. The VH sdAb scaffolds yielding the best-expressing CDR-shuffled variants were VH420, VH428, VHB82, VHB82SS, and VHM81SS (**Figure 1B**). CDR-shuffled variants of all five of these VH sdAb scaffolds were similarly monomeric (**Figure 1D**) but as expected, the two disulfide-stabilized sdAb scaffolds (VHB82SS and VHM81SS) yielded CDR-shuffled variants with higher *Tm*s (**Figure 1F**). Similar proportions of the CDR-shuffled variants of all five VH sdAb scaffolds showed minor turbidity upon heat denaturation (**Figure 1H**). On the basis of these data, we selected one VL sdAb scaffold (VL383SS) and two VH sdAb scaffolds (VH428 and VHB82SS) for library construction, reflecting a balance of their tolerance to CDR modification as well as their differing CDR3 lengths and germline gene rearrangements.

### Design and Construction of Phage-Displayed Human VH/VL sdAb Libraries

Three synthetic sdAb libraries (VL383SS, VH428, and VHB82SS) were constructed by limited *in vitro* randomization of the CDRs of these VH/VL sdAb scaffolds and cloning of the resulting DNA into the pMED1 phagemid vector (**Figures 2A,B**). CDR3 length was varied in each library (**Figures 2A,D**; VL383SS library: 8, 9, or 10 residues; VH428 and VHB82SS libraries: 10, 12, 14, 16, 18, 20, or 22 residues) while CDR1 and CDR2 lengths were constant. The major technical advances of these over our previously described phage-displayed synthetic human VH/VL sdAb libraries (12, 21, 31) were (i) trinucleotide mutagenesis allowed for incorporation of defined mixtures of amino acids at each randomization position, based on alignments of human and llama antibodies with additional bias toward solubilitypromoting residues (Asp, Glu) and against undesirable residues (Asn, Cys, and Met) and stop codons (**Figures 2B,C**; Figure S2 in Supplementary Material) and (ii) near-complete randomization of all three CDRs of each VH/VL sdAb scaffold resulted in much higher sequence diversity for the vast majority of library molecules (**Figure 2E**). All three VH/VL sdAb libraries were transformed into *E. coli* TG1 cells, yielding final library sizes (1.0–2.3 × 1010 independent transformants) that, as expected, vastly under-sampled theoretical library diversity (VL383SS: 2.9 × 1013 unique sequences; VHB82SS: 2.3 × 1033 unique sequences; VH428: 2.3 × 1035 unique sequences). At this stage, prior to helper phage rescue, no major clonality biases or deviations from library design were observed (data not shown).

# Isolation and Characterization of Antigen-Specific Human VH/VL sdAbs

All three VH/VL sdAb libraries were rescued with M13K07 helper phage and panned for four or five rounds against five model antigens (CEACAM6, CTLA4, GITR, HSA, and MBPintimin). All library pannings were performed in duplicate by two independent operators at the same time and using the same materials, except for panning against MBP-intimin, in which

Figure 3 | Properties of selected antigen-specific single-domain antibodies (sdAbs) isolated from the VL383SS, VH428, and VHB82SS synthetic human VH/VL sdAb libraries. The single highest affinity sdAb against each target is shown, except for MBP-intimin, where VLSS-3 is shown instead of VLSS-4 or VLSS-5 because of better fit to a 1:1 binding model. (A) Size exclusion chromatography (SEC) profiles of representative antigen-specific human VH/VL sdAbs. Arrows show molecular mass standards, from left to right: thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa). (B) Thermal unfolding of representative antigen-specific VH/VL sdAbs as determined by circular dichroism. Replicate unfolding curves are shown in red and blue; the sdAbs were then cooled to room temperature and remelted (shown in orange and green). (C) Titration ELISA of representative antigen-specific VH/VL sdAbs. Horseradish peroxidase mouse anti-c-Myc secondary antibody (clone 9E10, diluted 1:3,000) was used for detection. (D) Binding of antigen-specific VH/VL sdAbs to cognate antigen by SPR. Each antigen was immobilized on a CM5 sensor chip using amine coupling, then the indicated VH or VL sdAb was flowed over the surface at concentrations ranging from 25 to 1,000 nM (α-CEACAM6 VHB82SS-2), 25 to 500 nM (α-GITR VHB82SS-6), 2.5 to 50 nM (α-HSA VHB82SS-7), or 62.5 to 2,500 nM (α-MBP-intimin VL383SS-3). Black lines show data and red lines show fits.

*a Determined using steady-state analysis at 25°C.*

*bDetermined using multicycle kinetic analysis and fitting to a 1:1 binding model at 25°C. c Antigen was immobilized on a C1 sensor chip, evidence of complex binding. dKD is an estimate only due to lack of curvature in the steady-state plot.*

*e Size exclusion chromatography elution volume suggests VHB82SS-6 is a strict dimer. n.d., not determined.*

panning was done in triplicate. High attrition rates of VH/VL sdAbs showing false positive binding either as sdAb-phage or as soluble sdAb proteins (VL383SS: 53% attrition; VH428: 69% attrition; VHB82ss: 71% attrition; **Table 3**) resulted in a final yield of 20 unique antigen-specific VH/VL sdAbs against four targets. Of the 15 library-target screens we conducted, eight (53%) yielded at least one antigen-specific VH/VL sdAb (four screens yielded one sdAb, two screens yielded two sdAbs, one screen yielded three sdAbs, and two screens yielded five sdAbs). Most of the recovered sdAbs were monomeric (**Figure 3A**; **Table 4**; Figure S3 in Supplementary Material), thermostable (**Figure 3B**; **Table 4**), and had affinities for antigen ranging from 5 nM to ~12 μM (**Figures 3C,D**; **Table 4**). One notable exception was the α-GITR VH sdAb VHB82SS-6, whose elution volume by SEC suggested it existed in solution as a strict dimer (**Figure 3A**). Stable homodimerization has previously been reported for several human VH sdAbs and was critical for antigen binding (49–52); this phenomenon is distinct from the general tendency of some VH domains to forms soluble aggregates. Antigen-specific VH/VL sdAbs isolated from all three libraries had aggregation tendencies and thermostabilities reflective of the parental VH/VL sdAb scaffold from which they were derived, but generally poorer expression yields (**Figure 4A**). Reproducibility in the selection of specific sdAb sequences from the libraries was modest, with only 45% (9/20) of antigen-specific VH/VL sdAbs recovered by both operators (**Figure 4B**) and no apparent connection between antigen-binding affinity and consistency of isolation.

# Evaluation of Stability-Sequence Diversity Tradeoffs in Human VH/VL sdAb Libraries

To better understand potential constraints on the CDR sequences of monomeric and stable human VH/VL sdAbs, we examined the effects of stability selection (three rounds of protein A or L selection followed by amplification of the eluted phage overnight in *E. coli* TG1 cells; **Figure 5**) on randomized sequence diversity of two VH/VL sdAb libraries (VL383SS and VHB82SS). We performed this experiment in duplicate using either phage particles displaying monovalent sdAb (rescued with M13K07 helper phage; simultaneous expression of pIII from helper phage and pIII-sdAb from phagemid) or phage particles displaying multivalent sdAb (rescued with M13K07ΔpIII hyper phage; only pIII-sdAb expressed from phagemid). Out-of-frame and/or stop-codon-encoding VH/VL sdAb clones were rare in both libraries ("Library Cells"), but rose substantially in frequency upon phage rescue ("Library Phage") and upon amplification in *E. coli* ("Amplification in *E. coli*"), suggesting a growth advantage for non-sdAb expressing cells over sdAb-expressing ones (**Figures 5A,B**); this phenomenon was mitigated somewhat using hyper phage rescue. After three rounds of protein A/L selection and overnight amplification in *E. coli* with helper phage rescue, there were clear clonal biases observed in the resulting populations of sdAb-phage (**Figures 5C,D**). Using an arbitrary frequency cutoff (0.00006% for VL383SS and 0.00004% for VHB82SS; sdAbs at frequencies greater than these cutoffs were not present in any other dataset) to identify unique sdAb sequences enriched by stability selection, we found that different sdAb clones were selected in the two replicate pannings, although both sets of sdAbs were heavily biased toward the parental VH/VL sdAb scaffold's CDR3 length (9 residues for the VL383SS library and 10 residues for the VHB82SS library, which is the shortest CDR3 length possible in the library design and the nearest to the VHB82SS scaffold's CDR3 length of 6 residues; Figure S4 in Supplementary Material). However, stability selection reproducibly yielded VH/VL sdAbs with biased CDR amino acid composition (**Figures 5E,F**; Figures S5 and S6 in Supplementary Material). In order of magnitude, stability biases in the VL383SS library favored Asp, His, Pro, Ser and Thr and disfavored aromatic (Phe, Trp, Tyr) and hydrophobic (Ile, Leu, Val) residues. Similarly, stability biases in the VHB82SS library favored Asp, His, Pro, and Gln and disfavored aromatic (Phe, Trp, Tyr) and hydrophobic (Ile, Leu, Val) residues. These biases were especially acute at the C-terminus of CDR1 and N-terminus of CDR2, both of which flank FR2. Identical molecular signatures were observed to a lesser degree in the pools of all sdAb-phage (irrespective of frequency or mono- vs. multivalent display format) subjected to three rounds of stability selection (Figures S7 and S8 in Supplementary Material) as well as in the pools of sdAb-phage after phage rescue and eluted from protein A/L (data not shown), suggesting a common ontogeny.

#### DISCUSSION

The starting point for this investigation was the observation that several of our previously described synthetic human VH/VL sdAb libraries performed unpredictably (12, 21, 31), yielding monomeric antigen-specific binders against some targets but

not others. Thus, we tested the hypothesis that these issues might be solved by: (i) building libraries on ultrastable VH/VL sdAb scaffolds that maintain their biophysical properties upon CDR modification and (ii) randomizing the CDRs of the VH/VL sdAb libraries more extensively using trinucleotide mutagenesis. A few general lessons became apparent during the course of these experiments. First, although VH/VL sdAb scaffolds clearly vary in their tolerance to CDR modification, no "perfect" scaffold exists (i.e., some CDR-shuffled variants of every scaffold showed poor biophysical properties). Second, the trinucleotide mutagenesis approach used here was dramatically more effective at incorporating randomized sequence diversity in VH/VL sdAb CDRs compared to the Kunkel mutagenesis (21, 31) and splicing by overlap extension PCR mutagenesis approaches using degenerate oligonucleotides (12) that we have used previously. Third, as we showed previously (31), incorporating a stabilizing exogenous disulfide linkage spanning IMGT positions 54–78 into VH/VL sdAb libraries is clearly compatible with selection of antigen-specific binders, as 15/20 of the binders shown here bear this linkage. Finally, it is clearly possible, at least under some circumstance, to select monomeric, high-affinity binders from synthetic human VH/VL sdAbs libraries, as evidenced by the α-HSA VHSS-7 sdAb (*K*D: 5 nM) reported here.

However, the more general issues afflicting synthetic human VH/VL sdAb libraries (high attrition rates due to false positive binding, possibly caused by sdAb aggregation; inconsistency of isolation; variable expression yield and aggregation of isolated sdAbs) were not completely solved by using more stable VH/VL scaffolds or building more diverse and non-degenerate libraries. The additional randomized sequence diversity we achieved by the use of trinucleotide mutagenesis is almost certainly negligible when compared to the theoretical size of the libraries, and massive undersampling may account at least in part for inconsistent isolation of sdAbs. Other potential solutions to the problem

#### Figure 5 | Continued

Impact of stability selection on human VH/VL single-domain antibody (sdAb) library sequence diversity. After transformation of *Escherichia coli* TG1 cells with phagemid DNA, phage were rescued by superinfection of overnight cultures with M13K07 helper phage or M13K07ΔpIII hyper phage. The purified sdAb-displaying phage were bound and eluted from protein A (VHB82SS library) or protein L (VL383SS library), then amplified by reinfection of *E. coli* TG1 cells. Three rounds of panning were performed, and VH/VL sdAb sequences were interrogated by NGS at the stage of rescued phage, phage eluted after a single round of protein A/L selection, and phage amplified after the final round of panning. (A,B) Proportion of functional sdAb sequences (in-frame ORF, no stop codons) observed in VL (A) and VH (B) library phage and panning outputs. (C,D) Clonality of VL (C) and VH (D) library phage and panning outputs. (E,F) Complementarity-determining region (CDR) amino acid composition of enriched VL (E) and VH (F) sdAb clones after three rounds of stability selection. Crossed-out cells indicate a frequency of <0.1%. Analyses in (A–F) are representative of 7.7 × 104 to 7.9 × 105 sequences per sample.

of undersampling include increasing throughput (53), use of VH/VL sdAb-transgenic mice (54, 55) and use of *in vitro* VDJ recombination systems (56). Nonetheless, modest inter-operator reproducibility in isolating the same binders clearly demonstrates that antigen-specific human VH/VL sdAbs are not always isolated even when they were present in the library. We currently have no understanding of the factors influencing the number of VH/VL sdAb binders isolated, their affinities or biophysical properties, nor if these depend on the library, the target antigen quality or composition, or the panning methodology. One possibility, for example, is that the numbers of input library sdAb-phage used here, the target antigen surface size and density, and/or the numbers of eluted phage amplified from each round of selection were inadequate to consistently recover very rare sdAb specificities that may be present in the libraries. These factors could be investigated empirically in future studies.

It is virtually certain that some of the challenges of human synthetic VH/VL sdAbs relate to fundamental tradeoffs between stability and sequence diversity. There is no necessary reason why rare autonomous rearranged human VH/VL sdAbs should be compatible with any CDR sequence; rather, it should be expected that these molecules rely chiefly on particular CDR sequences for their solubility and stability, given that human VH and VL domains have evolved to be paired with one another, occluding a hydrophobic surface between the two domains. We expect that such challenges are much less of a problem for protein domains that naturally exist as soluble monomers such as camelid and shark sdAbs and non-antibody scaffolds (e.g., FN3 and SH3 domains). Some camelid VHHs rely on CDR residues for solubility as well (57), albeit likely to a lesser extent due to the presence of solubility-enhancing FR2 residues. One obvious explanation for the previously described bias toward negatively charged residues resulting in acidic overall pIs of VH/VL sdAbs (4, 12), especially in CDR1 and CDR2 (10, 11), is that this may enhance solubility and aggregation resistance. The role of charge and negative charge in particular in solubilizing VH/VL sdAbs with hydrophobic CDRs has been previously demonstrated (58, 59), and appears to be highly position and scaffold dependent (4). Bias in favor of His residues is less easy to explain, although its imidazole side chain may also have stabilizing and solubilizing effects near physiological pH (60). Notably, "camelized" human VH sdAbs and human VH/VL sdAbs selected *in vitro* to bear solubility-promoting FR substitutions (9, 61), may not be subject to the same stability-sequence diversity tradeoffs. We caution that although the growth advantages conferred by VH/VL sdAbs with the most stable and soluble CDR sequences were most apparent using helper phage rescue of phagemid libraries, forcing pIII-sdAb expression using hyper phage rescue or by using phage (not phagemid) or yeast display systems would not be expected to circumvent the issue of bias; instead, it might be expected to result in immediate loss of a large population of VH/VL sdAbs with poorly stable and soluble CDR sequences.

In conclusion, we have described three novel synthetic human VH/VL sdAb libraries as well as antigen-specific binders against a variety of target antigens selected from these libraries. Future work will seek to better understand the constraints imposed on human VH/VL sdAbs by stability-sequence diversity tradeoffs, and whether it is possible to circumvent them by *in vitro* engineering of human autonomous immunoglobulin variable domain folds. One possibility would be to identify and fix a minimal set of stability-enhancing CDR residues in human VH/VL sdAb libraries, which might allow for more effective randomization of the remaining CDR positions. However, doing so without significant divergence from human germline IGHV sequences, increasing the risk of immunogenicity, may be very challenging.

### AUTHOR CONTRIBUTIONS

DK, HK, and JT isolated and characterized human VH/VL sdAb scaffolds and conducted CDR-shuffling experiments and designed the libraries. JS conducted SEC-MALS experiments. ML, HK, and KH isolated and characterized antigen-specific sdAbs. ML and KH conceived and carried out stability selections and next-generation DNA sequencing analyses. QY, GH, and CRM designed, carried out, and analyzed surface plasmon resonance experiments. KH and GH made the figures and KH wrote the manuscript. All authors read and approved the final manuscript.

### ACKNOWLEDGMENTS

We gratefully acknowledge the excellent technical help of Camille Hebert-Martineau, Sonia Leclerc, Hongtao Qi, Ken Chan, Shannon Ryan, and Julie Champagne.

### FUNDING

This work was supported by funding from the National Research Council Canada.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2017.01759/ full#supplementary-material.

# REFERENCES

and bevacizumab and improved capacity over aflibercept. *J Biol Chem* (2016) 291:5500–11. doi:10.1074/jbc.M115.691162

**Conflict of Interest Statement:** JT and DK are inventors of US patents 8293233B2 and 9371371B2 as well as other patents and patent applications governing human VH/VL sdAb scaffolds.

*Copyright © 2015 Her Majesty the Queen in Right of Canada. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Expression Cloning and Production of Human Heavy-Chain-Only Antibodies from Murine Transgenic Plasma Cells

*Dubravka Drabek1 \*, Rick Janssens1 , Ernie de Boer1 , Rik Rademaker2 , Johannes Kloess3 , John Skehel3 and Frank Grosveld1,2*

*1Department of Cell Biology, Erasmus MC, Rotterdam, Netherlands, 2Harbour Antibodies BV, Rotterdam, Netherlands, 3WHO Influenza Centre, Frances Crick Institute, London, UK*

#### *Edited by:*

*Kevin A. Henry, National Research Council Canada, Canada*

#### *Reviewed by:*

*Johan Nilvebrant, Royal Institute of Technology, Sweden Dae Young Kim, Osong Medical Innovation Foundation, South Korea Andrew Hayhurst, Texas Biomedical Research Institute, USA*

> *\*Correspondence: Dubravka Drabek<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 28 September 2016 Accepted: 06 December 2016 Published: 19 December 2016*

#### *Citation:*

*Drabek D, Janssens R, de Boer E, Rademaker R, Kloess J, Skehel J and Grosveld F (2016) Expression Cloning and Production of Human Heavy-Chain-Only Antibodies from Murine Transgenic Plasma Cells. Front. Immunol. 7:619. doi: 10.3389/fimmu.2016.00619*

Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH–VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs.

Keywords: plasma cells, transgenic mice, human VH, HCAb, HEK cell library

# INTRODUCTION

*Camelidae* produce not only conventional antibodies, composed of two heavy and two light chains (H2L2), but also antibodies composed of heavy chains only. Although in the conventional antibodies both chains contribute to the antigen binding site, the antigen binding site of camelid heavy-chain-only antibodies (HCAbs) is formed by single heavy chain variable domain (VHH) (1, 2). We have previously generated transgenic mice containing hybrid llama-human antibody loci with two llama variable VHH regions and human D, J, and Cµ and/or Cγ constant regions. Such loci rearrange productively and rescue B cell development efficiently (3).

Heavy-chain-only antibodies are expressed at high levels in camelids (4) and in transgenic mice (3, 5), provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling, and antigen-specific heavy-chain-only IgGs are produced upon immunization (3). Camelid VHH segments are soluble and this is attributed to the presence of a germ line-encoded tetrad of specific hydrophilic amino acid substitutions at the hydrophobic interface of the conventional VH domain that normally interacts with a variable light Drabek et al. Human HCAbs from Transgenic Mouse

chain domain (VL) (6) and a CDR3 loop that folds over the VHH, covering the side of the domain that normally interacts with a VL domain (7).

In contrast, human VH domains usually aggregate and are less stable due to exposure of the hydrophobic amino acids at the former interface (8) and the loss of contacts between the V regions, respectively. This limits their applicability [see Rosenberg (9) and Fahrner et al. (10)]. However, extensive engineering and selection (7, 8) mainly by increasing the hydrophilicity of the VH domain (8) and by replacing exposed hydrophobic residues in the CDR3 region (7) will increase the solubility of the VH domain. These methods have the disadvantage that they require extensive work and that amino acid changes particularly in the CDR3 region could reduce or change the specificity and affinity of antigen binding.

We hypothesized that the mouse would be much more effective at such engineering *in vivo* through the natural process of selection. We, therefore, introduced a fully human HCAb locus into mice to generate fully human HCAbs of different classes or fragments thereof in response to antigen challenge for use as therapeutic agents in man. To this end, we replaced the llama VHH domains with human VH domains in the transgenic construct used by Janssens et al. (3), generated a number of transgenic lines, and derived a number of HCAb against different antigens by hybridoma and phage display technology. Both the hybridoma and phage display technologies have a number of disadvantages, are quite laborious, and in addition phage display needs additional full-format HCAb recloning in eukaryotic systems.

It has been known that long-term production of Abs is maintained by a combination of short-lived and long-lived plasma cells (PCs), usually defined functionally as Ab-secreting cells (ASC). Although short-lived ASC die within 3–5 days, Ab levels can be maintained by continuous proliferation and differentiation of memory B cells (MBC) into short-lived ASC upon continuous reactivation (11, 12), such as persistent antigen exposure. Alternatively, long-term production of Ab is maintained by long-lived ASC, which migrate to survival niches within the bone marrow (13, 14) and spleen (15). Thus, we used CD138<sup>+</sup> CD45R B220low/− CD19low/<sup>−</sup> antibody-secreting PCs (16), bone marrow, and spleen of immunized mice containing a human HCAb locus (4HVH) as the enriched RNA source for the production of an expression library.

Here, we describe an automatable alternative method for rapid cloning and identification of antigen-specific HCAbs from immunized transgenic mice (4HVH) carrying a fully human heavy chain locus by cloning the VDJ region of the HCAb cDNA directly into a mammalian expression vector and identifying the human embryonic kidney 293 T (HEK293T) clones secreting antigen-specific HCAb (See **Figure 1**).

### METHODS

#### Immunization

4HVH transgenic mice and control wild-type (WT) mice were immunized according to the protocol approved by the Dutch

Experimental animal committee DEC Nr EUR 1944. Briefly, mice were injected i.p. five times at 2-week intervals with the influenza virus X-31 hemagglutinin (HA), prepared as described by Ruigrok et al. (17), and dissolved in phosphate-buffered saline (PBS) at pH 7.4 using Stimune adjuvant (Prionics, Switzerland) according to the formulation provided by the supplier. The last injection was without the adjuvant. Four days after the last injection, 4HVH mice were sacrificed and PCs isolated.

### PC Isolation and Library Construction

A single-cell suspension was prepared from spleens and femurs in 0.5% BSA, 2 mM EDTA in PBS. Cells were counted (Burke chamber), and magnetic cell sorting of CD138<sup>+</sup> cells was performed using mouse CD138<sup>+</sup> plasma isolation kit (Miltenyi Biotec GmbH, Germany) according to the manufacturer's instructions. Basically, this consists of two steps: first a depletion of non-PCs by indirect magnetic labeling of CD49b and CD45R cells with a non-PC depletion cocktail and anti-biotin microBeads followed by magnetic separation using LD columns (Miltenyi Biotec GmbH, Germany), and the next step is a positive selection of PCs by direct labeling with CD138 MicroBeads followed by magnetic separation on a MS column (Miltenyi Biotec GmbH, Germany). PCs eluted from the column were spun down and the pellet resuspended in 400 µl of Ultraspec™ RNA reagent (Biotecx laboratories, Inc., Houston, TX, USA). Total RNA was made according to the manufacturer's instructions. The RNA was dissolved in 20 µl of dH2O and 3 µg was used in a 20 µl reaction volume for a first strand cDNA synthesis using SuperScript™ II RT (Invitrogen by Fisher Scientific, USA) according to the instructions using oligo dT priming with oligo (dT) 12-18 primer (Invitrogen by Life Technologies, USA). The transgenic mice contain four different VHs (1-46, 3-11, 3-53, and 3-23), and hence three different leader-specific primers were designed for the 5′-end. All of them contained a *Pvu*II site that is unique in the final expression vectors (pCAG hygro G2 and pCAG hygro G3 containing leader sequence from VH3-23). The following 5′-primers were used:

#### lib-3-23/53-S: 5′-GTGTCCAGTGTGAGGTGCAGCTG-3′, lib-3-11-S: 5′-GTGTCCAGTGTCAGGTGCAGCTG-3′, and lib-1-46-S: 5′-GTGCTCACTCCCAGGTGCAGCTG.

For the 3′-end, we used primer HINGEIgG2rv [previously used and described for phage display library (3)] and HINGEIgG3rv: 5′-AATTGTGTGAGCGGCCGCACCAAGTGGGGTTTT GAGCTC.

An additional 3′-end primer was used: lib-IgG2/3-CH3-AS: 5′-CTGACCTGGTTCTTGGTCATCTCCTC.

This primer from the CH3 constant region is common for both G2 and G3. However, in combination with any of the 5′-primers, it amplifies only IgG2. To make sure that all of the VHs were represented in the library, each of the 5′-end primers was used separately in combination with each of the 3′-end primers, and the products were mixed in an equimolar ratio later. PCR was performed with high-fidelity DNA polymerase Phusion™ (New England Biolabs, Inc., USA) using cycling conditions recommended by the manufacturer for the three-step protocol with an annealing temperature of 68°C and 35 cycles in total. PCR products for IgG2 were cut either with *Pvu*II/*Bst*EII or *Pvu*II/*Bsr*GI (unique site in the constant region) if the amplified fragment originated from using the combination with the Lib-IgG2/3-CH3 primer and cloned into *Pvu*II/*Bst*EII or *Pvu*II/*Bsr*GI cut and phosphatase-treated pCAGhygro G2 vector.

PCR products for IgG3 were digested with *Pvu*II/*Bst*EII or *Pvu*II/*Sac*I and cloned into *Pvu*II/*Bst*EII cut and phosphatasetreated pCAGhygro G2 or *Pvu*II/*Sac*I cut and phosphatasetreated pCAGhygro G3 vector.

pCAGhygro G2 and pCAG hygro G3 are depicted in **Figure 2**. In short, the vector contains an ampicillin resistance gene for bacteria selection and a hygromycin resistance for the eukaryotic cell selection. The HCAb expression is driven by ubiquitous CMV enhancer and chicken beta actin promoter. The human VH 3-23 leader is responsible for secretion of the antibody. Constant regions have a deleted CH1 exon.

### Transformation, Plasmid Preparation, and Transfection into HEK 293T Cells

All ligations were done overnight at 16°C in 10 µl total volume using 120 ng of vector alone (control) or in combination with 30 ng of insert using T4DNA ligase (Promega, Madison, WI, USA). Ligations were diluted 5× (40 µl of dH2O into 10 µl ligation), and 1 µl was used to transform 20 µl of electrocompetent MegaX DH10B™ T1® cells (Invitrogen by Life Technologies, Grand Island, NY, USA) according to the instructions provided by the manufacturer.

Plating was done on 2XTY/Amp agar plates, and 960 individual colonies were picked into 1.5 ml of 2XTY/Amp medium in 96-well format. A total of 960 DNA plasmid preparations were done using NucleoSpin R96 flash (Macherey-Nagel, Germany) designed for rapid manual and automated 96-well DNA preparation of high- and low-copy plasmid and Bac DNA from *Escherichia coli*. Each DNA was dissolved in 50 µl sterile dH2O. A total of 200 ng of DNA (estimated to be in10 µl of DNA preparations based on random sample concentration measurements) was used for transfection into HEK 293T cells. The HEK 293T cells were plated into 96-well plates and transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions for 96-well format. The following day, medium was removed and replaced with DMEM medium (Lonza, Belgium) supplemented with hygromycin (Roche, Diagnostics GmbH, Germany) at a concentration of 200 µg/ml, non-essential amino acids (NEAA, Lonza, Belgium), and 10% fetal calf serum (FCS). On day 4 posttransfection, 100 µl of medium was taken from each well for an antigen-specific enzyme-linked immunosorbent assay (ELISA) and replaced with a fresh medium.

#### ELISA Assay

Enzyme-linked immunosorbent assay plates were coated overnight at 4°C with 5 µg/ml of antigen in PBS or PBS only. Blocking was done for 1 h at room temperature (RT) with 1% milk 1% BSA/PBS (W/V). This was done to exclude possible "sticky" binders to plastic or non-specific binders to BSA/milk. Washing steps included 3× PBS/0.05% Tween-20 and 3× PBS. A total of 50 µl of transgenic mouse serum were diluted in PBS (for initial testing of immunized animals), or supernatants from HEK 293T cells were mixed with 50 µl of 2% milk 2% BSA/PBS (W/V) and incubated for 1 h at RT. After washing, the antigen-specific serum/supernatants were detected by incubation with goat antihuman IgG Fc coupled to horse radish peroxidase (HRP; Jackson Immuno Research Laboratories, Inc., USA) diluted 1:5,000 for 1 h at RT, followed by washing steps and incubation with peroxidase substrate BM Blue POD (Roche Diagnostics GmbH, Germany). The reaction was stopped with 1M H2SO4, and the absorption was measured at 450 nm (against reference wavelength 690 nm).

For the initial testing of immunized WT and 4HVH transgenic mice sera for the presence of antigen-specific mouse antibodies, polyclonal goat antimouse immunoglobulins, HRP (Dako, Denmark) diluted 1:2,000 was used.

#### Sequencing

Sequencing of the positive DNA clones was done using primer CAG seq2-s 5′-GCTGGTTATTGTGCTGTCTCATC-3′.

#### Initial Affinity Measurement Screen and Full Kinetics

Medium was collected from HEK 293T cells, stably transfected with an HCAb expression vector and grown to confluence. To screen for the clones that expressed the highest affinity HCAb for HA, 200 µl of HEK 293T cell medium from each clone was transferred to a single well of a black 96-well microtiter plate (Greiner Bio-One, Germany). The binding of the HCAbs to influenza HA was studied using the Octet QK (ForteBio, USA). Anti-human IgG-coated tips (ForteBio, USA) were incubated with PBS/0.05% Tween-20 (120 s) to establish baseline signals. To allow the capturing of HCAbs, the tips were then transferred to 200 µl HEK 293T medium containing HCAbs (600 s), HEK 293T medium without HCAbs and PBS/0.05% Tween-20 as a reference. Subsequently, the tips were transferred to 200 µl PBS/0.05% Tween-20 (480 s) to establish the binding levels after the dissociation of non-specific interactions. Next, the loaded tips were transferred to 200 µl 512 nM bromelain-released HA (BHA; 600 s) to allow HA binding to the captured anti-HA HCAbs and to 200 µl PBS/0.05% Tween-20 (1,200 s) to determine the dissociation rate of the HA from the HCAb.

The reference binding signal was subtracted from the binding curves, and the binding data were fitted to a 1:1 binding model using Octet 4.0 software. The HCAb clones that showed binding in initial affinity screen were selected for further analysis.

Full kinetics experiments were performed with purified HCAbs dialyzed against PBS. Anti-human IgG(Fc) sensors were dipped for 180 s in PBS/0.05% Tween 20. HCAb (10 µg/ml) was used for the loading step (300 s, 900 rpm), followed by 180 s in PBS/0.05% Tween 20. The association step with HA ligand in a concentration range from 0 to 512 nM was performed (600 s at 900 rpm), followed by dissociation step of 1,200 s in PBS/005% Tween 20 at 900 rpm. All steps were done in 200 µl volume. The buffer only well (PBS/0.05% Tween 20) was used as a reference well. The reference binding signal was subtracted from the binding curves, and the binding data were fitted to a1:1 binding model using Octet 7.1 software.

# Production and Purification of Anti-HA HCAbs

Positive clones were further expanded in medium containing 10% FCS. Alternatively, plasmid DNA was linearized with *Hin*dIII, stably transfected into HEK293T cells, and individual clones picked for the purpose of selecting the best expressors.

For production purposes, clones were grown in 15 cm Petri dishes in 25 ml of OPTI-MEM® (1×) + GlutaMAX™ (Gibco by Life technologies, CA, USA) medium. Medium was collected and replaced twice per week. Collected medium was spun down at 1,000 rpm (Eppendorf centrifuge 5810R) for 5 min to remove cell debris, and HCAbs were purified on Protein A agarose Fast Flow 50% (V/V; Sigma, USA). A total of 100 µl of Protein A beads were incubated with 50 ml of medium overnight at 4°C on a rotating wheel. After spinning at 1,000 rpm for 5 min, beads were washed in PBS/0.01% Tween-20 and loaded onto homemade columns (insulin syringe with cotton wool), washed 3× with PBS/0.01% Tween-20, 3× with PBS, and eluted with 3M potassium thiocyanate (KSCN). Eluted HCAbs were dialyzed for 5–6 h using Spectra/Por dialysis membrane MWCO 10000 (Spectrum Laboratories Inc., USA) against 1,000× volume excess of PBS at 4°C. The procedure was repeated three times.

# Production and Purification of VH Domains in *E. coli*

VH domains from HCAbs were cloned without a tag into pET SUMO vector (Champion pET SUMO Expression system, Invitrogen, CA, USA). Production of fusion protein, cleavage, and removal of SUMO and SUMO protease were done according to the manufacturer's instruction with recommended reagents.

VH domains in fusion with SUMO were purified from inclusion bodies starting with 200 ml BL21 transfected cells after 4 h of induction (1 mM IPTG). Cells were harvested, pellet frozen overnight, and lysed in 20 ml lysis buffer (50 mM KPO4, pH 7.8; 400 mM NaCl; 10 mM KCl; 10% glycerol; and 0.5% Triton X-100). After sonication 20 × 20 s ampl 12 and spinning for 15 min at 4,000 rpm at 4°C, pellet was taken in 1 ml PBS with addition of 10 µl benzonase for 30 min at RT. After washing in washing buffer (100 mM Tris pH 7.5; 5 mM EDTA; 2 M urea; and 2% Triton X-100), sample was spun for 15 min at 4,000 rpm, washed 2× in 10 ml of 100 mM Tris pH 7.5 and 5 mM EDTA. Pellet was taken into 8 M urea; 10 mM Tris pH 7.5; and 2 mM DTT, rotated for 2 h at RT, spun for 30 min, 15,000 rpm at 4°C, and the supernatant was used in refolding. Base refolding buffer 2 from Pierce refolding kit was used (440 mM 1-arginine; 55 mM Tris pH 8.2; 21 mM NaCl; and 0.88 mM KCl). Protein was diluted to 1 mg/ml; in 8 M urea, 10 mM Tris pH 7.5; and 2 mM DTT. To fold 0.5 mg of protein, we used 9 ml base refolding buffer, 100 µl 0.1 M EDTA, 6.75 mg GSH (reduced glutathione), 2.65 mg of GSSG (oxidized glutathione), and H2O up to 9.5 ml. Protein was added in 50 µl aliquots, well mixed, and left on ice for at least 1 min after each addition. After overnight incubation at 4°C, sample was dialyzed against PBS buffer.

# Solubility Test and FPLC

A total of 500 µg of each HCAbs was concentrated to an end point of 30 µl volume using Centriprep-10 K centrifugal filter device (Merck Millipore Ltd., Ireland) as suggested by the manufacturer. Concentrated samples were diluted 10× in 8M guanidine hydrochloride (Gdn-HCl) and the OD at 280 nm measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific, Ireland). The same procedure was used for selected VHs expressed in *E. coli*.

A total of 2 µg of purified and PBS-dialyzed HCAb in 50 µl of PBS was run on a Superdex 200 (3.2/30) column (GE HealthCare Life Sciences, USA) on the FPLC Smart system from Pharmacia. Samples were run in PBS. Bio-Rad's gel filtration standard as a mixture of molecular weight markers ranging from 1,350 to 670,000 Da was used as a control. The same procedure was done with selected VHs expressed in *E. coli* run on a Superdex 75 (3.2/30) column (GE HealthCare Life Sciences, USA).

#### Virus Reduction Assay

The microtiter plaque reduction assay, as described by Matrosovich et al. (18), was performed using MDCK-SIAT1 cells. Twofold dilutions of the HCAbs were incubated on cell monolayers prior to addition of virus. The neutralization titer is determined as the reciprocal of the dilution of HCAbs, which corresponds to 50% reduction in plaque formation, compared to the virus control.

### Hemagglutination Inhibition (HI) Assay

Hemagglutination inhibition assays were performed according to standard methods, Kendall et al. (19), using 0.75% and 1.0% of Turkey and guinea pig red blood cell suspensions, respectively. Four HA units and twofold dilutions of HCAbs were used in these assays. HI titers are reciprocals of the highest dilution of HCAbs, which inhibited hemagglutination.

# RESULTS

4HVH transgenic mouse lines contain four non-mutated human germ line heavy variable regions (VH3-11, VH3-23, VH3-53, and VH1-46) followed by all human D and J regions, the Cγ2 and Cγ3 constant regions, each with a deleted CH1 exon and the human immunoglobulin 3′LCR (**Figure 1**). All transgenic mouse lines rearranged the human HCAb locus and rescued B cell development in a mouse Cμ knockout background (20). They express HCAb dimers in the serum of the correct size (75–90 kDa). These mice were immunized and used as a source of antigen-specific antibodies in developing the method of cloning HCAbs directly into mammalian cells.

## Immunization of Transgenic Mice Leads to Successful Production of Antigen-Specific HCAbs

A total of 20 or 50 µg of influenza X-31 HA protein, prepared as described by Ruigrok et al. (17), per mouse was injected intraperitoneally in 2 weeks intervals into eight of 4HVH transgenic mice (two transgenic lines, 4HVH-B and 4HVH-C, originating from different founders, having different integration site of the transgene) and two WT mice as a control, using Stimune as an adjuvant. After the third injection, mice were bled, and an ELISA assay was performed on serum, using HA protein-coated plates (5 µg/ml) and anti-human IgG-HRP or anti-mouse IgG-HRP for the detection of HA-specific antibodies. In seven out of eight transgenic mice and two out of two WT mice, HA-specific antibodies were detected (Figure S1 in Supplementary Material). The higher amount of antigen did not lead to a better response, thus we concluded that 20 µg is sufficient for successful immunization. Seven transgenic ELISA positive mice were immunized three or more times with 20 µg of HA.

## HEK 293T HCAbs Library Construction and Subsequent Screening Result in a Diverse Repertoire of Antigen-Specific Soluble Antibodies

Four days after the last injection without adjuvant, mice were sacrificed, and CD 138<sup>+</sup> PCs were isolated from bone marrow and spleen. Total RNA was isolated, and cDNA was synthesized (depicted schematically in **Figure 1**). Human VDJ domains were amplified using a set of three different 5′-end primers specific for the leader sequences in the transgenic mouse construct in combination with two different 3′-end primers specific for either the human IgG2 or IgG3 hinge (3). Due to the high sequence similarity, the same 5′-end primer was used for amplification of both VH3-23 and VH3-53. All 5′-primers were designed to contain a *Pvu*II restriction site. A *Pvu*II site appears usually at the beginning of the DNA encoding VH regions (third to fourth codons at the amino acid level) and rarely occurs anywhere else in human VHs. Amplified PCR products were cut either with *Pvu*II/*Sac*I or with *Pvu*II/*Bst*EII restriction enzymes. The *Sac*I site is a unique site at the beginning of the IgG3 hinge, while the *Bst*EII site in frame 4 is unique in most of the VHs and is commonly used for constructing phage libraries (21, 22).

The VDJ fragments were cloned into bacteria using either pCAGhygro G2 or pCAGhygro G3 expression vectors, containing the ubiquitously expressed chicken β-actin promoter, the leader sequence from human VH3-23, and the constant region of human IgG2 or IgG3 (**Figure 2**). The ligated cDNA was transfected into electrocompetent *E. coli* cells, totaling 1 part out of almost 700,000 of the available RNA in the mice (PCs were isolated from half of the total number of cells, 1/4 of total RNA was made into cDNA, 1/30 of the gel purified PCR amplified cDNA was used in the ligations; 1/50 of the ligation was transformed, 1/32 of the transformed bacteria was plated, and 0.57 of total colonies counted were picked from those plates). A total of 960 colonies were picked from both the G2 and G3 library into 96-well plates filled with 2XTY medium. The resulting recombinant bacteria were grown overnight, and plasmid DNA was prepared in the same 96-well format. In parallel, HEK 293T cells were also grown in a 96-well format and transfected with the plasmid DNA maintaining the same 96-well format. A 96-well HA-specific ELISA

screen was performed with supernatants from each well 4 days after transfection. This yielded 66 positive supernatants/clones. The corresponding cDNAs were sequenced (**Figure 3**), which showed that two out of the four available variable segments were used preferentially (VH3-11 and VH3-23); the VDJ domains contained somatic hypermutations; J4 was predominantly used but J5 and J6 were also found; both IgG2 and IgG3 antibodies were present, confirming that class switching occurred in the transgenic mouse. Most prevalent are IgG2 HCAbs, and it is not surprising taking into account that the IgG2 constant region is the most proximal in the transgenic construct and thus the first to recombine. Out of 66 sequences, 45 were unique and these account for 33 different DJ regions. Based on the different CDR3 regions, the HCAbs were classified into 13 distinct groups represented in different colors (**Figure 3**). Positive clones were cultured further, and supernatants were collected for affinity screening, using the same HA antigen preparation. Twenty four clones showed significant binding. A selected number of clones (based on sequence diversity and affinity) were produced in serum free medium. The antibodies with low affinities were not tested at all in a functional assay (see below). Four groups represented by 7F2 (**Figure 3**, blue), 2F4 (green), 1F3 (red), and 3A8 (yellow) were left. Of these, 1F3 and 2F4 appear to bind the same epitope(s), while 3A8 and 7F2 appear to bind a different but overlapping epitope(s).

#### Characterization of Anti-HA Antibodies

Heavy-chain-only antibodies from medium were purified on protein A, eluted in 3M KSCN, and dialyzed against PBS. The average yield was estimated to be 2–4 µg/ml of medium. On SDS-PAGE gel, HCAbs are of expected size for a monomer (~40–45 kDa) under reducing and of a dimer (~80–90 kDa) under non-reducing conditions (**Figure 4A**). Size differences originate from different sizes and compositions of VH regions and different sizes (longer hinge) of IgG3 HCAbs, accounting for >5 kDa difference per monomer in comparison to IgG2 HCAbs. The profiles on size chromatography (smart columns) showed a single peak of the expected size; the peaks of the IgG3 HCAbs, 3A11, and 3B10 were of higher molecular weight than IgG2 HCAbs (**Figure 4B**). Solubility in PBS was tested with 500 µg of each HCAb by concentrating it to ~30 µl of final volume. The selected HCAb differed with concentrations up to 15.4 µg/µl for 1F1, the equivalent of 30 mg/ml of a normal H2L2 antibody (**Figure 4C**). Further concentration of the very soluble antibodies was not tested. Two of the VHDJ segments (3A8 and 2F4) were also expressed in bacteria and shown to be soluble in at least 5 mg/ml, which is

also equivalent to 30 mg/ml of a normal H2L2 antibody (Figure S2 in Supplementary Material). Binding affinities of the HCAb were determined on an Octet instrument with purified antibodies. A BHA preparation (23) that lacks a transmembrane anchor was used to prevent rosette formation and aggregation of antigen. The majority of KD values (9 of 17) for HCAbs were in the 10<sup>−</sup><sup>9</sup> molar range, 7 were in the 10<sup>−</sup><sup>8</sup> molar range, while the KD value for the highest affinity 1F3 HCAb was better than 10<sup>−</sup>10. HCAbs 1G7, 1G11, 2F4, 1F4, 1C12, 4H1, and 3A11 react on Western blots under both non-reducing and reducing conditions recognizing denatured BHA monomer and the BHA1 chain, respectively. 7F2 reacts with BHA only under non-reducing conditions, while 1F3 preferentially recognizes BHA1 under reducing conditions (data not shown).

# Anti-HA HCAbs Function in Virus Neutralization and HI

The HA HCAbs were first evaluated for their ability to neutralize X.31 (H3N2) influenza virus in a plaque reduction assay. Neutralization is reported as the reciprocal of the highest dilution of the antibodies corresponding to 50% plaque reduction compared to the virus control. A total of 18 of the antibodies tested show visible inhibition, and 50% reduction is observed for four HCAb, 1F3 > 2F4 > 3A8 > 4H1 = 7F2 (**Table 1**). A modified neutralization experiment was also performed, where antibodies were incubated for 30 min with the virus prior to addition to MDCK-SIAT1 cells. This led to an increased neutralizing effect especially in the case of 2F4 and 1F3 antibodies [2F4 neutralizing at ≥1.4 µg/ml (18nM) and 1F3 at ≥0.9 µg/ml (11.7nM)]. Ferret

*The numbers show best neutralizing capacity for 1F3 HCAb at* ≥*0.9 μg/ml (~11nM), followed by 2F4 at 1.4 µg/ml (~18nM).*

*NI, not preincubated with the virus; I, preincubated with the virus; NK, not known; neutralization titer, reciprocal of the 50% plaque reduction.*

anti X-31 serum and HC19, an anti-X31 HA mouse monoclonal antibody, were used as positive controls. 3A8 and 7F2 HCAbs also showed an HI performed with Turkey red blood cells (**Figure 5**) and with guinea pig blood (data not shown), suggesting that 3A8 and 7F2 neutralize infectivity by obstructing the binding of virus to the host cell.

### DISCUSSION

Here, we describe a very efficient method for obtaining fully human, antigen-specific, soluble, high-affinity HCAbs, which can easily be automated. Our approach is based on capturing the antibody repertoire of antibody-secreting PCs from both bone marrow and spleen of immunized transgenic mice. Recently, other laboratories have used mouse spleen PCs (24), mouse bone marrow PCs (25), human peripheral blood PCs (26, 27), or human MBC from patient recovered from infection (28) to obtain antigen-specific monoclonal antibodies. Basically, those methods utilize single-cell sorting, single-cell RT-PCR, and natural VH–VL pairing (27, 28). Screening methods have been developed to improve efficiency and enable detection of antigenspecific secreting cells before a single-cell RT-PCR step, such as enzyme-linked immunospot (29), immunospot array assay on chip or microengraving (30). Recently, a method was described (24) omitting a screening step, utilizing massive DNA sequencing and bioinformatics tools to analyze the VL and VH gene repertoires and to find several abundant VH and VL sequences that are paired based on their relative frequencies with 78% efficiency and a method that combines the next-generation sequencing and protein mass spectroscopy to obtain antigen-specific antibody repertoires (31). DeKosky and colleagues developed a low-cost, single-cell, emulsion-based technology for sequencing of antibody VH–VL repertoires with even better pairing precision of >97% (32).

The 4HVH transgenic mice produce human HCAb, without light chains, thus there is no pairing or the necessity for singlecell RT-PCR. The major concern was the solubility of human VH domains, that are not soluble *per se*, but as our results show, antigen-specific HCAbs selected through our screen show high solubility both as a full-length HCAbs and as VH fragments only, thus, the method allows easy isolation of soluble VH regions and the construction of multivalent soluble VH complexes.

The soluble human VH domains obtained from transgenic mice presented here do not possess the hallmark amino acid changes present in VHHs of camelid HCAbs (at positions 44, 45, and 47), which reduce the hydrophobicity of the former light chain interface. They remain as germ line VH being G, L, and W, respectively. As for replacing the V 37 of VH by more hydrophilic F or Y in VHHs, 1 out of 66 VH domains have mutated at this position to F 37, one to L 37. All V3-11-derived HCAbs have I-37 as in the germ line. We have analyzed many sequences from different immunizations with different antigens, beyond the restricted list of anti HA HCAbs shown in **Figure 3**. We have seen FR2 substitutions, which are not found in antibodies that comprise heavy and light chain. We found an increased net hydrophobicity within CDR1 and an increased number of charged amino acids present in CDR3, amino acid substitutions within the framework β-pleated sheet leading to increased net hydrophobicity within FR1, and increased number of charged amino acids present in FR3, all of which could lead to solubility of autonomous soluble VH domains obtained from transgenic mice (33).

This experiment was done in 4HVH transgenic mice bred into our own Cμ MT heavy chain knockout background. It was previously reported that Cμ MT knockout mice can produce low levels of IgG antibodies after prolonged time (34). In such a case, mouse light chains might theoretically attach to the human VHs and affect their solubility, which could cause a problem in using hybridoma fusions. If the reason for increased solubility is light chain attachment, the solubility will be hampered by cloning the heavy chain only (VH) in expression vectors. Once produced, such antibodies would have aggregation problems, which we do not see using HEK 293T libraries. Knocking out the loci completely would avoid the problem.

The choice of VHs in the transgenic construct was based on the VH usage in a human population. It is also known that soluble llama VHHs mostly resemble family three of human VHs (6). The fact that in this particular experiment most of the obtained antibodies originated from VH3-11, a few fromVH3-23, while none from V3-53 and VH1-47 could be explained by antigenrelated specific usage. A new class of influenza-neutralizing antibodies that target a conserved site in the HA stem, most of them being VH 1-69, have been described, but this VH is not present in our transgenic mouse (35). In other experiments, the other VH regions are also used (data not shown). The soluble human VH domains obtained from transgenic mice presented here do not possess hallmark amino acid changes present in VHHs of camelid HCAbs (at positions 44, 45 and 47), which function to reduce hydrophobicity of the former light chain interface. They remain as in germ line VH situation being G, L, and W, respectively.

The four human VHs obtained from transgenic mice were cloned into human IgG2 and IgG3 vectors to reproduce characteristics of antibodies circulating in transgenic mouse. For most therapeutic purposes, human IgG1 and IgG4 antibodies are preferred, due to their effector functions. Once obtained, selected VHs could be cloned and expressed as human IgG1 or IgG4 in appropriate vectors.

We are improving our transgenic mouse platform by increasing the numbers of germ line VHs in the construct, thus increasing the repertoire. The human constant regions in the new generation of transgenic mice have been replaced by the mouse constant region/s reported to be better suited to the mouse machinery (36), while direct cloning might be performed into human IgG vectors of choice.

The pilot experiment performed with 960 plasmid DNAs from the bacterial library and an estimated 7% vector contamination (plasmids without the cDNA insert) gave 66 ELISA-positive clones, 45 of these being unique. If we take into account that the mice carry only 4VH regions, that only a tiny fraction of the available material was used, and that 83% of retrieved antibody sequences were represented only once in the sample tested, a very

#### REFERENCES

high efficiency of obtaining many more high-affinity antibodies in an industrial scale automated/robotized process at low cost can be predicted.

# ETHICS STATEMENT

The animal study described was approved by the Animal ethical comity [**D**ieren**e**xperimenten**c**ommissie (DEC)] under the immunization protocol EUR 1944.

# AUTHOR CONTRIBUTIONS

DD, FG, and JS contributed to the design of the experiments. DD, EB, RJ, RR, JK, and JS contributed to acquisition and analysis of data. DD, FG, and JS contributed to interpretation of data and writing of the manuscript.

### ACKNOWLEDGMENTS

The authors thank Tao Chen for helpful discussions and Rose Gonzalves for help with HI assay. The modified Madin Darby canine kidney, MDCK-SIAT1, cells were kindly provided by Dr. M. Matrosovich, Marburg.

### FUNDING

This work was supported by the "Foundation Incubator ErasmusMC" and "Harbour Antibodies B.V."

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://journal.frontiersin.org/article/10.3389/fimmu.2016.00619/ full#supplementary-material.

leave lymph nodes. *Eur J Immunol* (2001) 31:609–16. doi:10.1002/1521-4141 (200102)31:2<609::AID-IMMU609>3.0.CO;2-9

memory B cells: potent neutralization of SARS coronavirus. *Nat Med* (2004) 10:871–5. doi:10.1038/nm1080

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Drabek, Janssens, de Boer, Rademaker, Kloess, Skehel and Grosveld. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

*Kathryn E. Tiller1 , Ratul Chowdhury2 , Tong Li2†, Seth D. Ludwig1 , Sabyasachi Sen1 , Costas D. Maranas2 and Peter M. Tessier1 \**

*<sup>1</sup> Isermann Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, United States, 2Department of Chemical Engineering, The Pennsylvania State*

#### *Edited by:*

*University, University Park, PA, United States*

*Kevin A. Henry, National Research Council Canada, Canada*

#### *Reviewed by:*

*Jennifer Maynard, University of Texas at Austin, United States Xin Ge, University of California, Riverside, United States Yongku Cho, University of Connecticut, United States*

#### *\*Correspondence:*

*Peter M. Tessier<EMAIL_ADDRESS>
#### *†Present address:*

*Tong Li, BASF Corporation, San Diego, CA, United States*

#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 07 June 2017 Accepted: 02 August 2017 Published: 04 September 2017*

#### *Citation:*

*Tiller KE, Chowdhury R, Li T, Ludwig SD, Sen S, Maranas CD and Tessier PM (2017) Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis. Front. Immunol. 8:986. doi: 10.3389/fimmu.2017.00986*

The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs) that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants) displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH) antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson's disease) with the greatest gains in affinity (>5-fold) have several (four to six) CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability) while other mutations enhance some of these properties (e.g., increased specificity) and display trade-offs in others (e.g., reduced affinity and/or stability). Computational modeling reveals that improvements in affinity are generally not due to direct interactions involving CDR mutations but rather due to indirect effects that enhance existing interactions and/or promote new interactions between the antigen and wild-type CDR residues. We expect that natural diversity mutagenesis will be useful for efficient affinity maturation of a wide range of antibody fragments and full-length antibodies.

Keywords: complementarity-determining region, stability, specificity, library, directed evolution, yeast surface display, protein design

# INTRODUCTION

The widespread interest in using antibodies in diagnostic and therapeutic applications has led to considerable efforts in developing methods for optimizing their properties (1–6). Methods for improving antibody affinity are particularly important because lead antibodies identified using *in vivo* (immunization) and *in vitro* (e.g., phage display) methods typically do not have high enough affinity for therapeutic applications. Moreover, improvements in antibody affinity are generally expected to enhance the performance of diagnostic antibodies due to improved specificity at reduced antibody concentrations. Methods such as phage, yeast surface and ribosome display are commonly used for *in vitro* affinity maturation because of their many attractive properties (7–13). These properties include the ability to precisely control antigen presentation, conformation, and concentration as well as the ability to perform negative selections against various types of non-antigens to eliminate non-specific variants (14–17). These display methods have been used to achieve large enhancements in affinity for a wide variety of antibody fragments and full-length antibodies (9, 18–23).

Nevertheless, there are several outstanding challenges related to *in vitro* affinity maturation that need to be addressed. First, while it is possible to use saturation mutagenesis to evaluate every possible single mutation in antibody complementaritydetermining regions (CDRs), single mutations typically do not result in large gains in affinity (1, 3, 24). Therefore, it is often necessary to generate sub-libraries to identify combinations of single mutations that result in large increases in affinity, which is a slow and laborious process. Second, it is not possible to test all combinations of single and multiple mutations in the CDRs of antibodies in a single library due to intractably large library sizes. For example, a library size of >1039 would be required to sample all possible combinations of single and multiple mutations at ~30 residues in the CDRs of typical variable domains. This means that only an extremely small subset of the possible single and multiple mutations can be tested using display methods, which is largely dictated by transformation efficiencies [~109 –1010 for phage (25, 26) and ~107 –108 for yeast (9, 27) using conventional transformation methods]. Therefore, it is important to develop smart library design methods that sample a relatively small number of residues at each CDR position that are most likely to generate antibodies with significant gains in affinity (28–41).

A third common challenge related to antibody affinity maturation is the identification of affinity-enhancing mutations that lead to reductions in antibody specificity (42–44). Highly interactive residues—such as arginine and aromatic residues—can be readily enriched in the CDRs during affinity maturation, which is concerning because they have increased risk for promoting non-specific interactions (43–47). While negative selections are useful for removing some non-specific variants, it is critical to use libraries with the highest possible fraction of specific variants to maximize the likelihood of isolating antibodies with not only increased affinity but also with high specificity. A related problem is that affinity-enhancing CDR mutations can lead to reductions in stability (48–51). Antibody affinity/stability trade-offs appear to be due to structural changes in the CDRs and frameworks that are necessary to increase affinity, and additional compensatory mutations are needed in some cases to maintain thermodynamic stability (48, 49, 51). Therefore, it is important to generate antibody libraries with the highest possible fraction of stable antibodies to minimize the frequency of isolating destabilized antibodies that require additional mutagenesis to restore stability.

To evaluate potential solutions to these challenges, we have sought to identify mutations that increase the affinity of a camelid single-domain antibody specific for the C-terminus of α-synuclein (52) (**Figure 1**). This variable (VHH) domain—originally referred to as NbSyn2 and herein referred to as N2—was previously isolated from an immune library. We selected this antibody domain for further optimization because its crystal structure is available in complex with antigen at high resolution (**Figure 1**), it is relatively simple to display on the surface of yeast for *in vitro* selections relative to more complex multidomain (scFv) and/or multichain (Fab or IgG) antibodies, it has intermediate affinity (*KD* of 58 ± 9 nM) that can be further increased, and it has relatively high stability (apparent melting temperature of 68 ± 0.3°C). We posit that efficient affinity maturation of antibody variable domains such as N2 can be accomplished in three steps: (i) identification of the most permissive sites in the CDRs that can be mutated without large (negative) impacts on affinity using

Tiller et al. Antibody Natural Diversity Mutagenesis

alanine scanning mutagenesis; (ii) sampling of a small number of mutations at each permissive CDR site that correspond to either the wild-type residue or residues most commonly observed in natural antibodies at each CDR site; and (iii) screening of all possible combinations of single and multiple natural diversity antibody mutations in a single library. Here, we test this methodology by identifying the most permissive CDR sites in N2 and use these findings to generate a single library that is based on natural antibody diversity and includes both single and multiple (up to 14) CDR mutations. We demonstrate how this library design approach can be used along with yeast surface display to identify stable and specific variable domains with increased affinity.

### RESULTS

# Alanine Scanning Mutagenesis Reveals Permissive CDR Sites

Toward our goal of developing systematic and robust affinity maturation methods, we first sought to identify permissive sites in the CDRs of N2 that weakly impact antibody affinity using both computational and experimental methods. Two of the CDRs (CDR2 and CDR3) are involved in mediating antigen binding (**Figure 1**). Our computational alanine scanning analysis of these CDRs identified two residues in CDR2 (N52 and K56) and two residues in CDR3 (Y100 and W100e) that are sensitive to mutation (Table S1 in Supplementary Material). We tested these observations using experimental alanine scanning mutagenesis at 18 sites in CDR2 and CDR3. Three sites in these CDRs (R50, P98, and C100a) were excluded from this analysis because they were either shown previously to be involved in mediating antigen binding (52) or suspected to be important for antibody structure and stability.

The alanine mutants were expressed in bacteria and purified using metal-affinity chromatography (purification yields of 0.7–2.6 mg/L). SDS-PAGE analysis revealed high purities (Figure S1 in Supplementary Material). The relative binding of each mutant was evaluated using fluorescence polarization at three VHH concentrations (44, 133, and 400 nM; **Figure 2**; Figure S2 in Supplementary Material). Consistent trends were observed at each VHH concentration. Eleven of the 18 mutants retained >50% of the wild-type binding activity, including three in CDR2 (L52b, G53, and V55) and eight in CDR3 (F96, S97, G99, G100b, G100c, S100d, S100f, and N100g). The other seven mutants that displayed greater reductions in binding included five CDR2 mutants (I51, N52, G52a, G54, and K56) and two CDR3 mutants (Y100 and W100e), which were not subjected to further mutagenesis. Four of the disruptive mutations (N52 and K56 in CDR2 and Y100 and W100e in CDR3) were identified in our computational alanine scanning mutagenesis (Table S1 in Supplementary Material). These and other previous results (39, 53, 54) highlight the value of alanine scanning mutagenesis to identify permissive CDR sites that can be mutated during antibody affinity maturation.

# Design of Antibody Libraries Using Natural Diversity Mutagenesis

We next sought to design a single antibody library with mutations in N2 at permissive sites in CDR2 and CDR3. We aimed

Figure 2 | Identification of VHH complementarity-determining region (CDR) residues involved in antigen binding *via* alanine scanning mutagenesis. The relative antigen binding of the VHH variants (400 nM) with single alanine substitution mutations in (A) CDR2 and (B) CDR3 was evaluated using fluorescence polarization (2 nM TAMRA-labeled αsynuclein peptide). Raw polarization signals were background subtracted (background signals were obtained using samples with only TAMRA α-synuclein peptide), and normalized signals are reported (signal for mutant divided by that for wild type). Error bars represent the SD for three independent experiments. The VHH sequence is defined using Kabat numbering. Alanine mutants that have modest impacts on antigen binding (mutant binding is at least 50% of wild-type binding) are highlighted in gray fill, while those mutants with larger negative impacts on antigen binding are indicated in white fill.

to accomplish multiple objectives in our library design. First, we limited the library size to ~107 variants to enable 10-fold oversampling of the library using yeast surface display given that our typical yeast transformation efficiencies are ~108 transformants. Second, we aimed to generate a single library with all possible combinations of wild-type residues as well as single and multiple mutations at the 11 permissive sites in CDR2 and CDR3 as well as at three additional sites not tested during alanine mutagenesis (A49, A94, and K95). This limits the number of possible mutations at each CDR site to typically one to two mutations in addition to the wild-type residue. Third, we sought to sample mutations that most closely correspond to those observed in the CDRs of natural antibodies in a site-specific manner. To accomplish this, we used the AbYsis database to identify the most common amino acids in camelid VHH and human VH domains at each site in CDR2 and CDR3 (55). We used an average site-specific amino acid frequency for camelid and human domains at each CDR site given that there are many more sequences for human domains than for camelid domains. Fourth, we aimed to use inexpensive primer synthesis methods to generate the libraries encoded by standard degenerate codons. Therefore, we sought to identify degenerate codons at each CDR site that encoded the wild-type residue and ~1–5 additional residues that maximize the coverage (sum of individual site-specific amino acid frequencies) of the combined camelid and human natural diversity at each site (**Figure 3**).

Based on these four key objectives, we designed the library shown in **Figure 3** and generated it using the process outlined in Figure S3 in Supplementary Material. The library contains 9.4 × 106 unique variants and includes wild-type residues at each position as well as all possible combinations of single and multiple mutations at 14 sites in CDR2 and CDR3. We sequenced several (22) members of the initial library, and the results are summarized in **Figure 4** and Figure S4 in Supplementary Material. All variants were found to be unique and contained mutations according to the proposed library design.

Figure 3 | VHH library design for N2 affinity maturation using natural diversity mutagenesis. A single VHH library was designed that involved mutating four sites in CDR2 (top) and 10 sites in CDR3 (bottom). The CDR sites selected for mutagenesis were identified primarily using alanine scanning mutagenesis (11 CDR sites). Each mutated CDR site involved sampling the wild-type residue and one to five of the most common natural diversity mutations. Degenerate codons were selected at each CDR site that maximized the natural diversity coverage and minimized the total number of mutations. It was not possible to sample the wild-type residue and the most common natural diversity mutations at each CDR site due to the limitations of degenerate codons. The resulting library (9.4 × 106 variants) theoretically encodes all possible combinations of single and multiple CDR mutations (up to 14 mutations per VHH). The reported CDR site-specific natural diversity statistics are averaged values for human (VH) and camelid (VHH) variable domains, as reported in the abYsis database (55). Boxed amino acids correspond to the selected natural diversity mutations.

CDR3 were generated from sequencing results for 22 (initial library) and 17 (enriched library) VHH variants. The CDR sequences are defined using Kabat numbering,

# Sequence Analysis of VHH Libraries after Sorting for Enhanced Antigen Binding

and the logos were generated using a web application (http://weblogo.berkeley.edu).

The library of antibody variable domains was displayed on the surface of *S. cerevisiae* and screened for variants with increased affinity for the α-synuclein peptide. The sorting process involved five rounds of selection *via* magnetic-activated cell sorting (MACS) with progressively reduced concentrations of α-synuclein peptide (starting at 50 nM peptide and ending at 5 nM) and one additional round of selection *via* fluorescence-activated cell sorting (FACS) (20 nM peptide). The sorting process was continued until the antigen binding of the library was increased by at least fivefold relative to wild type, as judged by flow cytometry. Selections were performed in a buffer (PBS) that contained both BSA (1 mg/mL) and milk (1% w/v). We have found previously that antibody selections in complex environments (e.g., buffers supplemented with milk) lead to identification of antibodies with improved specificity (56).

The enriched VHH library was sequenced after sorts 5 and 6, and 17 unique variants were identified and further analyzed (based on sequencing 23 clones) with 1–6 mutations in CDR2 and CDR3. Sequence logos in **Figure 4** summarize the general enrichment of amino acids in the CDRs, while the amino acid enrichment ratios are given in Figure S5 in Supplementary Material and the CDR sequences are given in Figure S6 in Supplementary Material. Most of the sites in CDR2 and CDR3 (11 out of 14) displayed either intermediate or strong preference for the wild-type residue (**Figure 4**). However, three sites (53 in CDR2, 96 and 100d in CDR3) either displayed similar preference for mutations as the wild-type residue (Arg, Gly, Ser, and Asn at position 53) or strong preference for a specific mutated residue (Ser at position 96 and Thr at position 100d). It is also notable that the four positions that were varied in CDR2 did not display strong preference for any single amino acid, while almost every residue in CDR3 (9 out of 10) displayed strong preference for a single residue. This result is unexpected based on alanine scanning mutagenesis, as the identified sites in CDR3 appeared to be as permissive (or even more permissive) to mutagenesis than those identified in CDR2.

# Identification of Affinity-Matured Variable Domains with High Stability and Specificity

To evaluate the effectiveness of the affinity maturation process, we next expressed and purified the unique VHH variants that were identified in the enriched library. The variable domains expressed at levels (purification yields of 0.1–2.0 mg/L) that were generally similar to wild type (1.0 mg/L), and also displayed purities similar to wild type (Figure S7 in Supplementary Material). We first used fluorescence polarization to evaluate the affinities of the variable domains for the α-synuclein peptide (**Figure 5A**). The equilibrium dissociation constant for the wild-type N2 variable domain (*KD* of 57.6 ± 9.0 nM) was approximately threefold lower than the previously reported value (*KD* of 190 ± 30 nM) that was measured by isothermal calorimetry (52).

We chose to characterize two VHH domains in more detail (N2.12 and N2.17). Both variable domains displayed improved affinity (*KD* of 7.6 ± 0.4 nM for N2.12 and 13.2 ± 4.8 nM for N2.17 relative to 57.6 ± 9.0 nM for wild type; **Figure 5A**). Interestingly, the improved affinity of the N2.12 variant came at the cost of reduced stability (apparent *Tm* of 59.7 ± 0.3°C relative to 67.8 ± 0.3°C for wild type; **Figure 5B**). By contrast, the N2.17 variant displayed similar stability as wild type (66.9 ± 0.1°C for N2.17 relative to 67.8 ± 0.3°C for wild type; **Figure 5B**). This finding demonstrates that our affinity maturation method can be used to identify antibody variable domains such as N2.17 with increased affinity without significant reduction in stability despite the common observation of affinity/stability trade-offs (such as those observed for N2.12) during affinity maturation (51, 58).

We also evaluated the specificity of the N2.12 and N2.17 VHH domains to evaluate if gains in affinity were offset by reductions

Figure 5 | Evaluation of the affinity and stability of select VHH mutants that were enriched after library sorting for improved antigen binding. (A) Fluorescence polarization analysis of VHH binding to labeled antigen (2 nM TAMRA-labeled α-synuclein peptide). The analysis was performed in a PBS buffer supplemented with BSA (0.001%) and Tween 20 (0.001%). Three independent experiments were performed, and representative binding curves are shown for wild type (black), N2.12 (red), and N2.17 (green). Each point shown is the average of two repeats and the error bars are SD. Data were fit with a binding model that accounted for the fact that the VHH antibodies were not in excess of the antigen at some of the VHH concentrations (57). (B) Extrinsic fluorescence analysis of apparent VHH unfolding as a function of temperature. The fluorescence data were obtained using an extrinsic dye (Protein Thermal Shift dye, Life Technologies). Three independent experiments were performed, and representative melting curves are shown for wild type (black), N2.12 (red), and N2.17 (green). The data were background subtracted using background signals obtained without antibody. Next, the fluorescence data were subtracted by the relatively low signal at 50°C, and divided by the maximum fluorescence signal (after the maximum signal was subtracted by the signal at 50°C). Finally, the pre- and post-transition regions of the normalized fluorescence data were flattened using linear fits.

in specificity (**Figure 6**; Figure S8 in Supplementary Material). A simple test of non-specific interactions is to evaluate the propensities of antibodies to interact with well plates coated with different types of non-antigen proteins (milk proteins and a panel of six

matured VHH domains. Non-specific binding of VHH variants was evaluated using well plates coated with milk proteins (left) and a panel of six non-antigen proteins (right). The non-specific binding analysis was performed at an antibody concentration of 1,000 nM. The reported non-specific binding values are the signals for antibody binding to well plates coated with milk proteins or other non-antigen proteins divided by the background signal without primary antibody (VHH). The reported binding values (right) are the averages for six non-antigen proteins (ovalbumin, BSA, KLH, ribonuclease A, avidin, and lysozyme). The values are averages of three independent experiments, and the error bars are SD. A two-tailed Student's *t*-test was used to determine statistical significance [*p*-values < 0.01 (\*\*)].

non-antigen proteins in **Figure 6** and Figure S8 in Supplemental Material) at relatively high antibody concentrations (~1 μM). Interestingly, the N2.17 variant displays significantly lower non-specific interactions than wild type (*p*-values of 0.003 for milk proteins and 0.009 for six non-antigen proteins), while the N2.12 variant displays similar non-specific binding as wild type (*p*-values of 0.129 for milk proteins and 0.342 for non-antigen proteins). These results demonstrate that the affinity-matured VHH domains display similar or improved specificity relative to wild type.

We next analyzed the affinity and stability of the other 15 unique VHH variants that were isolated during the sorting process (**Figure 7**; Figures S9 and S10 in Supplementary Material). All but one of the variable domains (N2.5) displayed a statistically significant increase in affinity relative to wild type (*p*-values <0.01; **Figure 7A**). This suggests that our library design and selection strategies enable robust identification of variable domains with improved affinity. Interestingly, variants with the greatest improvements in affinity (at least threefold) contained at least three mutations and up to six mutations. This highlights the inherent limitations of attempting to identify variable domains with large increases in affinity using single mutations.

The stability analysis of these variable domains also revealed interesting behaviors (**Figure 7B**). Most notably, the apparent stability of the VHH domains is much more variable than the affinity measurements. About one-third of variable domains (6 of 17) display similar stabilities as wild type (apparent melting temperature within 1°C of wild type). The variable domains with the largest reductions in apparent melting temperature (>7°C;