Table 1 | Binding kinetics of anti-hTNF-α variable new antigen receptor using Octet® RED96 system.

*For all measurements, streptavidin Dip and Read*™ *biosensors were used to immobilize biotinylated hTNF-*α*. All assessed domains were loaded at a top concentration of 100 nM followed by a twofold dilution series. Values obtained from BIAcore binding analysis are shown in italics, and constructs not tested are shown as "not available" (n/a).*

Table 2 | Neutralization of 0.3 ng/ml (LD80) hTNF-α-induced cytotoxicity in L929 cells.

minimal efficacy improvement (data not shown). In trivalent formats, where the spacing between the functional anti-TNF-α binding domains was increased further (**Figure 2**) through the inclusion of a well-characterized anti-HSA binding soloMER™ (humanized VNAR) domain BA11 (41), trimers D1–BA11–D1 and D1–BA11–C4 were capable of neutralizing hTNF-α at subnanomolar concentrations (**Table 2**).

# Affinity Determination

The equilibrium dissociation constant (*K*D) for lead VNAR domains was determined on the Octet® RED96 system as described above. As anticipated, the binding affinity of these multivalent domains significantly improved as a result of an enhanced avidity effect. The measured *K*D values were in the sub-nanomolar range for the multivalent VNAR constructs, demonstrating a 600-fold improvement in affinity following reformatting of parental domains (**Table 1**).

# TNF-**α** Neutralization in L929 Cell Line

Neutralization of hTNF-α-induced cytotoxicity in L929 cells remains one of the gold-standard *in vitro* efficacy experiments for determining the potency and therapeutic potential of antihTNF-α biologics. In this assay, the measured ND50 for the unformatted VNAR monomers D1 and C4 was ≈30 and 100 nM, respectively (**Table 2**). The control VHH monomer has an ND50 of less than 10 nM in our assay. The ability of the VNAR constructs to neutralize the cytotoxic effect of hTNF-α was significantly improved in the reformatted constructs from monomer to dimer and again to trimer with these trivalent domains showing the most enhanced potencies (**Table 2**). Of particular interest are the D1–C4 and D1–BA11–C4 formats. The D1–C4 had an ND50 value 10-fold better than its counterpart homodimer (D1–D1, ND50 7 nM), and D1–BA11–C4 had an ND50 value of only 20 pM which is almost 20-fold better than its counterpart D1–BA11–D1 (**Figure 3**; **Table 2**). These consistent and significant improvements seen for the mixed parental dimer clones (with D1 at the N-terminal end of the fusion protein) strongly suggest that D1 and C4 bind unique epitopes on the TNF-α molecule, delivering a bi-paratopic fusion protein of improved neutralizing potency.

# Reformatting Parental Domains As VNAR-Fc Constructs

Multivalent VNAR domains in the size range 25–40 kDa (D1–C4 and D1–BA11–C4, respectively) with picomolar neutralizing potency for hTNF-α were expressed successfully in *E. coli*. Although these small domains are well suited to site-specific delivery, they can also be administered systemically because they incorporate BA11, a half-life-extending domain (humanized VNAR), as part of the spacer region of the fusion protein (**Figure 2**). It is also possible to create fusion proteins that are closer to natural antibodies and incorporate an immunoglobulin Fc region. By carrying out these simple molecular fusions both N and C terminally to the human Fc region of an antibody it has been possible to generate the Quad-X™ and Quad-Y™ family of proteins both of around 50 kDa (**Figure 4**). When expressed in a mammalian system these proteins naturally and efficiently assemble as quadra-valent, bi-specific, and bi-paratopic proteins capable of both half-life extension and immune system recruitment. When examined in a "gold-standard" L929 assay the Quad™ family have proved to be super-sensitive neutralizers with 2–5 pM efficacy (**Figure 5B**) and significantly (10×) better than even the leading clinical anti-hTNF-α antibody adalimumab (Humira®). Even the least potent neutralizer amongst the characterized Quad™ constructs (C4–D1–Fc) is twice as potent as Humira® (**Table 2**).

Bivalent D1–Fc and C4–Fc formats show sub-nanomolar efficacies (**Figure 5A**). Interestingly if equimolar dosing of both D1–Fc and C4–Fc are mixed together then a significant enhancement of efficacy is seen over the use of D1–Fc or C4–Fc alone and provides additional evidence that the two parental VNARs are likely binding different epitopes on the hTNF-α molecule (**Figure 5A**).

linkers while their trimeric counterparts in addition to having flexible GlySer linkers incorporate an anti-human serum albumin (HSA) humanized VNAR (soloMER™), called BA11 (26, 41). This additional anti-HSA binder acts as a spacer and a half-life extension tool for prolonged systemic bioavailibity of the trivalent VNAR. The functional binding of BA11 to HSA was not compromised by the presence, in the expressed fusion protein, of the anti-TNF-α binding VNARs (data not shown).

Figure 3 | Efficacy assessment in standard L929 cell-based assay. Neutralization of 0.3 ng/ml hTNF-α-induced cytotoxicity in an actinomycin-D primed fibrosarcoma cell line (L929). (A) hTNF-α neutralization assay with anti-hTNF-α monomeric constructs at a maximum concentration of 500 nM. Results are the mean ± SD (*n* = 2) with two replicates per experiment. (B) Neutralization with multivalent anti-hTNF-α variable new antigen receptor (VNAR) constructs at maximum concentration of 100 nM. Results are the mean ± SEM (*n* = 3) with two replicates per experiment. Results (B) were analyzed statistically using a two-way ANOVA with Bonferroni's multiple comparison *post hoc* test. VNAR BA11 was included as a negative control domain in all assays where D1–BA11–D1 and D1–BA11–C4 were tested.

### Assessing Efficacy of Anti-hTNF-**α** VNARs in an *In Vitro* Model of Epithelial Barrier Dysfunction

While the Quad™ family would be suitable for parenteral administration, it is possible that the smaller dimer and trimer proteins may be ideal for topical or localized, site-specific administration avoiding the risk of systemic complications. One such clinical destination may be the lining of the gut to suppress or downregulate debilitating inflammation in conditions such as inflammatory bowel disease (IBD). While immunogenicity of VNAR domains would not be an important consideration in the intestine, their ability to tolerate and remain functional in this harsh environment would of course be vital if any therapeutic benefit is to be seen. VNARs are known to exhibit exceptional stability in harsh physiological conditions (28–30). Here, we investigated if reformatting of the parental VNAR monomers as multivalent domains impacted on this characteristic stability. In particular these studies focused on low pH 3 stability over extended periods of exposure (3–28 days). All formats tested showed an excellent tolerance to this extreme of pH even after 28 days with the dimer D1–C4 retaining almost full functionality (**Table 3**).

The utility of the VNAR formats was then tested in a series of bio-assays that mimic barrier dysfunction in IBD. Barrier dysfunction can be induced in IFN-γ primed Caco-2 monolayers by hTNF-α and results in disruption of tight junction morphology. Using the method described in Wang et al. (37), epithelial barrier dysfunction was created *in vitro* by treating fully differentiated Caco-2 cells with hTNF-α causing loss in barrier function (measured as leakage of solutes across tight-junctions and a decline in trans-epithelial resistance). The ability of anti-hTNF-α VNAR domains to block this dysfunction was assessed by studying

Figure 4 | Diagrammatic representation of variable new antigen receptor (VNAR)-Fc constructs. VNAR-Fc constructs of D1 and C4 were generated by direct fusion of VNARs to the hinge of a wild-type human IgG1 Fc. These directly fused VNARs contained a Glycine-rich residue (GGGSGGGGSG) at the end of framework 4 region, and it is this glycine-rich residue that is fused to the hinge. Quad-Y™ constructs are fused to the N-terminal position of the human Fc (as shown in the diagram). The VNARs in a Quad-Y format are linked by a flexible Gly4Ser linker (GGGGSGGGGSGGGGSGGGGS). The Quad-X™ are VNARs fused to both N- and C-terminal ends of the wild-type human IgG1 Fc. The C-terminally fused VNAR is linked to the CH-3 fragment *via* a flexible Gly4Ser linker (as previously described).

Figure 5 | hTNF-α neutralizing capacity of the Fc-based variable new antigen receptor (VNAR) constructs. (A) VNAR-Fc constructs demonstrating high neutralization potencies. D1–Fc + C4–Fc denotes equimolar dosing of both constructs. Results shown are the mean ± SEM (*n* = 3) with two replicates per experiment. (B) A demonstration of the superior potency of the Quad™ VNAR constructs over Humira® in neutralizing hTNF-α-induced cytotoxicity in L929 cells. Results shown are the mean ± SEM (*n* = 4) with two replicates per experiment (D1–Fc–C4) and mean ± SD (*n* = 2) with two replicates per experiment for the other constructs and Humira®.

Table 3 | Binding ELISA of variable new antigen receptor (VNAR) constructs post 28 days incubation at pH 3.

*VNAR protein samples incubated at pH 3 for 3–28 days were neutralized in phosphate buffered saline, pH 7.4 to a final concentration of 0.5 µg/ml before loading onto a 1 µg/ ml hTNF-*α *and/or human serum albumin (HSA)-coated ELISA plates. Binding activity was measured as percentage residual activity compared to an untreated control at each time point. Results shown are the mean* ± *SD (n* = *1 with multiple replicates per experiment). The control anti-human serum albumin monoclonal is clone HSA-11 produced in mouse (Sigma, A6684).*

both the: paracellular flux of large molecular weight hydrophilic molecules such as FITC-dextran and changes in the TEER of the treated Caco-2 cells monolayer. On average the dimer VNAR constructs restored TEER in cytokine treated cells to about 94.4% of the untreated control cells. Interestingly, VNAR D1–C4 restored TEER in cytokine treated cells to about 104.6 ± 5.34% of untreated control and was identical in potency to adalimumab (Humira®) which restored TEER to 103.8 ± 12.1% of untreated control (**Figure 6**).

Variable new antigen receptor monomers restored barrier integrity on average to 63% of untreated, while the VNAR multivalent constructs restored integrity to about 73% of untreated control (**Figure 7**). VNAR D1–C4 and VNAR D1–BA11–C4 restored barrier integrity to about 78 and 84%, respectively, while adalimumab at equimolar dose restored epithelial integrity by 83%. These efficacy data clearly indicate that the VNAR multivalent constructs are comparable in *in vitro* efficacy assays. The barrier leakage can be detected after 2 h of adding FITCdextran in the unprotected or negative control treated cells (BA11

treatment), while protection is evident even at 2 h and sustained over a 24 h period (**Figure 7**).

(33, 40). VNAR BA11 has been described elsewhere in this paper.

### DISCUSSION

IgNAR V regions (VNAR) have been successfully generated against a range of targets from immunized, naïve and semi-synthetic display VNAR libraries (16, 22, 23, 26, 31, 42–45). The notion that IgNAR are part of the adaptive immune system of sharks has been reported previously (16, 26), and although time consuming, immunization still remains our preferred route for the isolation of high-affinity binders or neutralizers. Shark immunization route can be particularly useful for cross-species conserved proteins as sharks are evolutionarily distant from humans, diverging from a common ancestor approximately 450 million years ago and, therefore, the likelihood of immune tolerance to such a protein antigen is much less likely (15, 20, 46).

In our pre-selected, but immunized VNAR library, 6% of randomly selected clones were specific to hTNF-α. We also observed a post-selection preference for the characteristic CDR3-protruding type II VNARs, suggesting that our anti-hTNF-α VNARs may be accessing recessed epitopes on the hTNF-α molecule.

Here, we demonstrate extensive multivalent reformatting of VNAR fragments, with significant improvement in *in vitro* efficacy (beyond that of clinical antibodies to the same target) and multifunctionality while retaining our unique selling point of "small size" (less than 50 kDa) and flexible expressibility. Using a human antibody Fc region as a spacer in molecular fusions, we have generated for the first time super-potent quadra-valent VNAR-Fc constructs, with two main classes named Quad-X™

added at a final concentration of 25 nM. FITC-Dextran flux was measured by taking media from the basolateral chamber at 2 h (A) and 24 h (B) post-addition of FITC-Dextran (i.e., 20 and 42 h post cytokine ± anti-TNFtreatment, respectively). Results shown are the mean ± SD (*n* = 1) with four replicates per experiment, and all treatment groups have been corrected for the fluorescence intensity measured in untreated cells. *y*-Axis on both bar charts are on different scales. VNAR 2V is an isotype control isolated from a naïve library, and has no inherent affinity for any known target antigen (33, 40). VNAR BA11 has been described elsewhere in this paper.

and Quad-Y™ after their shape when drawn in a stylized form (**Figure 4**). The significant improvement in binding affinity across the multivalent VNAR constructs (**Table 1**) is likely to be due to avidity effects. A similar stepwise trend was seen in all *in vitro* neutralization experiments, reaching a 50,000-fold efficacy improvement when comparing a single monovalent VNAR to Quad-X™ in the gold-standard L929 neutralization assay.

A number of diseases are associated with alterations in the intestinal barrier and its increased permeability. IBD, Irritable bowel syndrome, celiac disease, as well as extra-intestinal diseases such as type 1 diabetes, acquired immunodeficiency syndrome, multiple sclerosis, and rheumatoid arthritis have all been linked to intestinal barrier defects (47–51). For IBD patients that display increased intestinal paracellular permeability, TNFα levels become elevated in the intestinal mucosa, serum, and stools of these patients (47), with recent *in vitro* studies further suggesting that increased TNF-α impairs the intestinal tight junction barrier *via* upregulation of the myosin light-chain kinase protein expression (37, 52). Existing clinically available anti-TNF therapies (e.g., adalimumab, infliximab, and certolizumab) are an effective therapy for these pateints with remission seen for Crohn's disease and other related conditions (53). Despite the relative molecular and valency differences between the different anti-TNF-α biologic drug classes (95–150 kDa), both *in vitro* neutralization studies and clinical retrospective and non-randomized studies have demonstrated comparative efficacy (53–57). However, a common to all approach is a series of well documented systemic complications that include: immunogenicity and resulting ADA, profound issues with patient compliance because of a repeated injection dosing paradigm and serious side-effects associated with prolonged use (risk of life-threatening infections and/or lymphomas) (13, 14, 58). Therefore, the use of smaller molecular weight anti-TNF-α biologics through site-specific delivery may enhance clinical outcomes by reducing the level of systemic exposure to these powerful and pleiotropic biologic drugs (59, 60).

Intra-gastric delivery of an anti-TNF-α VHH constructs using an orally administered engineered *Lactococcus lactis* (*L. lactis*) significantly reduced inflammation in dextran sulfate sodiuminduced chronic colitis mice (61). This approach effectively delivers the single-domain VHH to the colon, thereby limiting or circumventing unnecessary exposure of the domain to the very acidic pH of the gastric environment and its associated gastric enzymes. In a preliminary study, VNAR D1–C4 efficiently expressed in *L. lactis* and neutralized hTNF-α in L929 assay with ND50 value of 105 pM.

To partially mimic the effects of site-specific delivery, we have utilized an *in vitro* cell-based intestinal epithelial dysfunction model, and have shown that even our monovalent VNAR domains elicit excellent protection against the inflammatory toxicity generated by the presence of human TNF-α. This protection was improved when the VNARs were used in dimer or trimer formats and the level of activity seen was equivalent to that of a clinical antibody control, Humira® (**Figures 6** and **7**). In a pH stability test, designed to mimic the environment of the gut, only the VNAR and VHH domains retained any activity (around 80%) after 7 days exposure (**Table 3**).

It was originally proposed, that unlike the VHH domains (62–64), VNARs might not efficiently form a dimeric fusion molecule, and even where dimerization of VNAR was achieved, the resulting dimeric construct showed a compromised expression level and binding activity thought to be due to steric hindrance or occlusion of the binding site by the incorporated linker (17). It was later shown that VNARs do in fact tolerate both N- and C-terminal molecular fusions to form either a dimer or trimer construct without significant loss of binding characteristics (25). In fact, improvements in binding kinetics, efficacy and pharmacokinetics of domains have been achieved through reformatting parental VNAR and VHH domains as multivalent and/or multi-specific domains (21, 25, 41, 63–66). Increasing the valency of binding domains such as from a monovalent Fab to a bivalent Fab fragment, monovalent scFv to bivalent scFv-Fc, or from a monovalent VHH to a bivalent, or trivalent VHH domain have shown in some cases up to a 500 fold increase in potency and/or affinity for a range of antigens and assays. Enhanced avidity and even additional mechanism of binding fragment-antigen interactions have been shown to be largely responsible for the improved efficacy seen with these multivalent binding domains (64, 65, 67). The improvements we have seen in potency is greater than one would have expected from just avidity (**Tables 1** and **2**) alone with some domains (e.g., D1–BA11–C4, Quad-X™ D1–Fc–C4) showing improvements of over 10,000-fold. As these jumps in potency are always associated with combinations of the D1 and C4 domains (and in that order), we postulate that some of this enhancement in neutralization may be due to bi-paratopic binding, not avidity alone. Further analysis would be required to confirm this hypothesis.

Any increase in valency of the VNAR domains increases the number of possible epitopes that can be assessed on a bioactive hTNF-α trimer, and this capacity has been optimized here using an empirically designed flexible linker (**Figure 2**). Previous studies have shown that multivalent constructs spaced by a short linker can achieve increased functionality by improving their antigen binding through avidity, increased antigen specificity or by cross-linking two hTNF-α trimers (64, 68, 69). We, therefore, hypothesize that the short but flexible 11 amino acid GlySer linker in the dimeric VNAR constructs (D1–C4) allow these domains to interact with two epitopes on one trimeric TNF-α molecules and/or cross-link the interaction of two epitopes on two trimeric TNF-α molecules. Similarly, the trivalent VNAR domains (D1–BA11–C4) were constructed with an increased spatial separation of the anti-TNF-α VNAR domains using two 20 amino acid flexible GlySer linkers and a 103 amino acid VNAR BA11 "spacer," which further enhanced the ability (reach) of these constructs to interact and cross-link bioactive trimeric TNF-α molecules. The VNAR BA11, is a humanized anti-HSA soloMER™ capable of both increasing the spatial separation of the anti-hTNF-α VNAR domains and providing an option for serum half-life extension if used in a systemic, therapeutic setting (25, 41). The logical extension of this approach is our Quad-X™ family of proteins (D1–Fc–C4). Here, we have replaced the BA11 spacer with a human Fc region (half-life extension and immune system recruitment) but have enhanced the neutralizing potency of the fusion protein further by increasing the valency of both D1 and C4 domains through natural and efficient dimerization of the expressed protein. This proved to be the most potent of all the constructs tested delivering complete neutralization at only 2 pM compared to 30 pM by the world's best-selling drug Humira®.

The simple molecular architecture of VNAR domains facilitates flexible reformatting options and the accommodation of additional functionality to deliver a panel of therapeutically useful formats optimized for administration both as systemic and/or site-specific drugs. This flexibility can also be used to enhance drug potency delivering a new class of biologics that can match and even surpass the activity of some of the best studied therapeutic antibodies and deliver this enhanced potency with easily expressed fusion proteins that are two-third the size of a whole antibody.

## ETHICS STATEMENT

This study was conducted in accordance with the recommendations of HMRC described in the project license—PPL 60/3799. Work conducted by the Institute of Marine and Environmental Technology (Baltimore, MD, USA) was under their ethical guidelines and authorized animal procedures. Sharks were kept in an extensive state of the art fish-holding facility as part of an environmentally responsible marine core facility that provides excellent experimental capacity for research with marine organisms. It is a completely contained, recirculating operation with large-scale mechanical and biological filtration and life support systems that enable safe and efficient re-use of tank water.

# AUTHOR CONTRIBUTIONS

OU: scientific lead on this paper who carried out the majority of the scientific work and wrote the manuscript. JS: assisted in the rational *in-silico* construction and expression of some of the VNAR-Fc constructs. MK and LF: assisted in the library construction and expression of multivalent VNAR proteins. CB: assisted

# REFERENCES

with the VNAR and monoclonal antibody stability assessment. CJB: led the science team at Elasmogen, and co-supervised this project alongside AP. Also both CJB and AP reviewed the manuscript.

# ACKNOWLEDGMENTS

The authors wish to acknowledge the funding support for this work from MSD/Scottish Universities Life Sciences Alliance (SULSA), Scottish Enterprise, the Biotechnology and Biological Sciences Research Council (BBSRC), and the University of Aberdeen.

# FUNDING

Grateful for support from Biotechnology and Biological Sciences Research Council (BB/K010905/1), Scottish Enterprise [VNAR\_001 (2012)], Scottish Universities Life Sciences Alliance/ MSD (MSD01\_A\_Porter-Teismann), and the College of Life Sciences and Medicine, University of Aberdeen (Fee bursary to OU).

from the psoriasis longitudinal assessment and registry (PSOLAR). *JAMA Dermatol* (2015) 151:961–9. doi:10.1001/jamadermatol.2015.0718

69. Smith LE, Crouch K, Cao W, Müller MR, Wu L, Steven J, et al. Characterization of the immunoglobulin repertoire of the spiny dogfish (*Squalus acanthias*). *Dev Comp Immunol* (2012) 36:665–79. doi:10.1016/j.dci. 2011.10.007

**Conflict of Interest Statement:** OU, JS, MK, LF, AP, and CJB are affiliated with Elasmogen Limited.

*Copyright © 2017 Ubah, Steven, Kovaleva, Ferguson, Barelle, Porter and Barelle. 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.*

# *In Vitro* Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development

*John Steven1 \*, Mischa R. Müller <sup>2</sup> , Miguel F. Carvalho3 , Obinna C. Ubah1 , Marina Kovaleva1 , Gerard Donohoe <sup>1</sup> , Thomas Baddeley4 , Dawn Cornock <sup>4</sup> , Kenneth Saunders5 , Andrew J. Porter1,6 and Caroline Jane Barelle1*

*1Elasmogen Ltd., Aberdeen, United Kingdom, 2Molecular Partners AG, Zurich, Switzerland, 3Pfizer, Aberdeen, United Kingdom, 4Department of Chemistry, University of Aberdeen, Aberdeen, United Kingdom, 5UCB BioPharma Sprl, Braine-l'Alleud, Belgium, 6Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom*

#### *Edited by:*

*Colin Roger MacKenzie, National Research Council Canada, Canada*

#### *Reviewed by:*

*Serge Muyldermans, Vrije Universiteit Brussel, Belgium Hans De Haard, argenx BVBA, Belgium Christian Cambillau, Aix-Marseille University, France*

> *\*Correspondence: John Steven<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 04 July 2017 Accepted: 04 October 2017 Published: 23 October 2017*

#### *Citation:*

*Steven J, Müller MR, Carvalho MF, Ubah OC, Kovaleva M, Donohoe G, Baddeley T, Cornock D, Saunders K, Porter AJ and Barelle CJ (2017) In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties to Facilitate Clinical Development. Front. Immunol. 8:1361. doi: 10.3389/fimmu.2017.01361*

Molecular engineering to increase the percentage identity to common human immunoglobulin sequences of non-human therapeutic antibodies and scaffolds has become standard practice. This strategy is often used to reduce undesirable immunogenic responses, accelerating the clinical development of candidate domains. The first humanized shark variable domain (VNAR) was reported by Kovalenko and colleagues and used the anti-human serum albumin (HSA) domain, clone E06, as a model to construct a number of humanized versions including huE06v1.10. This study extends this work by using huE06v1.10 as a template to isolate domains with improved biophysical properties and reduced antigenicity. Random mutagenesis was conducted on huE06v1.10 followed by refinement of clones through an off-rate ranking-based selection on target antigen. Many of these next-generation binders retained high affinity for target, together with good species cross-reactivity. Lead domains were assessed for any tendency to dimerize, tolerance to N- and C-terminal fusions, affinity, stability, and relative antigenicity in human dendritic cell assays. Functionality of candidate clones was verified *in vivo* through the extension of serum half-life in a typical drug format. From these analyses the domain, BA11, exhibited negligible antigenicity, high stability and high affinity for mouse, rat, and HSA. When these attributes were combined with demonstrable functionality in a rat model of PK, the BA11 clone was established as our clinical candidate.

Keywords: VNAR, soloMER, single chain binding domain, shark, humanization, half-life extension, pharmacokinetics

### INTRODUCTION

In recent years, different approaches such as complementarity-determining region (CDR) grafting, framework-homology-based, germline-homology-based re-surfacing, and epitope depletion have been adopted to reduce potential immunogenic responses from non-human therapeutic biologics (1–5). According to Reichert (6), of the 52 antibodies in late stage clinical trials in Europe and the

**Abbreviations:** IgNAR, immunoglobulin new antigen receptor; VNAR, variable domain of shark new antigen receptor; CDR, complementarity-determining region; FW, framework HV, hypervariable region; PK, pharmacokinetics; HSA, human serum albumin.

US, the majority, 43, are humanized or are fully human exemplifying the success and clinical validation of these processes. Furthermore, the six monoclonal antibodies (mAbs) approved for use in the European Union or the US in 2016, three were humanized, two were fully human, and one was a chimeric IgG1 with a further 10 human or humanized antibodies in review (6). Of those mAbs approved for therapeutic use, three are the blockbuster drugs for oncological indications, Rituximab (Rituxan), Trastuzumab (Herceptin), and Bevacizumab (Avastin), each of which generated in excess of \$6 billion in revenues in 2015 (7–9).

The development of next-generation therapeutic biologics has been gaining pace with over 50 products based on both immunoglobulin and non-immunoglobulin scaffolds at varying stages of development (6, 10). Much of this development reflects the drive to overcome the limitations of classical antibodies such as their complex structure, and large size resulting in high cost of manufacture from an economic perspective and limited tissue penetration from a biological perspective (11–14). The focus of this study was the improvement of the biophysical properties of a humanized VNAR, which is the variable binding antigen specific domain derived from the new immunoglobulin antigen receptor or IgNAR (15). As IgNAR forms an integral part of the adaptive immune system of sharks, high-affinity, highly selective IgNARs can be raised through immunization and target-specific VNAR domains selected through classic phage display technology (16–18). Although demonstrating the target-specific attributes of antibodies, VNARs do not originate from an immunoglobulin lineage but are postulated to have evolved from cell surface members of the Ig superfamily such as ICAMs and VCAMs (19–21). Interestingly, their sequence identity is closer to human light chain frameworks than heavy chains. As with other non-human sources of antibodies, this reduced relatedness to human mAbs is the driving force behind developing a humanization strategy for the development of these domains for clinical use. Elasmogen Ltd. has a proven, patented methodology for the humanization of VNAR domains, the endpoint of which are therapeutic clinical candidates known as soloMERs™ (22).

The subject of this work, hE06v1.10, is a humanized version of an anti-HSA clone called E06 that was originally isolated from an immunized dogfish (18, 23). E06 can be fused with many different therapeutic partners including but not limited to, VNAR domains, scFvs, peptides, and proteins to increase their systemic half-life and subsequent therapeutic window (23). With broad utility across multiple indications, the biophysical properties of the final humanized version of E06 were critical to negate any downstream production issues with aggregation or potential immunogenicity. The first steps toward humanization of VNAR domains were conducted by Kovalenko et al. (24) using the anti-hen egg (HEL) domain, 5A7 (16), as a model template. Given the evolutionary distance between VNARs and true IgGs, the foundation of the approach was based primarily on structural similarity to human framework scaffolds. Using this strategy, DPK9 was selected. The underlying methodology used to successfully humanize 5A7 was then transposed onto the anti-human serum albumin (HSA) binding VNAR domain, E06 resulting in the construction of v1.10. A second version of humanized E06, v2.4, was designed based on human Kappa germline framework DPK24.

This work has studied v1.10 and v2.4 in more depth and has revealed a propensity for the v1.10 domains to dimerize and in the case of v2.4 to bind to HSA with lower affinity, questioning their applicability for clinical development. To overcome the unfavorable characteristics of v1.10 and v2.4, these were used as templates for mutagenesis to select better performing humanized versions of these proteins.

### MATERIALS AND METHODS

### Random Mutagenesis of E06v1.10 and E06v2.4

E06v1.10 and E06v2.4 sequences were cloned into the phage display vector pWRIL-9 and tested as periprep extracts for binding to cognate target and relevant controls. This vector is comparable to pWRIL-1 (25) with the c-Myc tag replaced by an HA tag, and the leader sequence is derived from pelB. Sequences were mutagenized by error-prone PCR to deliver up to 9 substitutions/ VNAR sequence using a GeneMorph II random mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). Libraries were cloned into pWRIL-9 and transformed into TG1 electrocompetent cells (Lucigen, Middleton, WI, USA). Library diversity was analyzed by sequencing over 100 clones from each repertoire.

#### Selection from Libraries by Phage Display

Libraries were rescued and selected twice using Nunc Maxisorp immunotubes as previously described (26). For the first round of panning, tubes were washed five times with PBS containing 0.1% Tween 20 (PBS-T) and five times with PBS; for pan 2, the number of washes with and without Tween 20 was doubled. Following each round of panning, two 96-well plates of individual colonies were picked with a QPix2 XT (Genetix, San Jose, CA, USA) and grown for periplasmic protein extraction. Binding to HSA (and HEL control) was evaluated by ELISA using 50% of the crude periplasmic protein extract (27). VNARs were detected via their HA tag using a high-affinity mAb HRP conjugate (clone 3F10; Roche, Basel, Switzerland). All samples were processed with a Perkin Elmer MiniTrak robotic liquid handling system (Waltham, MA, USA).

### Reformatting to Eukaryotic Expression Vector and Analysis of Output by ELISA

Unique clones showing OD450 by periprep ELISA at least 25% higher than the readings obtained from parental huE06v1.10 or v2.4 were selected for batch conversion into the proprietary mammalian expression vector pSMED2. Equal amounts of pWRIL-9 plasmid DNA from clones were pooled, and VNAR sequences were amplified by PCR with primers inserting *Bss*HII and *Eco*RI at the 5′ and 3′ ends, respectively. After cloning and transformation of *Escherichia coli* TG1 electrocompetent cells, a fourfold over-representation of the starting number of clones was grown in 96-deepwell culture plates (Greiner Bio One, Frickenhausen, Germany) and plasmid DNA purified using a QIAprep 96 Turbo BioRobot Kit in a BioRobot 8000 (Qiagen, Hilden, Germany). In a 96-well plate format, each clone was expressed transiently in 200 µL of HEK293 cells previously adjusted to a density of 106 /mL. Each 200 µL culture was transfected with 200 ng of plasmid DNA using lipofectamine (Invitrogen, Carlsbad, CA, USA) and grown at 37°C and 8% CO2 while shaking at 250 rpm to maintain cells in suspension. After 24 h, cultures were supplemented with tryptone to a final concentration of 0.5% and expression continued for 6 days. Post-expression media samples from HEK 293 transfections were tested for binding to HSA and HEL by ELISA. Detection was achieved via an anti-6-His HRP conjugate (ab1187; Abcam, Cambridge, UK).

#### Off-Rate Selection Screening

Samples of the "best-performing" media from the small scale HEK293 transfections were subject to kinetic analysis using a T200 BIAcore instrument for off-rate ranking (28) (GE Healthcare Life Sciences, Little Chalfont, UK). For off-rate screening samples were diluted 1:5, 0.2 μm-filtered, then filtrates run over a research-grade carboxy-methyl-dextran chip (CM5) onto which HSA was immobilized using standard amine coupling chemistry. The association phase for all samples was 2 min, and the dissociation was monitored for 3 min at a flow rate of 100 µL/min, followed by two 10 µL injections of glycine pH 1.5 at a flow rate of 100 μL/min. All binding experiments were performed at 25°C in HBS/EP buffer. Analysis of the resultant sensorgrams made use of the 1:1 global Langmuir binding model. Those samples showing the slowest dissociation rates were then selected for larger-scale protein production and DNA sequences of VNARs determined.

#### ELISA Assay and EC50 Determination

Rat, mouse, and HSAs used in ELISA-binding assays were from Sigma-Aldrich. For direct ELISA formats Nunc Maxisorp 96-well plates were coated at 1 µg/mL antigen in PBS and then blocked with 4% non-fat milk in PBS. Purified 6-his-tagged control and humanized VNAR proteins in PBS were diluted 1/3 into wells and double-diluted further across the plate. After incubation for 1 h, plates were washed three times with 0.05% Tween 20 in PBS. The detection of antigen bound VNARs was achieved by incubation with an anti-6-His HRP mAb for 1 h or where appropriate with an anti HA tag mAb HRP conjugate (clone 3F10; Roche) and developed by adding TMB substrate. When fully developed, the reactions were halted by the addition of 1 M H2SO4 absorbance measured at 450 nM. Data were processed using SigmaPlot 9.

#### Affinity Measurements

Affinities of selected clones were determined on a T200 BIAcore surface plasmon resonance instrument essentially as described previously (24). As well as measuring affinity for HSA, the mutated anti-HSA VNARs were also assessed for binding to mouse serum albumin (MSA) and rat serum albumin (RSA). A CM5 chip was prepared in which the first flow cell was used as a reference to correct for bulk refractive index, matrix effects, and non-specific binding. Approximately 300 RU of HSA was immobilized onto flow cell 2, 350 RU of MSA was immobilized onto flow cell 3, and 600 RU of RSA onto flow cell 4. Prior to immobilization, the serum albumins were made up in 10 mM sodium acetate buffer (pH 4.5) and post-coupling the remaining activated groups was blocked with 1.0 M ethanolamine-HCl pH 8.5. For affinity measurements, purified anti-HSA VNAR monomers, dimers, and trimers were diluted to 1.56–100 nM in HBS/EP buffer and injected over the chip as above. Analysis of the resultant sensorgrams was performed using the 1:1 global Langmuir binding model fit analysis (BIAcore Evaluation Software).

#### Protein Expression and Purification

Expression of VNAR proteins for periprep screening and phage ELISA was carried out as described (29). For small-scale transient expression and off-rate screening, mutated VNAR genes were cloned into pSMED2 vector containing a CMV promoter and a C-terminal 6-His tag. Following off-rate screening plasmid preparations of selected clones were scaled up and used for 1 L PEI-mediated transfection and transient expression in HEK293 host cells (30, 31) using serum free FreeStyle™ 293 media (Invitrogen). Purification of expressed protein was achieved by immobilized metal chelate chromatography using Ni2<sup>+</sup> charged resin followed by cation exchange chromatography with buffer exchange as appropriate between steps. Final protein samples were buffer exchanged to PBS and stored frozen at −20°C. If required, proteins were then subjected to a final polishing step by preparative size-exclusion chromatography using a Superdex 200 26/60 size-exclusion column equilibrated with PBS. Eluted peaks from this chromatography were pooled, then concentrated using Amicon Ultra filtration units. Protein concentrations were determined by UV spectroscopy. Expression levels of VNAR proteins were generally in the region of 0.5–3 mg/L, and electrophoretic analysis of protein samples was performed on 12% NuPAGE BisTris gels using a MOPS buffer system (Invitrogen). Analytical size-exclusion chromatography was performed using an Agilent 1200 series HPLC system and ZORBAX GF 250 9.4 mm × 250 mm 4 μm column or TSK gel G3000PW 7.5 mm × 30 mm column equilibrated with phosphate-buffered saline at pH 7.4 with instrument set up and run parameters adjusted as required for each run.

# Cloning of VNAR Dimeric and Trimeric Constructs

VNAR E06 and lead humanized clones BA11, BB11, and BB10 were selected as the backbone of several dimeric and trimeric fusion protein constructs. Dimers were assembled using a standard PCR overlapping extension techniques joining the albumin binding domain, via a (GGGGS)4GAHS flexible linker to the carboxyl or amino terminal end of a control and naive VNAR domain known as 2V. The trimeric constructs were made by flanking the albumin binding domain with the same naive 2V domain at both terminal ends (23). Constructs were cloned into pSMED2, plasmid preparation scaled up and the resultant DNA used to transiently express protein in HEK 293 cells (30, 31).

# Dendritic Cell–T-Cell (DC–T) Assay

A DC–T proliferation assay was used to identify the presence or absence of possible T-cell epitopes within the wild-type E06 and humanized variants 2G, BA11, BB11 and BB10 (performed at ProImmune Ltd., UK using ProImmune's REVEAL® Immunogenicity System DC–T cell assay). For this study, test proteins and controls were incubated with CD8+ T-cell-depleted peripheral blood mononuclear cells (PBMCs) prepared for 7 days, from a panel of 20 different healthy human donors. Each PBMC sample was HLA-typed and donors were selected (by DRB1 allotyping) to approximately represent MHC class II allele frequency distributions across the global population.

In brief, adherent donor PBMCs were cultured with appropriate growth factors to generate monocyte-derived DCs. DCs were then loaded with either test or control antigen protein or left untreated. Test proteins were loaded at a final concentration of ~0.34 μM (5 µg/mL). Mature antigen-loaded DCs were then co-cultured (at a set ratio) with autologous CFSE-labeled T cells in multi-well plates. Each test condition was set up in octuplet and incubated for 7 days. Positive control antigens used for this assay were Tuberculin purified protein derivative (PPD from *Mycobacterium*) at a final assay concentration of 0.2–0.4 µM (~5 μg/mL) (70–100% of donors are expected to react to this protein as a result of previous vaccination, through a memory immune response). A second control antigen used, keyhole limpet hemocyanin (KLH) is a known potent naive protein immunogen. This was used at ~0.64 μM (0.25 mg/mL) in the assay (up to 70% of donor samples might be expected to react to this protein, driven by a naive immune response). At the end of the incubation period, cell samples were stained with anti-CD4 antibody, then washed and fixed for flow cytometric analysis. Proliferation was determined by measuring a decrease in CFSE intensity.

# Pharmacokinetic (PK) Studies

Three groups of four male Sprague Dawley rats received a single intravenous (bolus) administration of 2V-E06-2V, 2V-BA11-2V, or 2V-BB11-2V dosing at 1 mg/kg. Blood samples (0.3 mL) were collected into tubes containing Lithium Heparin as an anticoagulant before dosing and at the following times: 30 min and 4, 12, 24, 48, 84, 96 120, and 144 h. These were placed on ice and plasma collected from each after centrifugation at 3,000 × *g*, at 4°C for 10 min. Prior to analysis plasma samples were stored at −80°C.

### LC–MS/MS Methodology for the Bioanalysis of PK Samples

A quantitative LC–MS/MS method had been previously developed to specifically measure concentrations of 2V-E06 in rat plasma (23). In summary, the method utilizes the C-terminal 6x His tag present on these proteins molecules to provide sample enrichment using a magnetic Ni-NTA bead capture step. Following analyte enrichment and tryptic digestion of the sample, targeted LC–MS/MS is used to quantify specific signature peptides in all parts of 2V and the humanized E06 domains. Signature peptides within each partner were identified by *in silico* tryptic digestion. For E06 and humanized variants BA11 and BB11, the signature peptide EQISISGR was selected and AQSLAISTR for 2V. Control signature peptides and a labeled internal standard peptide EQI-[U13C3, 15N-Ser]-ISGRAQS-[U13C6, 15N-Leu]- AISTRHHHHHH were synthesized (by Cambridge Research Biochemicals, Billingham, UK). Assessment of peptides allowed the PK determination of the VNARs (serum half-life) as well as monitoring of (GGGGS)4GAHS linker stability.

# Plasma Pull Down and Trypsin Digestion

VNAR 2V-E06-2V protein was diluted into heparinized rat plasma at 30, 20, 10, 5, 3, 1, 0.3, 0.2, and 0.01 µg/mL. A total of 10 µL of each dilution was added to 40 µL of 0.1 M phosphate buffer pH 8 followed by 50 µL of 6 M guanidine hydrochloride containing 1 µg/mL of the heavy isotope labeled internal standard peptide EQI-[U13C3,15N-Ser]-ISGRAQS-[U13C6,15N-Leu]- AISTRHHHHHH. After mixing, these samples were each reduced and alkylated as previously described (23). For the PK sampling time points, 10 µL of plasma was added to 40 µL of 0.1 M phosphate buffer pH 8. The samples were then diluted by the addition of 100 µL 100 mM phosphate, pH 8, 0.1% CHAPS in preparation for sample enrichment. Sample enrichment and magnetic bead processing was performed in 1.5 mL capped tubes and magnetic capture blocks. Twenty-five microliters Ni2<sup>+</sup>-NTA magnetic beads plus 75 µL 100 mM phosphate, pH 8, 0.1% CHAPS were added to each sample. After mixing, the magnetic beads were trapped by magnetic block and the fluid containing the unbound proteins was removed. The beads were washed three times with 500 µL 100 mM phosphate, pH 8, 0.1% CHAPS with magnetic immobilization of beads between each wash before a final 500 µL wash with 100 mM phosphate, pH 8, 0.1% CHAPS containing 20 mM imidazole. Bound proteins were eluted in 100 µL of 0.5 M Imidazole, 10 mM CaCl2, 50 mM Tris HCl, pH 8. Eluted protein samples were then trypsin digested by addition of 20 µL of proteomics grade trypsin (made up at 20 µg/mL in 10 mM CaCl2, 50 mM Tris HCl, pH 8) followed by incubation at 37°C for 18 h prior to LC–MS/MS analysis.

### LC–MS/MS Analysis

The samples were loaded into the auto-sampler module of a Waters Acquity UPLC (chilled at 6°C) and 5 µL of the extracted samples injected onto the LC–MS/MS system. The native and stable-labeled signature peptides were separated on Acquity UPLC BEH C18 1.7 µm 2.1 mm × 50 mm column equilibrated with solvent A water/acetonitrile/formic acid (95/5/0.1) and eluted with a stepped gradient as follows. At a flow rate of 0.5 mL/min, the column was washed with buffer A for 4 min, then from 4 to 8 min buffer was changed by gradient mixing to 20% solvent B + 100% acetonitrile + 0.1% formic acid. From 8 to 8.5 min solvent B increased to 55% then back to 45% solvent B at 9 min and back to 100% solvent A at 10 min in preparation for the next sample injection. Chromatography runs were carried out at ambient temperature, and under these conditions, the retention time for the analytical and reference peptides was 6.11 min for EQISISGR and 6.27 minutes for AQSLAISTR (±0.5 min). The peptide analytes EQISISGR and AQSLAISTR and control internal standard labeled peptides were detected by atmospheric pressure electrospray ionization MS/MS using a Xevo TQS MS/ MS detector. The analytical column eluate was delivered into the source operated at an IonSpray voltage of 3,300 eV with settings as follows: cone (V) 25, source offset (V) 45, source temperature 150°C, desolvation temperature (600°C), cone gas flow (L/h) 0, desolvation gas flow (mL/min) 0, collision gas flow (mL/min) 0.15, and nebulizer gas flow (Bar) 7.00.

For the analytes and control labeled internal standard peptides, MRM transitions were 445.2–519.3 for EQISISGR, 473.8–747.4 for AQSLAISTR, 447.2–523.3 for EQISISGR-IS, and 477.3–754.4 for AQSLAISTR-IS. In all experimental runs, a system suitability test was performed by the injection of a 50 µg/mL AQSLAISTR and EQISISGR mixed standard in buffer. The ion chromatograms were quantified by reference to standard curves spiked into fresh control plasma and analyzed over the range 0.01–30 µg/mL and a calibration curve constructed by plotting the peak area ratio of the calibration standards *vs.* the concentration of peptide fragments in a control matrix and determine the linear regression parameters of the curve, using a 1/*x*<sup>2</sup> weighting factor. The concentration of peptide fragments in the quality control and test samples were determined by interpolation of the peak area ratios from the calibration curve.

#### Stability Assay

The stability of VNAR E06 and humanized E06 BA11 was assessed after exposure to extremes of temperature and pH. Samples of E06 and humanized E06 BA11 proteins were prepared at 10 µg/mL working concentration and placed on a 100°C preheated block. Samples were withdrawn from experimental conditions at 0, 5, 10, 20, 30, 40, 50, and 60 min time points and transferred into wells containing appropriate volume of PBS pH 7.4 to obtain a final concentration of 0.5 µg/mL. Boiled samples recovered in PBS were kept on ice for 1 h before assessing VNAR E06 and humanized VNAR BA11 in an has-binding ELISA. In a similar assay the pH stability of VNAR domains was assessed. Samples were prepared and incubated at a working concentration of 10 µg/mL in a final volume of 50 µL at the designated pH value. For acidic conditions, pHs 1.5, 3, and 5.5 protein samples in PBS pH 7.4 were adjusted using 1 M HCl or 0.1 M citric acid and for basic pHs 8.5, 10 and 11 samples in PBS were titrated to required pH value using borax buffer or 1 M NaOH. Samples were incubated at room temperature, and aliquots withdrawn at stipulated time points and neutralized in PBS pH 7.4 to a final concentration of 0.5 µg/mL. Samples were incubated at designated pH values for up to 28 days, and the HSA-binding activity of the treated samples was determined using ELISA.

# RESULTS

# Affinity Maturation of Humanized VNARs by Random Mutagenesis, Library Screening and Selection

Analysis of E06 clones humanized (hE06) by targeted insertion of residues and sequences from human V Kappa germ lines led to two "parental" molecules (v1.10 and v2.4 **Figure 1A**) with specific but lower binding affinities and undesirable biophysical properties (**Figures 1** and **2** respectively). In order to make improvements to these two humanized variants, two randomly mutated libraries were constructed by error-prone PCR using DNA from these clones as the initial template (v1.10 and v2.4 **Figure 1A**, based on DPK9 and DPK24 human Kappa germline sequences, respectively). To ensure full coverage of the hE06 sequences and to reap any potential benefits of multiple substitutions in the protein sequences, conditions were optimized to deliver a maximum of nine nucleotide mutations per VNAR. Phagemid libraries of ~5 × 107 clones for both hE06v1.10 and v2.4 were constructed

and diversity determined by randomly selecting and sequencing of over 100 individual clones from each library. Results indicated a good coverage of mutations along the full sequence length, with an overall mutation rate of ~90% (90/103 and 94/103 clones had a changed amino acid residue, for hE06v1.10 and v2.4, libraries respectively).

In order to recover molecules with improved binding properties two rounds of phage display selection were performed using HSA as a target. Approximately 200 individual clones were randomly picked from each library and round of panning (total of 800 clones) and tested for binding by ELISA using VNAR protein derived from crude periplasmic extracts. Additional control screening was performed using non target antigens (HEL and blocking agent) to confirm that there was no overall gross VNAR misfolding leading to polyreactivity or non-specific stickiness. An initial examination of the sequences of the enriched clones did not offer up a clear pattern or any obvious positional bias but placed the mutations randomly throughout the parental templates. In addition, parental hE06v1.10 was recovered a total of 53 times from pan 1 and 80 times from pan 2, while hE06v2.4 was observed only once in pan 2 and did not occur in the pan 1 sequences at all. The remaining "new" clones were subjected to further analysis including their expressability and any propensity to form dimers (results not shown). **Figure 1C** shows the isolation of a final panel of mutated clones based on periprep ELISA binding from which 37 humanized E06v1.10 (boxed) and 12 humanized E06v2.4 clones (circled) were selected for further characterization. These 49 clones were transferred to a eukaryotic expression vector and small scale transient expressions performed in HEK 293 cells.

# Protein Expression, Assessment, and Characterization

Media samples from HEK 293 expressions were screened for specificity using an HSA-binding ELISA and off-rate ranking was performed on the same samples using a T200 BIAcore surface plasmon resonance instrument. Clones that showed a slow-off rate nearing that of parental E06 and were positive by ELISA, giving signals similar to the parental hE06v1.10 or v2.4 clones, and were selected for further study (**Figure 3A**). The sequences of these selected clones were determined and those from v1.10 and v2.4 derived libraries are shown in **Figures 3B,C,** respectively. The positions of the mutations identified, even in this focused sub-panel of lead clones, were typically one or two amino acid mutations randomly located along the length of the VNAR proteins (**Figures 3B,C**).

Transient expression of these 15 lead clones was scaled up in HEK 293 cells to produce proteins for further study. After scale up and growth in serum free media and post-purification, expression levels of between 3 and 10 mg/L were obtained for the humanized monomeric, dimeric, and trimeric constructs. Purified protein samples were quantified by UV spectroscopy and concentrations determined. For the best expressing clones, together with suitable controls (E06 wild-type, v1.10, AH7, AD4, AG11, BA11, BB11, BB10, and v2.4 derived clones 1H, 2G, 5F, 8C, and 8D), ranking of the purified proteins was determined by ELISA (**Figures 4A,B**) and EC50 values calculated using SigmaPlot 9.0 (**Figures 4C,D**). Based on expression levels and EC50 values, a final panel four of clones was selected. From the mutated v1.10 library BB10, BA11, and BB11 and from the v2.4 derived library, clone 2G was characterized in more detail.

Figure 3 | ELISA and off-rate screening analysis of related and humanized mutant. The intensity of binding of media samples from small scale transient expressions in HEK 293 cells were screened and compared by kinetic off-rate analysis, using a T200 BIAcore instrument, and HSA-binding ELISA. (A) Selected lead clones derived from v1.10 library (data for v2.4 selection not shown). Off-rate as a percentage of the parental E06 off-rate plotted together with ELISA binding. (B) The amino acid sequence of selected clones from the v1.10 mutagenesis library. (C) The amino acid sequence of selected clones from v2.4 mutagenesis library. Mutated residues differing from parental sequence are highlighted. See Figure 1 legend for list of abbreviations.

# SDS PAGE and Analytical SEC of Final Four Purified Leads

Post-purification proteins were analyzed by SDS PAGE and analytical SEC. On SDS PAGE, the selected clones ran as a single band (**Figure 5A**), with an expected molecular mass of 14 kDa. Analytical size-exclusion chromatography of the purified monomeric BB10, BA11, BB11, 2G, and E06 control appeared mainly as single peaks showing minimal signs of dimerization (**Figure 5B**) unlike the parental clone v1.10 (**Figure 2C**). The purified BB11 monomer showed a small peak eluting from the column ahead the of the main monomeric protein peak. It is unclear if this is dimerized BB11 protein or low level higher molecular weight contaminating protein. The remaining humanized VNAR monomers eluted as single peaks.

# Antigenicity Assessment of E06 and Four Lead Humanized VNAR Clones

Immunogenicity of lead clones was assessed in a T-cell proliferation assay using ProImmune Ltd REVEAL® Immunogenicity System DC–T cell assay. The immunogenicity of each protein was determined by measuring the extent of T cells proliferation and by determining the number of immune-responsive individual

(B) Binding curves of selected lead clones derived from mutagenesis of the v2.4 backbone. (C) EC50 values of lead v1.10 derived clones. (D) EC50 values of lead v2.4 derived clones. With the exception of E06 v2.4-2G, clones obtained by mutagenesis of E06 v1.10 performed better with several selected clones showing single digit nanomolar or lower EC50 values.

donors treated with test proteins. Stimulation above background was determined using flow cytometric evaluation to count unlabeled, therefore proliferating T cells in each of the eight replicates for each sample. These values were then used to calculate percentage stimulation above background. Thus, the strength and frequency of response to each test sample could be used to calculate a response index (RI) value for each test protein (E06, BB10, BA11, BB11, and 2G) and control antigens (**Figures 6B,C**). Both the wild-type E06 and humanized variants had a very low RI compared to positive controls. Of the four humanized leads, clone 2G had the highest response. Clone BB11 gave a response, which was slightly higher than wild-type E06 with clones BA11 and BB10 having a slightly lower RI. These RI values were considered suitable for further development and similar to those of other therapeutic antibodies currently in the clinic and assayed using an identical protocol. Campath, Avastin, Humira, and Remicade had RI values of 0.26, 0.21, 0.14, and 0.49, respectively, and control RI values of 4 for KLH and 27.5 for PPD.

Based on this antigenicity assessment, clone 2G was not chosen for further study while clones BB10, BA11 BB11, and wild-type E06 were reformatted as dimer or trimer genetic fusions and expressed with a second control VNAR called 2V. Originally isolated from the dogfish *Squalus acanthias*, 2V is part of a sequence database from this species and has no known target, making it an ideal control for these and other studies (23). When reformatted and expressed as amino or carboxyl terminal end dimeric fusion proteins (e.g., 2V-hE06), samples ran as single non-aggregated peaks when analyzed by analytical SEC (**Figure 7A**) with the exception of samples BB11-2V and 2V-BB10 that showed a minor peak eluting before the larger main peak, which may indicate the presence of some dimer in these samples. When the parental clone v1.10 was reformatted as a dimer with 2V, the resultant v1.10-2V protein retained its propensity to run as two peaks (**Figure 7A**). Formatting to produce the trimeric versions, 2V-E06-2V, 2V-BA11-2V, and 2V-BB10-2V also resulted in proteins which after purification

ran predominantly as single peaks (**Figure 7B**) with BB11 containing trimeric construct, 2V-BB11-2V, showing a minor peak which eluted from the SEC column before the main peak. SDS PAGE analysis of monomer BA11, dimers 2V-BA11 and BA11-2V, and trimer 2V-BA11-2V confirmed that all migrated as single bands with significantly improved biophysical properties (**Figure 7C**).

### Affinity Measurements

Affinity measurements of purified anti-HSA VNAR and humanized VNAR monomers E06, 2G, BA11, BB11, and BB10 were determined by surface plasmon resonance using a T200 BIAcore instrument. The affinities for HSA, presented as a kinetic distribution plot (6A), were in the range of 0.58–24.9 nM. Affinities for rat and MSAs were in the range of 2.74–130 and 2.01–99 nM, respectively (**Figure 6A**). For all species tested, wild-type E06 had the highest affinity. The loss of binding of clone BB10 to rodent albumin could be attributed to the mutation in CDR3 and was therefore not taken forward as a candidate for further study. The eight dimeric constructs (BB11-2V, 2V-BB11, BB10-2V, 2V-BB10, BA11-2V, 2V-BA11, E06-2V, and 2V-E06) and four trimeric constructs (2V-BB11-2V, 2V-E06-2V, 2V-BA11-2V, and 2V-BB10-2V) bound HSA with high affinity including low picomolar values, 0.08–8.41 nM. In addition, binding and affinities for rat, mouse, and cynomolgus macaque albumins confirmed the utility of BB11 and BA11 as possible candidates for further clinical development (**Figure 8**).

# Rat PK Profile and Half-Life Determination of Candidate Clones

A previous PK study, in three animal species, demonstrated the ability of an albumin binding wild-type VNAR domain, E06, to extend the half-life of other proteins when administered as a molecular fusion (23). The present study looked at the ability of the humanized versions of E06, BB11, and BA11 to extend the circulating half-life of fusion proteins. Trimeric fusion proteins, 2V-BB11-2V, 2V-E06-2V, and 2V-BA11-2V, were expressed, purified, and characterized (**Figures 6** and **8**) prior to administration to rats at 1 mg/kg body weight. For this study, the sensitive and quantitative LC–MS techniques developed previously (23) were used to detect the presence of the test trimer VNAR fusion proteins in plasma samples. Circulating half-lives were determined of 11, 15, and 10 h for 2V-E06-2V, 2V-BA11-2V, and 2V-BB11-2V, respectively (**Figure 9**). Peptides derived from the albumin-binding domains E06, BA11, and BB11 as well as the 2V VNAR domain were detected, indicating that the domains remained stably linked for the duration of the study (results not shown).

### Stability

The robust nature of VNAR monomeric proteins has been well documented (16, 32, 33). This robustness is characterized by the ability of VNARs to refold and bind cognate antigens after heating to high temperatures, up to 100°C, for extended periods of time. In this study, the ability of the shark VNAR parental protein E06 and candidate humanized variant BA11 were subjected to a challenge at both elevated temperatures and extremes of pH. Incubation at 100°C for 60 min (**Figure 10A**) resulted in both E06 and BA11 proteins retaining a high percentage (~80%) of antigen specific binding activity. When challenged by incubation at extremes of pH for up to 2 weeks, both proteins showed robust stability and retained the capacity to bind HSA after neutralization to pH 7.4 (**Figures 10B,C**). Only prolonged treatment of BA11 at pH 1.5 showed any drop off in antigen binding. Humanization of the E06 VNAR domain appears not to have had any marked detrimental or deleterious effect on the ability of the protein to refold correctly after denaturing challenge.

Figure 6 | Affinity measurement and antigenicity assessment of lead humanized VNAR albumin binding domains. (A) Kinetic distribution plot and BIAcore affinity values for purified monomeric proteins E06, BB10, BA11, BB11 and 2G specific for human serum albumin, rat serum albumin, and mouse serum albumin. Values are the average of at least two experimental runs. (B) Proliferative T-cell response used to assess immunogenicity of monomeric E06, BB10, BA11, BB11, and 2G. (C) Table of calculated response index values. Positive control antigens used for the assay were (i) Tuberculin purified protein derivative (PPD from *Mycobacterium*) at a final assay concentration of 5 µg/mL (70–100% of donors are expected to react to this protein) and (ii) keyhole limpet hemocyanin, a recognized highly immunogenic protein.

# DISCUSSION

The utility of proteins as therapeutic agents is often compromised by their biophysical or biochemical properties which can lead to downstream processing issues including a propensity to precipitate or aggregate during purification and/or subsequent storage. Even if bio-processing is not an issue then a predisposition to elicit an adverse or immune response *in vivo* may also prevent further rapid development (34–36). While *in silico* modeling of VNAR domains predicts a low level of immunogenicity with similar values for those seen for a human Fc region (results not shown), we still embarked on a humanization strategy to accommodate any possible concerns from regulators. Immunogenicity testing of the parental E06 domain (**Figure 6B**) did in fact confirm these original *in silico* predictions. Earlier published efforts to humanize the HSA-binding, VNAR domain, E06 (24) produced a lead clone hE06v1.10, which unfortunately spontaneously dimerized (see **Figure 1**). A further version of humanized E06, based on human germline Kappa sequence DPK24, was also assessed, and although it was monomeric in solution and expressed well in recombinant systems, it unfortunately showed a greatly reduced affinity for target antigen. In an effort to improve these characteristics, both these humanized versions were subjected to a program of random mutagenesis (using error-prone PCR) and functional screening.

A panel of 15 clones (**Figures 3B,C**), demonstrating slow-off rates and strong ELISA binding, were chosen for further study and sequenced. Monomeric proteins for these clones were produced by transient expression in HEK 293 cells and EC50 values determined (**Figures 4C,D**). Clones BA11, BB11, BB10, and 2G emerged as the preferred "leads." Only the EC50 values of clones generated by mutation of hE06v1.10 were close to the values of the starting HuE06v1.10 and E06 (picomolar range). The 2G clone however saw the greatest improvement in binding to 1.2 nM from a parental hE06v2.4 EC50 value of 171 nM (also confirmed by BIAcore). This 2G clone also had the greatest number

Chromatograms show trimeric proteins running as a single peak. (C) SDS PAGE analysis and Coomassie blue staining of approximately 5 µg of purified lead humanized protein BA11 as monomer and multimers. Lane M, Thermo Scientific Spectra Broad Range Protein Ladder; Lane 1, monomeric BA11; Lane 2, 2V-BA11; Lane 3, BA11-2V; Lane 4, 2V-BA11-2V.

of amino acid changes (4), although it is unclear from this work whether the improvement is the result of a single change or the four changes in concert.

Binding kinetics of the monomeric anti-HSA clones E06, BA11, BB11, BB10, and 2G was measured by surface plasmon resonance. The affinity of E06 was determined as 0.58 nM and that of huE06v1.10 at 6.4 nM (24). The affinities of the candidate humanized and mutated clones all fell within this range. For accelerated clinical development of the humanized clones, it is also important that they have retained the animal species crossreactivity of E06 with comparable affinities. BA11, BB11, and 2G bound rat and MSAs with affinities between 2 and 130 nM. Interestingly, monomeric BB10 was no longer able to bind rodent albumin. This is due to the substitution of an isoleucine residue at position 90 in CDR3 with a larger hydrophobic phenylalanine residue. This hypothesis is supported by the published crystal structure (24) as isoleucine 90 contributes to hydrophobic intermolecular bonding between huE06 v1.10 and HSA. This lack of species cross-reactivity precluded BB10's use as a candidate clone for future clinical development.

Analysis of a larger number of mutated sequences from both libraries, performed as a part of their quality control, revealed that multiple changes of up to four amino acid positions were observed (data not shown). The four lead clones had a change in CDR3, indeed in clones BB10 and BB11, this was the only region where a change of residue occurs. For BB11, the glycine at position 87 was replaced by an alanine residue. BA11 also has a change at position 87 to a serine and also at position 78 where a phenylalanine is replaced by a serine. Clone 2G, which comes from the v2.4 background, has four mutations from the parental v2.4: asparagine 45 to a lysine, serine 52 to a glycine, leucine 73 to a methionine and a change in CDR3 where glycine 87 is mutated to a serine. The result of these mutations was a recovery of binding affinity but may have also contributed to the increased immunogenicity seen for this clone (**Figure 6**). Interestingly, clones 1H and 5F, from the v2.4 background, both had a substitution at position 88 from a valine to an alanine. Clone 5F also had a mutation at position 63 in HV4 from a threonine to an asparagine. Previous work has noted that although containing no contact residues, the HV4 region of E06 contributed to the binding of albumin by packing against contact residues of CDR1 (24). This second mutation in the HV4 region of clone 5F may explain the improvement noted in EC50 values, which decreased from 82 nM (1H) to 18 nM (5F)

Figure 8 | Affinity measurement of monomeric and reformatted dimeric and trimer humanized VNAR lead clones. BIAcore analyses of E06-2V, 2V-E06, 2V-E06-2V, BB10-2V, 2V-BB10, 2V-BB10-2V, BB11-2V, 2V-BB11, 2V-BB11-2V, 2V-BA11, BA11-2V, and 2V-BA11-2V against serum albumin from different species, human serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin, using a BIAcore instrument as described in the methods section. Kinetic measurements are summarized as averages from multiple runs.

and supports the theory that HV4 packs against the CDR1 region, thereby positioning amino acid residues of the CDR1 loop in contact with albumin. The highly selected change of CDR3 residue 87 (BB11, BA11, and 2G) is not an antigen specific contact residue (24), and therefore, retention of binding specificity and affinity is unsurprising. However, it does appear that a change at this position (or close by at position 90 for BB10) has resolved the dimerization bio-processing issue identified as a problem in the parental backbone v1.10 (**Figure 2**). While this phenomenon has been seen previously in antibody CDR regions, where particular motifs contribute to the multimerization of antibody proteins in solution (37, 38), we were surprised that such a small change from a glycine to a serine or even alanine could result in the dramatic improvement in manufacturability of the expressed protein.

Human immune response to bio-therapeutic proteins is notoriously difficult to predict and varies from individual to individual. It has been reported that proteins having high homology with human proteins or humanized proteins have reduced immunogenicity (35), but this cannot simply be presumed to be the case. No assay is capable of definitively predicting *in vivo* antigenicity with the only real test being observations from "in human" clinical studies (35). However, *in vitro* testing combined with bench-marking against existing clinical assets can provide a reasonable level of comfort when selecting candidate molecules for costly late stage pre-clinical studies. Humanized E06 VNAR domains BA11, BB11, BB10, and 2G showed very low RI values in a human donor T-cell proliferation assay using ProImmune Ltd. REVEAL® Immunogenicity System DC–T cell assay. Importantly, the RI values were comparable to those obtained when Campath, Avastin, Humira, and Remicade were investigated using the same assay. In fact, when control values are also compared, the humanized VNAR, BB10, had a relative RI slightly below those determined for this panel of well-known clinical agents and chosen lead BA11 has a lower RI than Remicade and is similar to humanized antibodies Campath and Avastin.

Based on a combination of affinity, antigenicity, species crossreactivity, and expressability, BA11 and BB11 (together with E06 and BB10 controls) were reformatted as dimeric and trimeric fusion proteins using a naive control VNAR 2V. By SDS PAGE, these proteins migrated as single bands with predicted molecular masses (**Figure 7C**) and confirmed that the bio-processing and affinity improvements seen for the monomeric humanized mutants was retained when they were assembled in a more relevant therapeutic format (fusions at the carboxyl terminal,

for 14 days shown for E06 (B) and BA11 (C).

amino terminal fusion, or both). Indeed 2V-BA11 protein was concentrated in PBS to therapeutic levels of over 50 mg/mL without precipitation, a favorable property for drug formulation, and retained a single-peak, SEC profile (data not shown).

Albumin half-life in the systemic circulation is prolonged by the FcRn recycling process (39, 40). In an early publication, monomeric and multimeric VNAR constructs containing the wild-type E06 albumin-binding domain showed a very impressive extended half-life equivalent to that of the species-specific (rodent or non-human primates) serum albumin (23). In this study, the functional ability of the humanized albumin-binding domains BA11 and BB11 and the wild-type E06 to extend circulating half-life, formatted as fusion proteins with the 2V VNAR domain (as trimers), was examined in a suitable rodent model. The humanized variants had a half-life (2V-BA11-2V 15 h and 2V-BB11-2V 10 h) equivalent to the parental control (2V-E06-2V 11 h), thereby confirming that the humanized domains remained bound to RSA during the FcRn recycling process.

Based on the accumulated data from affinity, analytical SEC profile, expressability, protein bio-processing quality, immunogenicity, and rodent-half-life studies, BA11 emerged as the clear candidate molecule for further study. To avoid confusion, it is worth noting that recent commercial literature often refers to humanized VNAR as soloMERs™ (22) and BA11 as NDure™.

Single-domain antibodies from shark (16, 32, 33) and camelid (41–43) species have an ability to refold and bind antigen after thermal and chemical denaturation. Here, the candidate clone BA11 was subjected to extreme thermal and pH challenge. Both the monomeric wild-type E06 and candidate clone showed a remarkable ability to bind cognate antigen after temperature or pH induced unfolding, confirming that functional binding and surprisingly functional stability (which had been ignored as part of clone "culling") had been retained through the mutation and selection process.

Naturally occurring protein binding domains with the potential for development as bio-therapeutic drugs are being investigated many research and development laboratories of the pharmaceutical industry. In this article, we have detailed the successful humanization of an albumin binding domain isolated from shark VNAR library. This domain, E06, was humanized in a manner that facilitated the retention of high affinity and specificity for cognate antigen with low immunogenicity and the hallmark high stability of native VNARs. *In vivo* PK studies have proven this final clinical domain BA11 (NDure™) to have broad utility as an enabling product for increased systemic exposure across multiple therapeutic modalities while retaining the benefit of small size and can be formulated at concentrations concurrent with clinically approved mAbs.

### ETHICS STATEMENT

All animal studies were conducted by Charles River (Elpinstone Research Centre, Elphinstone, Tranent EH33 2NE) in accordance with their regulated procedures.

### REFERENCES

#### AUTHOR CONTRIBUTIONS

CB is the PI on the project and managed and led the team. AP is the co-PI. JS, MM, MC, OU, MK, and GD are the senior scientists who conducted the work. TB, DC, and KS developed and ran the LC/MS.

#### ACKNOWLEDGMENTS

The authors would like to acknowledge the funding support for this work from Scottish Enterprise [VNAR\_001(2012)] and the Biotechnology and Biological Sciences Research Council (BB/K010905/1).

**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 © 2017 Steven, Müller, Carvalho, Ubah, Kovaleva, Donohoe, Baddeley, Cornock, Saunders, Porter and Barelle. 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.*

# Development and Characterization of a Camelid Single Domain Antibody– Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2

*Baomin Tian1 , Wah Yau Wong1 , Marni D. Uger1 , Pawel Wisniewski2 and Heman Chao1 \**

*1Helix BioPharma Corp., Toronto, ON, Canada, 2Helix ImmunoOncology, Warsaw, Poland*

#### *Edited by:*

*Colin Roger MacKenzie, National Research Council Canada, Canada*

#### *Reviewed by:*

*Peter Timmerman, Pepscan Therapeutics B.V., Netherlands Patrick Chames, UMR7258 Centre de recherche en cancérologie de Marseille (CRCM), France*

*\*Correspondence:*

*Heman Chao<EMAIL_ADDRESS>
#### *Specialty section:*

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

*Received: 28 April 2017 Accepted: 26 July 2017 Published: 21 August 2017*

#### *Citation:*

*Tian B, Wong WY, Uger MD, Wisniewski P and Chao H (2017) Development and Characterization of a Camelid Single Domain Antibody– Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2. Front. Immunol. 8:956. doi: 10.3389/fimmu.2017.00956*

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody–urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MSE peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the *E. coli* BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[(*N*-maleimidopropionamido)-diethyleneglycol] ester (SM(PEG)2), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM(PEG)2), which targets the cysteine added to the antibody C-terminus. V21H4-DOS47 was determined to be the superior conjugate as the antibody is easily produced and purified at high levels, and the conjugate can be efficiently generated and purified using methods easily transferrable for cGMP production. In addition, V21H4-DOS47 retains higher binding activity than V21H1-DOS47, as the native lysine residues are unmodified.

Keywords: antibody–drug conjugates, conjugation sites, conjugation ratios, camelid antibodies, urease, vascular endothelial growth factor receptor 2, cancer

### INTRODUCTION

As tumors outgrow local oxygen diffusion gradients, angiogenesis is triggered and new capillaries sprout from pre-existing blood vessels to support tumor cell demands for nutrients and oxygen and spread to distant sites (1, 2). Growth factors secreted by tumor cells mediate the induction of angiogenesis and control the inflammatory infiltrate. Multiple ligand–receptor signaling networks have been associated with tumor angiogenesis. The vascular endothelial growth factor (VEGF)/ vascular endothelial growth factor receptor 2 (VEGFR2) ligand– receptor complex is one of the most important signaling pathways identified and extensive research has been done on its roles in vascular functions (3, 4). During cancer-induced angiogenesis, cancer cells secrete VEGF that binds to VEGFR2, triggering a tyrosine kinase signaling cascade *via* the dimerization of VEGFR2. Consequently, the kinase signaling cascade stimulates the production of factors that variously stimulate vessel permeability, proliferation/survival, migration, and finally differentiation of mature blood vessels. Tumor blood vessels are structurally and functionally abnormal and are present at high density. Therefore, targeting tumor vasculature is a rational strategy with great promise and different anti-angiogenic agents targeting the VEGF/VEGFR2 signaling cascade have been developed as cancer therapeutics. Small chemical drugs, such as Sunitinib (SU11248, Sutent, Pfizer), Axitinib, and Sorafenib (BAY43-9006, Nexavar, Bayer) were approved by the FDA as anti-angiogenic agents that directly inhibit the kinase activity of the VEGFR2 intracellular kinase domains (5, 6). Monoclonal antibodies such as bevacizumab (Avastin, Genentech/Roche) and ramucirumab were developed to suppress angiogenesis by binding, respectively, to VEGF and VEGFR2, interrupting VEGFR2 dimerization and consequently inhibiting autophosphorylation (7, 8).

We have developed an antibody–drug conjugate (ADC) approach to suppress angiogenesis. Unlike most of the antiangiogenic agents that interrupt the kinase signaling cascade by blocking the dimerization of VEGFR2 or by inhibiting kinase activity, our ADC, V21-DOS47, kills VEGFR2-expressing cells by inducing cytotoxic activity at the target cells. Similar to our previous antitumor immunoconjugate, L-DOS47 (9), V21-DOS47 is composed of a camelid single domain antibody and the enzyme urease (derived from jack beans, *Canavalia ensiformis*): the V21 antibody binds to VEGFR2, thus targeting the complex to VEGFR2-expressing cells, whereas the urease enzyme converts endogenous urea into ammonia *in situ* to induce cytotoxicity. Since VEGFR2 is not only expressed in the tumor vasculature but has also been identified on the surface of a variety of tumors (4, 10, 11), V21-DOS47 targets both VEGFR2<sup>+</sup> vascular endothelial cells and VEGFR2<sup>+</sup> tumor cells. The elevated local concentration of ammonia also neutralizes the acidic environment surrounding the tumor microvasculature, which is otherwise favorable to cancer cell growth (12). As urease is a plant product with no known mammalian homolog, it is likely to be immunogenic, although an auto-immune reaction is not expected. L-DOS47 is currently being tested in clinical trials and results show that anti-urease antibodies are formed, but no known severe immune toxicity is observed. The full impact of urease immunogenicity is still being studied.

One advantage of camelid single domain antibodies is their relatively small size (approximately 15 kDa) compared to conventional immunoglobulins (approximately 150 kDa). This is particularly important when coupling antibodies to urease, as urease is a large protein with a molecular weight of 544 kDa. By using llama single-domain antibodies, multiple antibodies can be coupled to each urease molecule with a relatively minor increase in overall molecular weight. This allows for the generation of a high avidity therapeutic reagent that retains an acceptable biodistribution profile. Other benefits of single domain camelid antibodies (13–15) are that they are easy to clone and express recombinantly (16, 17), they are generally more thermally and chemically stable than conventional IgG (18, 19), and they bind to epitopes that are not recognized by conventional antibodies (20). In addition, they are not particularly immunogenic as human VH and camelid VHH domains share approximately 80% sequence identity (21) and renal clearance is high (22).

Antibody–urease conjugates are complex and large proteins: with multiple antibodies per urease, the molecular weight of the conjugate can reach 680 kDa. This provides a challenge to large-scale production. In our previous report, we described conjugation chemistry and separation procedures designed to address these challenges (9). In this study, we evaluated additional antibody production and conjugation chemistry methods to generate a novel antibody–urease conjugate, V21-DOS47.

In order to produce high-affinity antibodies to VEGFR2, a llama was immunized with recombinant VEGFR2 and a VHH phage display library was generated. The V21 antibody was isolated by panning this library with recombinant VEGFR2. Additional amino acid residues were added to the C-terminus of the V21 antibody in order to fulfill multiple objectives: to optimize the antibody pI, to target antibody expression to bacterial inclusion bodies, and to provide a unique target for cross-linking chemistry. In this report, we describe two versions of the V21 antibody, designated V21H1 and V21H4, and the different methods used to conjugate each antibody to urease. Both antibody–urease conjugates were characterized with a variety of analytical techniques, including size exclusion chromatography (SEC) (to evaluate protein purity), SDS-PAGE (to determine the average number of antibodies conjugated per urease), and ESI mass spectrometry (to identify conjugation sites on both the antibody and urease). The effects of conjugation ratio (CR) were examined, and the binding of the two conjugates with the same CR were compared. Binding to VEGFR2 expressed at the cell surface was confirmed by flow cytometry.

#### MATERIALS AND METHODS

#### Purification of High Purity Urease (HPU)

Crude urease (cat #U-80, 236 U/mg) was purchased from BioVectra Inc. (Charlottetown, PE, Canada). Prior to use in conjugation, crude urease was purified to remove jack bean matrix protein contaminants such as canavalin and concanavalin A. One million units of crude urease were dissolved in 430 mL of high purity (HP) water at room temperature. The solution was brought to pH 5.15 with 10% (v/v) acetic acid and then centrifuged at 9,000 rcf and 4°C for 40 min. The urease-containing supernatant was cooled to 4°C and fractionated by adding chilled ethanol to a final concentration of 25% (v/v) while maintaining the temperature at 0–8°C. The mixture was stirred overnight and then centrifuged at 9,000 rcf and 4°C for 40 min. The pellet was resuspended in 150 mL of acetate–EDTA buffer (10 mM sodium acetate, 1 mM EDTA, 1 mM TCEP, pH 6.5) and then centrifuged at 4°C and 9,000 rcf for 40 min. The supernatant was concentrated to 75 mL using a Minimate TFF system (Masterflex Model 7518-00 with a Minimate TFF capsule, MWCO 100 kDa), diafiltered three times with 200 mL of acetate–EDTA buffer, and then concentrated down to 100 mL. The diafiltered urease solution was collected, and the strained solution in the capsule and tubing connections was expelled from the system with 50 mL acetate–EDTA buffer and added to the collected solution (total volume ~150 mL). The ethanol fractionated urease solution was further purified by anion exchange chromatography using a Bio-Rad Biologic LP system. The urease solution was loaded at a flow rate of 3.5 mL/ min onto a 35 mL DEAE column (DEAE Sepharose Fast Flow, GE Healthcare, cat #17-0709-01) which was pre-equilibrated with 150 mL of IEC Buffer A (20 mM imidazole, 1 mM TCEP, pH 6.5). The column was washed with 100 mL of IEC Buffer A, followed by 80 mL of 40% Buffer B (Buffer A with 0.180 M NaCl). The urease was eluted with 100% Buffer B at a flow rate of 3.5 mL/min and fractions with A280 >0.1 were pooled. The pooled fractions were concentrated to a target protein concentration of 6–8 mg/mL using a Minimate capsule with a 100 kDa MWCO membrane and then diafiltered against acetate–EDTA buffer (20 mM sodium acetate, 1 mM EDTA, pH 6.5). The HPU was stored at −80°C. The yield from this purification protocol is typically >55% of the starting activity.

### Expression of V21H1 and V21H4

Both antibodies were expressed in the *E. coli* BL21 (DE3) pT7 system with kanamycin as the selection antibiotic. Transformation of BL21 (DE3) competent *E. coli* cells (Sigma, B2935-10 × 50 µL) was according to the manufacturer's instructions. One colony from a transformation plate was aseptically inoculated to 200 mL of LB broth (LB media EZ mix, Sigma cat #L76581, 20 g/L) supplemented with 50 mg/L kanamycin. Cultures were incubated at 200 rpm and 37°C. Once the culture reached an OD600 greater than 0.6, 50 mL of culture was transferred to four 2 L flasks, each containing 1 L of LB broth with 50 mg/L kanamycin. Flasks were incubated in a shaker incubator at 200 rpm and 37°C. Once the culture reached an OD600 of 0.9–1.0, antibody expression was induced by the addition of 1 mM IPTG and overnight incubation at 200 rpm and 37°C. The cells were harvested by centrifugation into aliquots, one per 2 L culture.

### Purification of V21H1

The majority of the V21H1 protein was expressed in the *E. coli* cytosolic solution, not in the inclusion bodies. An aliquot of cell pellet was lysed in 100 mL of lysis buffer (50 mM Tris, 25 mM NaCl, pH 6.5) by sonication in an ice-water bath for 10 min (Misonix 3000 sonicator, tip part #4406; each sonicating cycle: sonicating 30 s, cooling 4 min, power 8). The lysate was centrifuged at 9,000 rcf and 4°C for 30 min. In order to remove the most abundant bacterial matrix proteins, the supernatant was mixed with ice-cold ethanol to a final concentration of 10% (v/v) and incubated in an ice-water bath for 30 min, followed by centrifugation at 9,000 rcf and 4°C for 30 min. The supernatant was mixed with ice-cold ethanol to a final concentration of 45% (v/v) and stirred in an ice-water bath for 60 min, followed by centrifugation at 9,000 rcf and 4°C for 30 min. The pellet was resuspended in 200 mL of wash buffer (50 mM acetate, 0.1% Triton X-100, 1 mM DTT, 25 mM NaCl, pH 5.0). After centrifugation at 9,000 rcf and 4°C for 30 min, the pellet was resuspended in 100 mL of SP Buffer A (50 mM acetate, 8 M urea, pH 4.0) supplemented with 2 mM DTT, and filtered through a 0.45-µm filter. The filtered solution was loaded on to a 1 mL SP FF column (GE Healthcare, cat #17-5054-01) with a peristaltic pump at 2 mL/ min, and the column was then connected to an ACTA FPLC system (Amersham Bioscience, cat #UPC-920). After washing the column with 10 mL of SP Buffer A at 1 mL/min, the V21H1 antibody was eluted by a gradient of 0–50% SP Buffer B (SP Buffer A with 0.7 M NaCl) over 30 min at a flow rate of 1 mL/min. The OD280 of the peak fraction was determined and the concentration was calculated with an extinction coefficient of 1.967 mg/ mL. DTT was added to the SP column peak fraction to a final concentration of 1 mM and the pH of the solution was adjusted to 8–8.5 with 2 M Tris-base. The refolding of the antibody was performed by adding the pH adjusted SP peak fraction drop by drop to refolding buffer (100 mM Tris, 10 µM CuSO4, pH 8.8) and continuous stirring at 4°C until the refolding was completed. The refolding process was monitored by intact protein LC-MS. After refolding, the solution was centrifuged at 9,000 rcf and 4°C for 30 min before loading on to a 1 mL QHP column. The column was connected to a FPLC system and washed with 10 mL of Q Buffer A (50 mM HEPES, pH 7.0) at a flow rate of 1 mL/min. The antibody was eluted by a gradient of 0–40% Q Buffer B (Q Buffer A with 0.7 M NaCl) in 40 min at a flow rate of 1 mL/min. The peak fractions from 8 L of cell culture were pooled, concentrated to 2–4 mg/mL, and dialyzed against 20 mM HEPES, pH 7.1 overnight (MWCO 5–8 kDa, volume ratio 1:50) at 4°C. The final V21H1 antibody solution was filtered through a 0.22 µm syringe filter and stored at 4°C.

### Purification of V21H4

In contrast to V21H1, the majority of the V21H4 protein was expressed in the *E. coli* inclusion bodies. The cell pellet from each 2 L culture was resuspended in 100 mL of lysis buffer (50 mM Tris, 25 mM NaCl, pH 6.5) and mixed with lysozyme to a final concentration of 0.2 mg/mL. The cell suspension was incubated at room temperature for 30 min, then lysed by sonication in an ice-water bath for 10 min (Misonix 3000 sonicator, tip part #4406; each sonicating cycle: sonicating 30 s, cooling 4 min, power 8). The lysate was centrifuged at 9,000 rcf and 4°C for 30 min. The pellet was washed twice with 400 mL of Pellet Wash Buffer (50 mM Tris, 25 mM NaCl, pH 6.5, 1% Triton X-100, 2 mM DTT) and once with 50 mM of acetic acid containing 2 mM DTT. The pellet was resuspended in 100 mL of SP Buffer A (50 mM acetate, 8 M urea, pH 4.0) supplemented with 2 mM DTT and centrifuged at 9,000 rcf and 4°C for 30 min. The resulting supernatant was filtered through a 0.45-µm filter and loaded on to a 5 mL SP-XL column (GE Healthcare, cat #17-1152-01) at a flow rate of 5 mL/min. After washing the column with 50 mL of SP Buffer A, the protein was eluted by a gradient of 0–50% SP Buffer B (SP Buffer A with 0.7 M NaCl) over 30 min at a flow rate of 5 mL/min. Peak fractions were collected when A280 >700 mU. DTT was added to the pooled SP peak fraction to a final concentration of 1.0 mM and the pH was adjusted to pH 8.6–8.7 with saturated Tris-base. Refolding was initiated by mixing the SP peak fraction with refolding buffer (50 mM Tris, 2 M urea, 1.0 mM DTT, pH 8.6–8.7). After stirring at room temperature for 2 h, 1.2 mM cystamine was added to the refolding mixture. Refolding continued at room temperature and was monitored by RP-HPLC [Agilent 1100 system; ZORBAX-C3 column, PN883750-909; Solvent A: 0.025% (v/v) TFA in water; Solvent B: 0.025% TFA in acetonitrile; gradient: 20–60% B over 30 min at a flow rate of 0.25 mL/min. 100 µL of sample was collected at various time points and acidified by immediately adding 1.0 µL of neat formic acid. 30 µL of each sample was injected to the column to record the chromatogram]. The resulting refolding mixture was centrifuged at 9,000 rcf and 4°C for 30 min before loading to a 5 mL QHP column (GE Healthcare, cat #17-1154-01) at a flow rate of 5 mL/min. After washing the column with 50 mL of Q Buffer A (50 mM HEPES, pH 8.7), the protein was eluted by a gradient of 0–70% Q Buffer B (Q Buffer A with 0.7 M NaCl). Peak fractions with A280 >700 mU were pooled. The Q peak fractions were pooled, concentrated to 6–10 mg/mL, and buffer exchanged with 10 mM HEPES, pH 7.1. The final V21H4 antibody solution was filtered through a 0.22-µm filter and stored at 4°C.

#### Conjugation of V21H1 to Urease

10 mg of V21H1 antibody was activated with cross-linker at an antibody to cross-linker molar ratio of 1:2.4 by adding 70.4 µL of SM(PEG)2 (10.0 mg/mL in DMF) stock solution to the V21H1 antibody while vortexing. The reaction solution was incubated at room temperature for 90 min. The reaction was quenched by adding 300 mM of Tris buffer (pH 7.6) to a final concentration of 10 mM and incubating at room temperature for 10 min. The unconjugated, hydrolyzed, and quenched cross-linker was removed with a 20 mL G25 desalting column pre-equilibrated with 50 mM Tris buffer containing 50 mM NaCl and 1 mM EDTA, pH 7.1. After removing the excess cross-linker, the desalting column fraction was pooled and a 100 µL sample was collected for intact protein mass spectrometric analysis and peptide mapping analysis to evaluate the activation sites on the V21H1 antibody. The remaining pooled fraction was chilled in an ice-water bath for 5 min. 20 mg of HPU was thawed and incubated in another ice-water bath for 5 min. The chilled HPU solution was poured into the activated V21H1 antibody solution while stirring. The stirring continued in an ice-water bath for 5 min, and then the reaction solution was moved to a bench at room temperature. After the conjugation reaction solution was incubated at room temperature for 90 min, cysteine solution (200 mM in 300 mM Tris, pH 7–7.5) was added to a final concentration of 5 mM to quench the reaction. The reaction solution was concentrated down to approximately 4 mL by centrifugation in a 15 mL centrifuge filter (MWCO 100 kDa) at 4°C and 2,000 rcf. The resulting concentrated reaction solution was divided into three aliquots before SEC separation. The separation was performed by loading each aliquot of reaction solution to a Superose 6 100/300 GL column (GE) connected to an AKATA FPLC system. The protein was eluted by an isocratic flow at 0.5 mL/min with SEC buffer (50 mM NaCl, 0.2 mM EDTA, pH 7.2) and the major peak fractions of A280 >200 mU were pooled. The peak fractions from all three SEC separations were pooled and dialyzed against 1 L of formulation buffer [10 mM histidine, 1% (w/v) sucrose, 0.2 mM EDTA, pH7.0]. The resulting conjugate solution was filtered through a 0.22-µm filter and divided into 0.8 mL aliquots. Aliquots were stored at −80°C.

#### Conjugation of V21H4 to Urease

20 mg of V21H4 was mixed with TCEP (100 mM in 300 mM Tris buffer, pH 7–7.5) to a final concentration of 1.5 mM and incubated at room temperature for 60 min. The excess TCEP and the resulting cysteamine were removed by a 25 mL G25 desalting column using Tris-EDTA buffer (50 mM Tris, 1 mM EDTA, pH 7.1). The resulting desalting fraction was pooled in a 40 mL beaker and diluted with Tris-EDTA buffer to a total volume of 30 mL. The activation reaction was performed by quickly dispensing 0.420 mL of BM(PEG)2 stock solution (10 mg/mL in DMF) into the V21H4 antibody solution in the beaker while stirring. After incubation at room temperature for 10 min, the reaction solution was transferred to a 200 mL Amicon diafiltration concentrator with a filter membrane (MWCO 5 kD) and mixed with Tris-EDTA buffer up to 100 mL. The excess cross-linker was removed by connecting the diafiltration concentrator to a 70 psi nitrogen source and concentrated down to 20 mL while stirring. After five cycles of dilution and concentration, the diafiltration concentrator was detached from the nitrogen source and a 100 µL sample was collected to determine the antibody activation sites (using intact protein mass spectrometric analysis and peptide mapping analysis). Tris-EDTA buffer was added to the concentrator to dilute the solution up to the 50 mL marker. The concentrator with the activated V21H4 antibody was chilled in an ice-water bath for 10 min while stirring. After completely thawing at 4°C, 80 mg of HPU was incubated in another ice-water bath for 5 min and then poured into the activated V21H4 antibody solution in the concentrator while stirring in its ice-water bath. After stirring in the ice-water bath for 5 min, the concentrator with the reaction solution was moved to a lab bench and incubated at room temperature for 90 min. The conjugation reaction was quenched by adding cysteine (100 mM in 300 mM Tris, pH 7–7.5) to a final concentration of 5 mM. After quenching the reaction at room temperature for 5 min, the reaction solution was transferred to another container and the concentrator was cleaned and re-installed with a new filtration membrane (MWCO 100 kDa). The reaction solution was transferred back to the concentrator and formulation buffer [10 mM histidine, 1% (w/v) sucrose, and 0.2 mM EDTA, pH 7.0] was added to the 160 mL marker. The concentrator was connected to a 10 psi nitrogen source and concentrated down to 20 mL while stirring. After the dilution–concentration cycle was repeated four times, the diafiltration concentrator was detached from the nitrogen source and the V21H4-DOS47 conjugate solution was transferred to a new container and diluted to 40 mL. The conjugate solution was filtered through a 0.22-µm filter and divided into 0.8 mL aliquots. The aliquots were stored at −80°C.

#### Size Exclusion Chromatography

A Waters 2695 HPLC system with a 996 PAD was employed with Empower 2 software for data acquisition and processing. Chromatograms were recorded over 210–400 ± 4 nm with the signal at 280 nm extracted for processing. Separation was performed on a Superose 6 100/300 GL column (GE). Proteins were eluted in 10 mM phosphate, 50 mM NaCl, and 0.2 mM EDTA, pH 7.2. Separation was carried out with an isocratic flow at 0.5 mL/min after injection of a certain volume of neat samples. The column temperature was kept at room temperature, while the sample temperature was controlled at 5 ± 2°C.

### SDS-PAGE

A Bio-Rad Mini Gel Protein Electrophoresis kit and a Bio-RAD Molecular Imager Gel Doc XR+ with ImageLab software were employed to analyze V21-DOS47 CRs. 10 µg of protein samples were mixed with 60 µL of protein gel loading buffer and the mixture was heated to 70°C for 10 min. Denatured samples were loaded (10 µL/well) to a 4–20% Tris–Glycine gel (Invitrogen, cat #XP04200) and electrophoresis was performed at a constant voltage of 150 V with current <40 mA until the electrophoresis front reached the gel bottom. After washing, staining, and destaining, the gel image was scanned with the Gel Doc XR+ imager for analysis. SDS-PAGE was also used to calculate the average number of antibodies conjugated per urease molecule. This was determined by interrogating the intensities of the five bands in the main cluster [see Ref. (9) for further details]. All CRs reported are average values.

### ELISA Assays

A 96-well plate was coated with 100 µL/well of goat anti-human IgG-Fc (Sigma, 5 µg/mL in PBS) at room temperature for 6 h and then blocked with 200 µL/well of 3% BSA/PBS at 2–8°C overnight. After washing 2× with T-TBS (50 mM Tris, 0.15 M NaCl, pH 7.6, containing 0.05% Tween-20), 100 µL/well of VEGFR1/ Fc, VEGFR2/Fc, or VEGFR3/Fc [R&D Systems, 0.25 µg/mL in TB-TBS (0.1% BSA/T–TBS)] was added and the plate was incubated at room temperature for 1 h with gentle shaking. After washing 3× with T-TBS, 100 µL/well of antibody–urease conjugate or biotinylated antibody dilutions (in TB-TBS) were added and the plate was incubated at room temperature for 2 h with gentle shaking. For antibody–urease conjugates, plates were washed 3× with T-TBS, 100 µL/well of rabbit anti-urease (1/6,000 or 1/10,000-fold dilution in TB-TBS, Rockland) was added and the plate was incubated at room temperature for 1 h with gentle shaking. For all samples, the plate was washed 3× with T-TBS and 100 µL/well of goat anti-rabbit-AP (1/8,000-fold dilution in TB-TBS, Sigma) was added to detect antibody–urease conjugates or streptavidin–alklaine phosphatase (0.5 µg/mL in TB-TBS, Sigma) was added to detect biotinylated antibodies, and the plate was incubated at room temperature for 1 h with gentle shaking. After washing 3× with T-TBS, 100 µL/well of substrate (4-nitrophenyl phosphate disodium salt hexahydrate, Fluka, 1 mg/mL in diethanolamine substrate buffer, Pierce) was added to each well and incubated at room temperature for 5–15 min with gentle shaking. The absorbance at 405 nm (A405) of each well was acquired by scanning the plates with a UV–Vis spectrophotometer.

# Urease Activity Assay

Urease catalyzes the hydrolysis of urea to ammonia. One unit of urease activity is defined as the amount of enzyme that liberates 1 µmol of ammonia per minute at 25°C at pH 7.3. V21H4-DOS47 samples were diluted in sample dilution buffer [0.02 M potassium phosphate containing 1 mM EDTA and 0.1% (w/v) BSA, pH 7.3]. 100 µL of the diluted sample was mixed with 2.00 mL of 0.25 M urea (in phosphate buffer containing 0.3 M sodium phosphate and 0.5 mM EDTA, pH 7.3), and incubated at 25 ± 0.1°C for 5 min, then the reaction was quenched by adding 1.00 mL of 1.0 N HCl. To determine the concentration of ammonium ion produced in the enzyme reaction solution, 100 µL of the quenched reaction solution was mixed with 2.00 mL of phenol solution (0.133 M phenol containing 0.25 mM sodium nitroferricyanide) in a 15 mL testing tube. After 30 s, 2.50 mL of NaOH–NaOCL solution (0.14 N NaOH containing 0.04% sodium hypochlorite) was added to the testing tube, mixed, and incubated at 37°C for 15 min. The absorbance of the solution was determined at 638 nm with the reagent reaction solution (without sample) as the blank. The urease enzyme activity was calculated according to the following equation: U/mL = *D* × (*A* × Tc × Te)/(5 × *E* × Sc × Se) where *A* = absorbance at 638 nm, Tc = total volume of color reaction (4.60 mL), Te = total volume of enzyme reaction (3.10 mL), *E*= molar extinction coefficient of indophenol blue per assay condition (20.10 mM<sup>−</sup><sup>1</sup> cm<sup>−</sup><sup>1</sup> ), Sc = sample volume for color reaction (0.10 mL), Se = sample volume for enzyme reaction (0.10 mL), and *D* = dilution time. The protein concentration of each sample was determined with a Sigma total protein kit (cat #TP0200) following the manufacturer's instructions. Urease activity/milligram of conjugate was calculated by dividing the urease activity (units per milliliter) by the amount of protein tested (milligrams per milliliter). Specific urease activity was calculated by dividing the activity/mg conjugate by the proportion of the conjugate's mass which was composed of urease.

# Western Blot

V21H4-DOS47 test samples and controls were resolved by SDS-PAGE gel electrophoresis and then transferred to a nitrocellulose membrane using a Bio-Rad blot kit. 1.2 µg of HPU and 4.0 µg of V21H4 as controls and 2.0 µg of V21H4-DOS47 samples were mixed with 60.0 µL of protein gel loading buffer. The resulting sample mixtures were denatured by heating to 60°C for 10 min and 10 µL of each sample was loaded per lane. Duplicate blots were made from gels run in parallel for urease and V21H4 antibody probing. For urease detection, a rabbit anti-urease IgG (Rockland) was used. To detect the V21H4 antibody, a rabbit anti-llama IgG (ImmunoReagents Inc.) was used. A goat antirabbit IgG conjugated to AP (Sigma) was used as the secondary visualization antibody. Final development of the Western blots was performed with AP buffer containing NBT/BCIP.

# Mass Spectrometry

A Waters Xevo G2 QTOF mass spectrometer and an Acquity UPLC system H class were employed for all mass spectrometry analyses. A lock mass of 785.8426 Da was applied for real time point to point mass calibration. LC-MS data acquisition was controlled by Masslynx V4.1 software.

# Intact Protein Mass Spectrometry Analyses

Cross-linker-activated antibody samples were reacted with 5 mM cysteine at room temperature for 30 min, diluted to 0.5–1 mg/mL in water, and acidified by adding neat formic acid to a final concentration of 1% (v/v). A BEH300 C4 (1.7 µm, 2.1 × 50 mm) column was used. The column temperature was set at 60°C and Solvent A (0.025% v/v TFA in water) and Solvent B (0.025% TFA in acetonitrile) were used for UPLC separation. The UPLC was performed with a flow rate of 0.15 mL/min with a gradient from 20 to 60% Solvent B over 30 min. LC-MS TIC (total ion counts) data acquisition was carried out in an *m/z* range of 500–3,500 Da in resolution mode with a scan rate of 0.3/s, capillary voltage 3.0 kV, sample cone voltage 40 V, extraction cone voltage 4.0 kV. Ion source temperature was set at 100°C and desolvation temperature was set at 350°C. Desolvation gas flow rate was 600 L/h. A real time lock mass TIC raw data set (scan/20 s) was acquired with 100 fmol/μL Glu-Fib B at a flow rate of 6.0 µL/min. Mass spectrometric raw data were processed with BiopharmaLynx software (v 1.2) in intact protein mode with a resolution of 10,000. Mass match tolerance was set at 30 ppm, and the protein sequence of each antibody containing one disulfide bond was input as the match protein for protein match searches.

### Tryptic Digestion of V21H1-SM(PEG)2-Cys and V21H4-BM(PEG)2-Cys

The cross-linker-activated antibody samples were reacted with 10 mM cysteine at room temperature for 30 min and then diluted to 0.5 mg/mL with 100 mM ammonia hydrogen carbonate. Neat acetonitrile was added to the diluted sample solution to a final concentration of 20% (v/v). Trypsin/Lys-C mix (Promega, cat #V507A) was added at a protein:protease ratio of 20:1 and digested at 37°C for 16–20 h. DTT was added to the digested sample to a final concentration of 10 mM and samples were incubated at 37°C for 30 min to reduce the core disulfide bond. The digestion was stopped by adding neat formic acid to 1% (v/v) before mass spectrometry analysis.

# Tryptic Digestion of V21H4-DOS47

100 µg of V21H4-DOS47 was mixed with DTT to a final concentration of 10 mM and neat acetonitrile was added to a final concentration of 20% (v/v). To reduce the disulfide bond and denature the conjugated proteins, the sample mixture was heated at 60°C for 30 min. The denatured protein precipitate was pelleted by centrifugation at 16,000 rcf at room temperature for 5 min. 5.0 µL of 0.20 M iodoacetamide and 100 µL of water were added to the pellet then mixed by vortexing. The suspension was centrifuged at 16,000 rcf at room temperature for 5 min and the supernatant was discarded. The resulting pellet was dissolved in 100 µL of Tris–guanidine buffer (4 M guanidine chloride, 50 mM Tris, 10 mM CaCl2, and 10 mM iodoacetamide, pH 8.0). After this alkylation reaction was performed at room temperature in the dark for 30 min, the reaction was quenched with 5 mM DTT. The resulting solution was diluted four times with Tris buffer (50 mM Tris, 10 mM CaCl2, pH 8.0). Trypsin/Lys-C mix was added to the diluted sample solution at a protein:protease ratio of 25:1. After the digestion was performed at 37°C for 16–20 h, the reaction was stopped by adding neat formic acid at a final concentration of 1% (v/v).

# LC-MSE Peptide Mapping of V21H1- SM(PEG)2-Cys, V21H4-BM(PEG)2-Cys, and V21H4-DOS47 Tryptic Digests

A BEH300 C18 (1.7 µm, 2.1 mm × 150 mm) column was used for UPLC separation. The column temperature was set at 60°C. Solvent A (0.075% v/v formic acid in water) and Solvent B (0.075% formic acid in acetonitrile) were used for peptide elution. UPLC was performed with a flow rate of 0.15 mL/min. A gradient of 0–30% Solvent B in 50 min was used for the separation of the tryptic digests of V21H1-SM(PEG)2-Cys and V21H4-BM(PEG)2- Cys samples. For the tryptic digests of V21H4-DOS47, a gradient of 0–45% Solvent B in 150 min was used. LC-MSE TIC (total ion counts) data acquisitions were carried out in an *m/z* range of 50–2,000 Da in resolution mode with a scan rate of 0.3/s, capillary voltage 3.0 kV, sample cone voltage 25 V, and extraction cone voltage 4.0 kV. Ion source temperature was set at 100°C and desolvation temperature was set at 350°C. Desolvation gas flow rate was 600 L/h. A real time lock mass TIC raw data set (scan/20 s) was acquired with 100 fmol/μL Glu-Fib B at a flow rate of 3.0 µL/ min. With the instrument setup, two interleaved scan functions are applied for data acquisitions. The first scan function acquires MS spectra of intact peptide ions in the sample while applying no energy to the collision cell. The second scan function acquires data over the same mass range; however, the collision energy is ramped from 20 to 60 eV. This scan is equivalent to a non-selective tandem mass spectrometric (MS/MS) scan and allows for the collection of MSE fragment spectra from the ions in the preceding scan. The high energy collision induced fragmentation randomly cleaves peptide backbone bonds. For each C-N peptide backbone bond cleaved, the amino-terminal ion generated is called the "*b*" ion and the C-terminal ion generated is called the "*y*" ion. In **Tables 1**–**3**, the column entitled "MS/MS *b*/*y* Possible" indicates the theoretical maximum number of *b* and *y* ions that would be produced for each peptide if all peptide bonds in the protein were equally likely to be broken. The column entitled "MS/MS *b*/*y* Found" indicates the actual number of *b* and *y* ions identified for each peptide. The identification of *b*/*y* ions provides unambiguous confirmation of peptide identity. Mass spectrometric raw data were processed with BiopharmaLynx software (v 1.2) in peptide map mode with a resolution of 20,000. A lock mass of 785.8426 Da was applied for real time point to point mass calibration. The low energy MS ion intensity threshold was set at 3,000 counts and the MSE high energy ion intensity threshold was set at 300 counts. Mass match tolerances were set at 10 ppm for MS and at 20 ppm for MSE data sets. Peptides with one missed cleavage site were included in mass match searching. V21H1, V21H4, and urease (Uniprot P07374)

*Thick boxes (also shaded blue) around sets of tryptic peptides indicate related groups of peptides used to calculate percentage of activation for each activation site. nd, not detected.*

*\*indicates the modified version of the peptide.*

protein sequences were, respectively, input into the sequence library for peptide matching/identification. Variable modifiers, including deamidation N, deamidation succinimide N, oxidation M, +K, +Na, and carbamidomethyl C (for alkylated cysteine) were applied for peptide map analysis. SM(PEG)2-Cys (429.1206 Da) was set as a variable modifier to identify the activation sites of V21H1 conjugation, whereas BM(PEG)2-Cys (431.1362 Da) was input as a variable modifier to identify the activation sites of V21H4 conjugation. For the V21H4-DOS47 tryptic digests, GGGEEDDGC-BM(PEG)2 (1,145.3453 Da) was set as a variable modifier to identify the conjugation sites on urease.

#### Flow Cytometry

293 or 293/KDR cells were detached from flasks using nonenzymatic cell dissociation buffer (Sigma). Cells were centrifuged at 300 × *g* for 5 min and then resuspended in staining buffer at 106 cells/mL (PBS with Ca2+ and Mg2<sup>+</sup>, 0.02% NaN3, 2% FBS). 100 µL of cells was added to wells of a 96-well plate. The plate was centrifuged at 350 × *g* for 4 min, buffer removed, and then cells were resuspended in 50 µL of antibody–urease conjugate or biotinylated antibody (diluted in staining buffer) and then incubated at 2–8°C for 1 h. For cells stained with antibody–urease conjugates, cells were washed 3× with staining buffer and then resuspended in mouse anti-urease (Sigma, cat #U-4879) at 5.8 µg/mL (diluted in staining buffer) and incubated for 30 min at 2–8°C. For all samples, cells were washed 3× with staining buffer and then resuspended in AF488-anti-mouse IgG (Jackson, cat #115-545-164) at 3 µg/mL (diluted in staining buffer) for antibody–urease samples or with PE-SA (Biolegend, cat #405204) at 133 ng/mL (diluted in staining buffer) for biotinylated antibodies. All cells were incubated for 30 min at 2–8°C in the dark, washed 3× with staining buffer, then resuspended in 1% paraformaldehyde (diluted in PBS). The plate was incubated for 15 min at room temperature, covered with tin foil. The plate was then centrifuged as above, paraformaldehyde removed, and the cells were resuspended in staining buffer. The plate was covered in tin foil and stored at 2–8°C until analysis using a Guava flow cytometer and guavaSoft software (Millipore). S/N values are the ratio of V21H4-DOS47 binding to 293/KDR cells vs V21H4-DOS47 binding to 293 cells or the ratio of biotin-V21H4 vs biotin-isotype control antibody (anti-CEACAM6) binding to 293/KDR cells.

#### Table 2 | List of identified peptides and activation sites of V21H4-(PEG)2-Cys.

*Thick boxes (also shaded blue) around sets of tryptic peptides indicate related groups of peptides used to calculate percentage of activation for each activation site. nd, not detected.*

*\*indicates the modified version of the peptide.*

Table 3 | ESI LC-MSE peptide mapping analysis: Identification of urease cysteine residues modified by V21H4-(PEG)2-Cys.

#### Conjugation sites searched from the urease side

Conjugation sites searched from the antibody side

*na, not applicable.*

*\*indicates the modified version of the peptide.*

#### RESULTS

#### Production and Purification of V21H1

When generating single domain antibodies for immunoconjugate drugs, HP antibodies must be produced at high yield and with controllable processes, including expression, protein refolding, and purification. Other considerations include the following: the pI of the antibody should be such that the antibody conjugate is stable and soluble at physiologic pH, the properties of the antibody should be suitable for the conjugation chemistry, and the modifications of the antibody residues during conjugation reactions should not compromise the affinity of the antibody binding to its antigen.

The V21 camelid antibody consists of 122 amino acids (ending at S122, **Figure 1A**). Eleven amino acids were added to the C-terminus of the V21 antibody in order to generate V21H1. By

adding these amino acids, the pI of the antibody was changed from 8.75 to 5.44, as required for conjugate stability and solubility. The heterobifunctional chemical cross-linker SM(PEG)2 reacts with amine and sulfhydryl groups and was selected for use in conjugating V21H1 to urease (**Figure 1B**). There are five lysine residues in the core V21 sequence, two of which (Lys66 and Lys101) are located in the CDR2 and CDR3 sequences, respectively. As these amino acids could be modified by the amine conjugation chemistry utilized by SM(PEG)2, potentially altering antibody activity, two extra lysine residues were added to the antibody C–terminus to minimize this probability.

V21H1 was expressed primarily in the cytosolic solution of BL21(DE3) bacteria, with virtually no expression in inclusion bodies. Therefore, after cell lysis, the antibody was separated from bacterial proteins by ethanol crystallization and cation exchange chromatography. After antibody refolding, the native antibody was further purified by anion exchange chromatography. To confirm that the molecular mass of the purified antibody matched the designed protein sequence, LC-MS intact protein analysis was performed. No impurity proteins were detected from the LC-MS TIC chromatograms and the detected molecular mass of V21H1 matched the theoretical value calculated from its protein sequence within 30 ppm mass match error (data not shown). However, the yield of purified V21H1 was very low (4–6 mg/L of culture) and the purification processes used are not suitable for large-scale cGMP production.

#### Cross-Linker Activation of V21H1

V21H1 was activated by SM(PEG)2 at pH 7.0 using conditions previously found to be optimal for activation of AFAIKL2 antibody with SIAB in the production of the antibody–urease conjugate L-DOS47. Since the NHS-ester reaction is the same for SIAB and SM(PEG)2 and the LC-MS spectra are similar for AFAIKL2 and V21H1 reaction products (data not shown), these conditions should also be optimal for activation of V21H1 with SM(PEG)2.

Only the NHS-ester group of SM(PEG)2 can react with V21H1. The two cysteine residues in the V21H1 antibody form a disulfide bond and are, thus, unavailable to react with the maleimido end of the cross-linker. The primary amines from the antibody N-terminus and the lysine residues from the protein sequence can all potentially react with the NHS-ester of the cross-linker. The maleimido end of the antibody-carrying cross-linker then reacts with cysteines on the surface of urease molecules. The probability of each amine being activated depends on its accessibility due to its surrounding native structure. To avoid urease dimer and polymers forming in the second reaction step, ideally only one amine per antibody would be activated by the NHSester. However, since multiple primary amines are present in each antibody, it is statistically inevitable that some V21H1 antibodies will be activated by more than one cross-linker molecule. The optimal activation condition was selected, which minimizes the percentage of antibodies that are activated by more than one crosslinker while maximizing the total amount of activated antibody. To assess the activation distribution, the SM(PEG)2 activated V21H1 was reacted with excess cysteine and evaluated by intact mass spectrometric analysis. The mass spectrum is shown in **Figure 2**. Approximately 50% of the V21H1 was activated by SM(PEG)2 and of the activated antibody, approximately 30% was activated by two cross-linkers. Thus, only 35% of the V21H1 antibody is optimally activated for cross-linking with urease.

In order to determine which lysines of V21H1 were targeted by SM(PEG)2, V21H1-SM(PEG)2-Cys was subjected to tryptic digestion followed by LC-MSE analysis. Trypsin cleaves peptide backbone bonds at the C-terminal side of arginine and lysine residues (unless proline is immediately C-terminal to K or R). If a lysine is activated by SM(PEG)2, the polarity and side-chain structure of the lysine is altered and spatially blocked. Thus, this tryptic site is no longer accessible to the protease. For example, if K66 of V21H1 is activated by SM(PEG)2, it is linked to -SM(PEG)2-Cys and is no longer available for tryptic digestion; therefore, a peak with a molecular mass of 2,862.3018 (2,431.1656 + 431.1362) Da should be observed, which represents the -SM(PEG)2-Cys linked lysine-in-middle peptide (ELVAAISWSDDSTYYANSVK66GR)- SM(PEG)2-Cys. In the LC-MSE peptide mapping analysis, all possible activation sites can be identified by searching all the lysine carrying peptides and the N-terminal peptide with the -SM(PEG)2-Cys (431.1362 Da) as a variable modifier. The detected tryptic peptides along with conjugation sites are listed in **Table 1**.

All tryptic peptides were detected with mass match errors of less than 5 ppm and the amino acid sequence recovery was 100%. Assuming that ESI sensitivity is not affected by the linkage of the modifier, an activation percentage was assessed by comparing the intensity of the cross-linker modified peptide with the sum intensity of all the related peptides. Under the activation conditions used, lysine residue K66 in CDR2 was substantially (~25% of the entire activated V21H1 antibody) activated by the cross-linker; however, K101 in CDR3 was not modified during cross-linker activation. Surprisingly, the two C-terminal lysine residues that were intentionally added for conjugation chemistry purposes were not modified by the cross-linker.

#### Production and Purification of V21H4

The antibody V21H4 was designed to improve upon the issues identified during production, purification, and cross-linker activation of V21H1. The amino acid sequence of the V21H4 antibody is shown in **Figure 3A**. As for V21H1, a number of amino acid residues were added to the antibody C-terminus (G123–C136) and the pI of the antibody was adjusted from 8.75 to 5.43. With V21H1, the presence of SM(PEG)2 cross-linker activated K66 in the antibody CDR2 region was a concern as this could impair antibody binding affinity. Thus, a cysteine residue (C136) was added to V21H4 for sulfhydryl-to-sulfhydryl cross-linking using a different cross-linker, BM(PEG)2 (**Figure 3B**). The inclusion of a C-terminal cysteine also allowed the antibody to be expressed in bacterial inclusion bodies. As the two core cysteine residues of the V21 antibody form a disulfide bond and are unavailable for chemical conjugation, the additional C-terminal cysteine residue provides a unique activation site for targeted conjugation.

As expected, V21H4 was expressed at high levels in inclusion bodies. After cell lysis, antibody was separated from bacterial matrix proteins by centrifugation. The denatured antibody was purified by cation exchange chromatography to remove nucleic acids and other proteins. The refolding of the V21H4 antibody was performed in an easily controllable manner and was monitored by HPLC (**Figure 3C**).

The refolding process was initiated by mixing the peak fraction of the cation exchange column with refolding buffer. While the folding process was very slow without cystamine, folding was complete in 2 h at room temperature after cystamine was added to a final concentration of 1.2 mM. Anion exchange chromatography was used to isolate the properly folded protein and yields of greater than 80% were generally observed. The typical yield of purified V21H4 is 20–40 mg/L culture, which is considerably higher than that of V21H1. In addition, the method used to produce and purify V21H4 is amenable to scale up and cGMP procedures.

#### Cross-Linker Activation of V21H4

The C-terminal cysteine of V21H4 is required for conjugation to urease. However, as cystamine was included in the V21H4 refolding buffer, the C-terminal cysteine was modified by forming a disulfide bond with a half cystamine (cysteamine-H). This was confirmed by LC-MS intact protein analysis (**Figure 4A**). Thus, the half cystamine must be removed and the cysteine must subsequently be available for activation by cross-linker. In addition, this removal must occur using a controllable mild reduction under the native conditions to be used for conjugation purposes and it must not reduce the antibody's internal disulfide bond. As shown in **Figure 4B**, after reducing V21H4 with 2 mM TCEP at pH 7.1 for 1 h at room temperature, the detected antibody molecular mass was 14,667.94 Da, suggesting that the protective half cystamine had been removed. In order to confirm that the de-protected cysteine residue was active to the cross-linking reagent, 10 mM iodoacetamide was added to the de-protected V21H4 antibody. After 30 min at room temperature at pH 7.5–8.0, the resulting detected molecular mass was increased to 14,724.83 Da (**Figure 4C**), suggesting a carboxymethyl group (57.05 Da) was alkylated to the cysteine residue. In summary, the C-terminal half cystamine can be removed and the resulting de-protected cysteine is available for chemical conjugation. The alkylated antibody was also digested with trypsin and evaluated by LC-MSE peptide mapping. The LC-MSE peptide map (data not shown) covered 100% of the amino acid sequence and the C-terminal cysteine was specifically and effectively alkylated, confirming the specificity of the de-protective reduction reaction and the suitability of the C-terminal cysteine in targeted sulfhydryl cross-linking chemistry.

Figure 3 | (A) Amino acid sequence of the V21H4 antibody. (B) Description of conjugation strategy used to conjugate V21H4 to urease to generate V21H4- DOS47. Step 1 is the activation of the antibody using BM(PEG)2. Step 2 conjugates the activated antibody to urease. (C) RP-HPLC chromatograms of V21H4 samples at different refolding time points. Blue line: sample at refolding time 0, immediately after the SP pooled fraction was mixed with refolding buffer. Red line: refolding time point 2 h after mixing. Green line: refolding sample 4 h after time 0 and 2 h after addition of 1.2 mM cystamine. Unfolded antibody elutes at 12.513 min and folded antibody elutes at 10.958 min.

The V21H4 antibody was activated by the cross-linker BM(PEG)2. As BM(PEG)2 is a homobifunctional cross-linker, it is possible that both maleimido groups of BM(PEG)2 could react with and link two V21H4 molecules, leading to the generation of antibody dimers that cannot conjugate to urease. The frequency of antibody dimers generated depends upon the molar ratio of the reactants, the native hydrophobicity environment of the cysteine residue and the relative mobility of the molecules in the reaction solution. This reaction was performed with a 10:1 cross-linker to antibody molar ratio. In addition, the molecular weight of the cross-linker is 308.29 Da, which is approximately 50-fold less than the molecular weight of the antibody. To evaluate the activated V21H4 antibody, 100 µL of the activated antibody solution

was reacted with excess cysteine and evaluated by intact mass spectrometric analysis (**Figure 4D**). Under the experimental conditions used, more than 99% of the V21H4 was coupled to a single cross-linker, leaving the cross-linker's other maleimido group available for the subsequent reaction to urease.