Appl Microbiol Biotechnol (2015) 99:2693–2703 DOI 10.1007/s00253-014-6268-4

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Facile domain rearrangement abrogates expression recalcitrance in a rabbit scFv B. Vijayalakshmi Ayyar & Stephen Hearty & Richard O’Kennedy

Received: 25 August 2014 / Revised: 21 November 2014 / Accepted: 24 November 2014 / Published online: 24 December 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Rabbit-derived recombinant antibodies have traditionally been viewed as intractable molecules due to the presence of a cysteine in position 80 of the VL domain that becomes rendered ‘aberrant’ when present in the ‘unpaired’ context of a single chain Fv (scFv) and chimeric Fab formats. This aberrant Cys80 can severely impinge on the achievable expression levels when rabbit recombinant antibodies are produced in prokaryote systems. The unpaired Cys residue also renders purification problematic. Consequently, researchers often disregard rabbit antibody libraries due to perceived limitations in accessible repertoire diversity. We have shown that by switching the orientation of the VH and VL domains in an aberrant-Cys-containing rabbit scFv isolated in a bona fide screening campaign, it was possible to substantially increase the expression and purification yields of this clone. Furthermore, by incorporating a novel rabbit C-kappa constant fusion domain, we were able to potentiate a further increase in expression level and purify this antibody to a high degree of homogeneity, hitherto impossible to achieve using B. Vijayalakshmi Ayyar and Stephen Hearty contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-6268-4) contains supplementary material, which is available to authorized users. B. V. Ayyar : S. Hearty : R. O’Kennedy Biomedical Diagnostics Institute, National Centre for Sensor Research, Glasnevin, Dublin City University, Dublin 9, Ireland B. V. Ayyar : S. Hearty : R. O’Kennedy (*) School of Biotechnology, Glasnevin, Dublin City University, Dublin 9, Ireland e-mail: [email protected] Present Address: B. V. Ayyar Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

the aberrant-Cys-containing wild-type scFv. Cumulatively, these findings demonstrate that facile re-formatting can help make the rabbit antibody repertoire, a very valuable resource, more accessible to researchers in the field. Keywords Rabbit antibodies . scFv . scAb . Aberrant cysteine . Phage display

Introduction Rabbit polyclonal antibodies are established diagnostic workhorses, which have prevailed since the earliest immunodiagnostic travails and continue to be used as factories for many diagnostic and research-grade polyclonal antibody reagent generation campaigns. Logistically, rabbits are ubiquitously accessible in traditional immunology laboratories, or they are at least readily accessible through a multitude of commercial contractors, and purification of rabbit antibodies is relatively straightforward with high yields returned. Moreover, the quality of antibodies obtained is often superior to rodent-derived polyclonal and monoclonal antibodies, particularly against some of the more intractable target antigens such as haptens (Li et al. 2000a), peptides/phosphopeptides (Epstein et al. 1992; Coghlan et al. 1994) and possibly also conserved human proteins (Rossi et al. 2005). Human and mouse immune antibody repertoires are characterised by multiple variable (V) gene diversity arising from the large number of germline V gene singularities and further augmented by antigen-driven somatic mutation. Rabbits, birds and ruminants (e.g. cattle and sheep) typically have a much more limited number of V genes. In particular, VH gene usage is extremely restricted within this group. Chickens, rabbits and cattle all employ an additional V gene diversification mechanism called gene conversion (GC). Although rabbits have the more heterogenous VH repertoire

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(Sitnikova and Su 1998), only a very small and restricted preferential fraction of these (Knight 1992) are truly ‘functionally expressed’ and they exhibit lower GC frequency (Lavinder et al. 2014). The rabbit appears to compensate for this restriction by exhibiting heightened VkJk light chain (VL) diversity mechanisms (Sehgal et al. 1999) to increase overall diversity. Their typically extended VH CDR3 loop diversity combined with the plasticity of VL CDR3 contribute to high affinity which may be especially significant in binding minimalist antigenic species such as haptens. The lack of universally accessible rabbit plasmacytoma cell lines or efficient immortalization protocols and exclusionary intellectual property in this area undoubtedly contribute to the paucity of reported research papers using fully rabbit hybridoma-derived antibodies. However, it is more difficult to reconcile the preponderance of rabbit polyclonal antibodies referenced in the global research setting with the dearth of publications reporting recombinant rabbit antibodies when compared to murine, human and more recently, avian recombinant antibodies. Antibodies generated using in vitro display and selection recombinant technologies have the advantage of incorporating additional combinatorial diversity (Hearty and O’Kennedy 2011). The seminal work of Rader et al. (2000) in demonstrating reliable isolation of rabbit single chain Fvs (scFvs) using phage display was tempered by a strong implication that the presence of an aberrant cysteine residue in the K1 light chain of rabbit scFvs and chimeric Fabs compromised the expression fitness of the recombinant antibodies and thus, concomitantly reduced the accessible repertoire diversity in rabbit allotypes exhibiting this trait (Popkov et al. 2004). We sought to elucidate the practical manifestation of this impediment and investigate facile strategies to overcome these process limitations and combat expression recalcitrance. To this end, the technology was challenged in a diagnostic antibody generation campaign undertaken to isolate recombinant rabbit scFv antibodies against cardiac troponin I (cTnI), considered the ‘gold standard’ cardiac biomarker (McDonnell et al. 2009).

Materials and methods Rabbit immunisation A single New Zealand White (NZW) rabbit (B4 allotype) was immunised with 200 μg cTnI (Life Diagnostics, West Chester, USA) in a 1:1 emulsion of phosphate-buffered saline (PBS) and Freund’s complete adjuvant (FCA) administered by 6point subcutaneous injection. Three weeks postimmunisation, the rabbit was boosted with 100 μg cTnI in a 1:1 emulsion of PBS and Freund’s incomplete adjuvant (FICA) administered as above. Additional boosts containing

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50 μg cTnI in a 1:1 emulsion of PBS and FICA were administered at successive 2-, 3- and 4-week intervals, respectively. The animal was humanely euthanised 6 days after the final boost, and the spleen and bone marrow were harvested. Serum titration A blood sample was drawn after 10 days following the penultimate booster injection and the cTnI-specific serum titre was measured by enzyme-linked immunosorbent assay (ELISA). Briefly, the serum fraction was serially diluted in PBS-TBM (PBS containing 0.5 % (v/v) Tween®20, 0.5 % (w/v) bovine serum albumin (BSA) (Sigma-Aldrich Co., Dorset, UK) and 0.5 % (w/v) skimmed milk protein (Premier Brands UK Ltd., Stafford, UK). This was incubated on a MaxiSorp™ ELISA plate coated with 1 μg/ml cTnI and ‘blocked’ with PBS-BM (PBS containing 2.5 % (w/v) BSA and 2.5 % (w/v) skimmed milk protein). The cTnI-specific serum IgG component was detected by subsequently incubating the wells with horseradish peroxidase (HRP)-labelled antirabbit IgG (whole molecule) goat polyclonal antibody diluted 1/2000 (#A0545, Sigma-Aldrich Co., Dorset, USA) in 1 % (w/v) PBS-TBM. Library construction The freshly harvested spleen and bone marrow were processed independently. Tissue was first mechanically homogenised in TRIzol® reagent (Invitrogen, Carlsbad, USA) and total RNA was extracted by standard phenol/ chloroform phase separation. Complementary DNA (cDNA) was reverse-transcribed with oligo-dT primers using a SuperScript III kit (Invitrogen, Carlsbad, USA). The scFv library construction was ostensibly carried out as described by Rader et al. (2000) and Andris-Widhopf et al. (2000), with minor modifications. The variable kappa (VΚ) and variable heavy (VH) gene pool were amplified independently using specific 9-primer and 4-primer permutations, respectively (Table S1 in the Supplementary Material). Equimolar mixtures of VΚ and VH genes were assembled into scFv composite genes (incorporating a sequence for an 18 residue amino acid linker—SSGGGGSGGGGGGSSRSS) using splice-byoverlap extension (SOE) PCR, digested with SfiI enzyme (New England Biolabs Ltd., Hitchin, UK) and purified from a 1 % (w/v) agarose gel. The phagemid vector pComb3XSS (Barbas Laboratory, The Scripps Research Institute, La Jolla, USA) was correspondingly digested with SfiI, and the resultant SfiI-excised stuffer sequence was then further fragmented with XhoI and XbaI (targeting intra-sequence XhoI and XbaI sites) and finally dephosphorylated with Antarctic Phosphatase (New England Biolabs Ltd., Hitchin, UK). These latter steps were included to prevent library adulteration with reannealed phagemid or stuffer contamination, thereby

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preserving library integrity. The scFv gene pool was ligated into the pComb3XSS phagemid and transformed into electrocompetent Escherichia coli XL1-Blue (genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F’ proAB lacIqZΔM15 Tn10 (Tet )] (Stratagene, La Jolla, USA). The library was propagated in super broth (SB) supplemented with 1 % (w/v) glucose, carbenicillin (100 μg/ml) for phagemid retention and tetracycline (10 μg/ml) for F′ pilus maintenance. Selection of cTnI-specific candidate scFv clones The phage-displayed scFv library was ‘rescued’ using VCSM13 helper phage (Stratagene, La Jolla, USA), which was then resuspended in PBS containing 1 % (w/v) BSA and 0.02 % (w/v) NaN3 and subjected to five rounds of biopanning on MaxiSorp™ wells coated with successively decreasing concentrations (10, 5, 2.5, 1 and 1 μg/ml, respectively) of cTnI. Selection stringency was further exerted by incrementally increasing the number of washing steps through rounds 1 to 5. Antigen-bound phage particles were recovered after each panning step by trypsin elution (10 mg/ml porcine trypsin (Sigma-Aldrich Co., Dorset, USA) in PBS) and then reinfected into log phase E. coli XL1-Blue cells. The polyclonal phage outputs from each successive round were monitored for target-specific enrichment by phage ELISA on cTnI-coated MaxiSorp™ plates using an anti-M13 HRP-labelled mouse monoclonal antibody (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Screening candidate scFv clones by monoclonal ELISA A sample (10 μl) of round-5 phage output was used to infect a 4-ml mid-logarithmic culture (O.D.600nm ∼0.6) of the nonsuppressor strain E. coli Top10F' (genotype: F'{lacIq, Tn10 (TetR)} mcrA Δ (mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ (ara leu) 7697 galU galK rpsL (StrR) endA1 nupG) (Invitrogen, Carlsbad, USA) which was then subjected to static incubation at 37 °C for 30 min. The infected cells were serially diluted across the range 10−1–10−8 in SB media, and 100 μl of each dilution was plated onto Luria-Bertani (LB) agar (supplemented with 100 μg/ml carbenicillin and 1 % (w/v) glucose). Following growth overnight at 37 °C, 180 discrete colonies were selected for further screening. These were inoculated across 3×96 well plates (inner 60 wells only) containing 200 μl SB media (supplemented with 100 μg/ml carbenicillin and 1 % (w/v) glucose) and grown overnight at 37 °C. The following day, the cultures were sub-cultured into replicate plates containing 200 μl SB media (supplemented with 100 μg/ml carbenicillin and 1× ‘505’ supplement (0.5 % (w/v) glycerol +0.05 % (w/v) glucose). The sub-cultured cells were induced (at O.D.600nm ∼0.6) with 1 mM final concentration of isopropyl β-D-1-

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thiogalactopyranoside (IPTG) and expressed overnight at 30 °C. The induced cultures were subjected to three cycles of freeze-thawing, and the lysates were collected following centrifugation (3220×g for 30 min at 4 °C). The clarified lysates were diluted 1:5 in PBS-TBM and tested for binding to cTnI-coated ELISA (Maxisorp®, Nunc, Wiesbaden, Germany) plates (1 μg/ml in PBS) blocked with 5 % (w/v) PBS-BM. The binding was probed with a 1/2000 dilution (1 % (w/v) PBS-TBM) of HRP-conjugated mouse anti-HA monoclonal (clone 3F10) antibody (Roche, Mannheim, Germany). Following incubation with tetramethylbenzidine dihydrochloride (TMB) substrate, the reaction was quenched with 10 % (v/v) HCl and the absorbance was read at 450 nm. A second ELISA screen was performed to confirm solutionphase binding of cTnI. The lysates were diluted 1:3 in 1 % (w/v) PBS-TBM and mixed in a 1:1 ratio with 5 μg/ml (arbitrary ‘high’ concentration) cTnI in 1 % (w/v) PBS-TBM solution prior to incubating on cTnI-coated ELISA plates (1 μg/ml in PBS). The remainder of the ELISA was as described above with the degree of solution-phase binding inversely proportional to the degree of peroxidase activity returned on the substrate-treated wells. Ranking of clones by ‘off-rate’ analysis using Biacore 3000 Surface plasmon resonance (SPR)-based ‘off-rate’ ranking (Ayyar et al. 2010; Hearty et al. 2012) was performed using a BiacoreTM 3000 instrument (Biacore, Uppsala, Sweden), and data analysis was completed using BIAevaluation 4.1 softwareTM (Biacore, Uppsala, Sweden) and a 1:1 Langmuir binding model. All experiments were conducted using freshly filtered and degassed HEPES-buffered saline (HBS), pH 7.4, (10 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES); 150 mM NaCl; 3.4 mM ethylenediaminetetraacetate (EDTA) and 0.025 % (v/v) Tween®20) as both running and basal sample preparation buffer. Research-grade CM5 sensor chips (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used throughout. The chip was prepared by immobilising an anti-HA capture mAb using standard amine-coupling chemistry (Johnsson et al. 1991; O’Shannessy et al. 1992). The surface was activated by injecting 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M N-ethyl-N-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), mixed at 1:1 (v/v) ratio, at a flow-rate of 5 μl/min for 7 min over the sensor chip surface. The anti-HA mAb (Affinity Bioreagents Inc., Golden, USA) was diluted in 10 mM sodium acetate buffer (pH 5.0) to 25 μg/ml concentration and passed over the activated chip surface at flow-rate of 5 μl/min for 30 min. The surface was capped by injecting 1 M ethanolamine hydrochloride solution (pH 8.5) for 20 min (flow-rate 5 μl/min) and regenerated twice with 30 s pulses (flow-rate 30 μl/min) of 10 mM NaOH.

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Crude bacterial lysates were supplemented with 12 mg/ml BSA and 12 mg/ml CM-dextran to reduce non-specific binding during the analysis cycles. Candidate scFvs were captured on the anti-HA surface by injecting the lysates at a flow-rate of 10 μl/min for 1 min. A fixed concentration of cTnI (30 nM) was passed over the captured scFvs for 3 min (flow-rate 30 μl/ min), and the association and dissociation times were calculated at 2 and 10 min, respectively. The surface was regenerated by injecting a 30-s pulse of 25 mM NaOH for at a flowrate of 30 μl/min. The complete data analysis incorporated double referencing of each data point against the sample buffer response in addition to the online referencing against a blank flow-cell to avoid anomaly in results due to nonspecific interactions. DNA sequencing of scFv clones Plasmids were purified from 10 ml overnight culture of the scFv clones using SV Miniprep ™ kit (Promega, Madison, USA) and sequenced (Eurofins MWG Operon, Ebersberg, Germany) using primers RSC-F and RSC-B (Barbas et al. 1992) (Table S1 in the Supplementary Material). The generated sequences were submitted to GenBank and were assigned accession numbers KM044274 (MG4), KM044275 (MG5), KM044276 (MB6), KM044277 (MB10), KM044278 (MF11), KM044279 (MF7), KM044280 (MF4), KM044281 (MC8), KM044282 (MD3) and KM044283 (ME4). Comparative functional expression and purification of different scFvs Large volumes (250 ml) of the scFv cultures were grown and expressed by inducing overnight at 30 °C with 1 mM final concentration of IPTG. The cultures were pelleted by centrifugation at 3220×g for 15 min at 4 °C and the pellets were resuspended in lysis buffer (1× PBS [pH 7.4], 0.5 M NaCl and 20 mM imidazole). The cells were lysed by sonication, and the resultant cell debris was removed by centrifugation (3220×g, 4 °C, 15 min). A sample of each lysate preparation (500 μl) was retained and analysed for expression by ELISA, and the remaining lysate was subjected to purification by immobilised metal affinity chromatography (IMAC). A 4 ml aliquot of NiNTA resin was added to a 20-ml disposable column and equilibrated with 30 ml of running buffer (1× PBS, 0.5 M NaCl, 20 mM imidazole and 1 % (v/v) Tween®20). The supernatant was filtered through a 0.2-μm filter, and a 20mM final concentration of β-mercaptoethanol was added to reduce the free cysteine in the expressed scFv. The supernatant was further diluted 1:2 with the lysis buffer, the clarified lysate was passed through the equilibrated resin followed by washing with 50 ml running buffer to remove any loosely bound non-specific proteins. Bound scFv was eluted using 100 mM sodium acetate (pH 4.4) which was neutralised with

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neutralisation buffer (100 mM NaOH and one-tenth volume of 10× PBS). The neutralised scFvs were buffer exchanged with five volumes of PBS and concentrated using a VivaSpin column (VWR International, Lutterworth, UK) with a 10-kDa cut-off. The degree of purification was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), and the functional activity returned was assessed by standard ELISA titration on cTnI-coated plates. Site-directed mutagenesis The Cys80 residue was targeted for substitution by sitedirected mutagenesis using inverse, ‘whole plasmid’ amplification PCR. A Cys-Ala80 substitution was introduced using the primer pair Ala_For (5′-GTG GAG GCT GAC GAT GCT GCC-3′) and Ala_Rev (5′-GTC AGC CTC CAC GCC GCT GAT G-3′), and Cys-Ser80 substitution was imparted by the primer pair Ser_For (5′-GTG GAG AGT GAC GAT GCT GCC-3′) and Ser_Rev (5′-GTC ACT CTC CAC GCC GCT GAT G-3′). The reaction was carried out using an initial denaturation temperature at 98 °C for 10 min, followed by 35 cycles each of 1 min denaturation at 98 °C, 1 min annealing at 62 °C and 3 min extension at 72 °C and a final extension for 10 min at 72 °C. The mutated scFv composite was excised from the amplified phagemid using SfiI and re-cloned into freshly SfiI-digested and purified pComb3XSS phagemid. The cloned mutant scFv gene was transformed into Top10F′ cells, for soluble expression of scFvs, and the transformants were screened by indirect ELISA on cTnIcoated plates (1 μg/ml) and probed with anti-HA-HRP secondary antibody. Expression analysis was carried out by titering the wildtype (MG4) and mutant scFv lysates on cTnI-coated ELISA plate. Twenty-millilitre cultures were propagated in SB carbenicillin media supplemented with 1× ‘505’, from an overnight culture, at 37 °C while shaking at 200 rpm. All the cultures were induced at an O.D.600nm ∼0.8, with a 1-mM final concentration of IPTG followed by an overnight incubation at 30 °C and 200 rpm. The pellets were collected by centrifugation at 3220×g for 15 min at 4 °C. The supernatant was decanted, and the pellet was resuspended in 1 ml PBS. The cell suspension was transferred into a sterile 1.5 ml microfuge tubes. The cells in the suspension were lysed by alternating freeze-thaw cycles and the debris from the lysis was removed by centrifugation at 22,000×g for 15 min. Re-formatting of scFv for assessing comparative functional expression Rabbit scFvs were engineered by reversing the domain orientation and were further converted into ‘scAb’ formats by cloning within the context of the pMoPac16 (Hayhurst et al.

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2003) plasmid framework (kindly provided by Dr. Andrew Hayhurst, Southwest Foundation for Biomedical Research, San Antonio, USA). This plasmid confers a C-terminal human constant kappa (huCk) domain on scFv genes inserted within the upstream SfiI-defined cloning site. We used a 3-stage process to generate ‘MG4’ scAb variants. (i) Reversing variable domain orientation The VH and VK genes were amplified independently from the anti-cTnI scFv ‘MG4’ (GenBank accession no. KM044274) (VL-VH configuration). Primers RABVH_For (5′-GCG GCC CAG CCG GCC ATG GCG CAG CAG CAG CTG ATG GAG TCC GG-3′) and RABVH_Rev (5′GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC GCC CGA GCC ACC AGA GGA ACT AGT GAC TGA TGG TGG AGC CTT AGG TTG CCC-3′) were used for amplifying the heavy chain and primers RABVK_For (5′GGT GGT TCC TCT AGA TCT TCC GAG CTC GAT CTG ACC CAG ACT CCA-3′) and RABVK_Rev (5′-GGC CCC CGA GGC CGC TTT GAC GAC CAC CTC GGT CCC-3′) were used for amplifying the light chain. The VH and VK chains were reconfigured (VH-VL order) using standard SOE-PCR with the primers RabSWSOE2_For (5′-GGA ATT CGC GGC CCA GCC GGC CAT GGC GCA G-3′) and RabSWSOE1_Rev (5′-TTA CTC GCG GCC CCC GAG GCC GCT TTG-3′) primers. Composite scFv product was then re-cloned into freshly prepared SfiI-digested pComb3XSS vector and transformed into E. coli Top10F′ for expression and analysis. (ii) Generation of constant domain scAb plasmid variants The pMoPac16 vector was modified by excising the native huCk domain gene and replacing it with either rabbit Cκ or chicken Cλ light chain gene. Rabbit Cκ gene was amplified from NZW rabbit cDNA as prepared for the scFv library construction using the primer pair RABCLAMB_FOR (5′ATA AGA ATG CGG CCG CTG GTG ATC CAG T-3′) and RABCLAMB_REVtrp (5′-CCG GCA ATG GCC GAC GTC GAC CCA GTC A-3′). The chicken Cλ gene was amplified from a pooled chicken spleen/bone marrow cDNA preparation using the primers CLAMB_FOR (5′-ATA AGA ATG CGG CCG CTC AGC CCA AGG TGG CCC CCA C-3′) and CLAMB_REV (5′-CCG GCA ATG GCC GAC GTC GAC GCT CTC GGA CCT CTT CAG GG-3′). The respective chicken or rabbit constant chain genes were cloned into the pMoPac16 vector by sequential digestion with NotI and SalI and subsequent ligation with T4 DNA ligase. Ligated product was transformed into chemically competent E. coli Top10F′ cells (Invitrogen, Carlsbad, USA), and the clones were screened by ELISA using HRP-labelled anti-chicken and anti-rabbit antibodies.

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(iii) Assembly of ‘MG4’ variant scAb panel The SfiI restriction sites (GGCCNNNNNGGCC) on the pMoPac and pComb vectors contained different intrapalindromic sequences (5′-GGCCCAGCCG GCC————————GGCCTCGGGGGCC-3′ and 5′GGCCCAGGCGGCC————————GGCCAGGCCG GCC-3′, respectively) and thus, it was necessary to harmonise these for shuttling between both vectors. Cloning of VH-VL scFv configurations from pMoPac16 into the pComb3XSS vector was facilitated by re-amplifying the VH-VL scFv gene from the former with primers RABVH-VL_pComb_For (5′-GGA ATT CGC GGC CCA GGC GGC CAT GGC GCA G-3′) and RABVH-VL_pComb_Rev (5′-TTA CTC GCC TGG CCG GCC TGG CCG CCT TTG-3′). Primers RABVL-VHPAK_For (5′-GGA ATT CGC GGC CCA GCC GGC CAT GGC GGA GCT C-3′) and RABVL-VHPAK_Rev 3′ (5′-TTA CTC GCG GCC CCC GAG GCC GCA CTA GTG-3′) facilitated shuttling into pMoPac16 vector. Functional expression analysis of anti-cTnI antibody in different formats Expression efficiency of the modified antibody formats was checked by titering the scAbs and scFvs containing Cys80 on a cTnI-coated plate (1 μg/ml). Twenty-mililitre cultures, for all the antibody formats, were grown in SB carbenicillin media with 1× ‘505’ supplement. The cultures were incubated at 37 °C, shaking, until the O.D.600nm ∼0.8, after which they were induced with IPTG (1 mM final concentration) overnight for expression. All the cultures were subjected to identical conditions for growth and expression. On the following day, the lysates were prepared and analysed for expression by assaying the antibody titre (dilutions 1/10, 1/100, 1/1,000, 1/10,000 and 1/100,000) by ELISA. The antigen-antibody reaction was probed with 1/2,000 dilution of anti-HA antibody.

Results Isolation of candidate rabbit scFv panel For the construction of the anti-TnI rabbit scFv library, in addition to the VH genes, we opted to selectively amplify only the kappa light chain (Vκ) since the main objective was to isolate a candidate panel of natural and aberrant Cys80bearing clones, such as are peculiar to the kappa chains of rabbit antibody repertoire. The estimated library size was approximately 2×107, which although might be considered relatively small in the case of a naïve library, was deemed sufficiently large for the construction of the immune anti-TnI library based on the high

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Fig. 1 Rabbit serum titre against cTnI. The solid line represents the cTnI-specific antibody response obtained from the rabbit after two successive boosts with cTnI in Freund’s incomplete adjuvant and the broken line represents the control sera obtained from the animal before primary immunisation

TnI-specific serum response achieved with the immunisation regimen employed (Fig. 1). A panel of 50 clones was selected based on the ELISA binding signals observed in preliminary screening (data not shown), and these were then subjected to offrate ranking as previously described (Ayyar et al. 2010; Hearty and O’Kennedy 2011; McDonnell et al. 2011) and the top 10 clones (based on their apparent off-

rates) selected for further analysis. Sequence comparison based on CDR and Vκ position 80 traits confirmed two distinct lineages (Fig. 2), one of which comprised Cys80bearing clones (4 out of 10 clones) and the other comprised Arg at position 80 (6 out of 10 clones). It thus represented an ideal candidate panel for further critiquing of the soluble expression characteristics of Cys80-bearing clones.

MB10 MF11 MF7 MB6 MC8 MF4 MG5 MG4 ME4 MD3

CDR L1 CDR L3 VL80 CDR L2 ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDMTQTPSSTSAAVGDTVTINCQASESISSWLSWYQKKPGQPPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISDLERDDAATYYCQNNGDSSRHGYT ELDLTQTPSSVSAAVGGTVTIKCQASEDIYGFLGWYQQKPGQRPKLLIYSASKLASGVPSRFRGSGSGTEYTLTISGVECDDAATYYCQQYGRYDGVDNT ELDLTQTPSSVSAAVGGTVTIKCQASEDIYGFLGWYQQKPGQRPKLLIYSASKLASGVPSRFRGSGSGTEYTLTISGVECDDAATYYCQQYGRYDGVDNT --------------CGRQVTIKCQASEDIYGFLGWYQQKPGQRPKLLIYSASKLASGVPSRFRGSGSGTEYTLTISGVECDDAATYYCQQYGRYDGVDNT ELDLTQTPSSVSAAVGGTVTIKCQASEDIYGFLGWYQQKPGQRPKLLIYSASKLASGVPSRFRGSGSGTEYTLTISGVECDDAATYYCQQYGRYDGVDNT

MB10 MF11 MF7 MB6 MC8 MF4 MG5 MG4 ME4 MD3

Linker CDR H1 CDR H2 FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSS-QSLEESGGDLVTPGTPLTLTCTVSGFSLSTYAMNWVRQAPGKGLEWIGVTHSGGYKYYASWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSSQQQLMESGGGLVTPGGTLTLTCTVSGFSLSNYYIVWVRQAPGKGLEWIGAIHPNGNRYYANWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSSQQQLMESGGGLVTPGGTLTLTCTVSGFSLSNYYIVWVRQAPGKGLEWIGAIHPNGNRYYANWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSSQQQLMESGGGLVTPGGTLTLTCTVSGFSLSNYYIVWVRQAPGKGLEWIGAIHPNGNRYYANWAKGRFTISKA FGGGTEVVVKSSGGGGSGGGGGGSSRSSQQQLMESGGGLVTPGGTLTLTCTVSGFSLSNYYICWVRQAPSKGLEWIGAIHPNGNRYYTNWAKGRFTISKA

MB10 MF11 MF7 MB6 MC8 MF4 MG5 MG4 ME4 MD3

CDR H3 His-Tag HA-Tag S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS S-TTVDLKIVSPTTEDTATYFCARGSGITGLGLWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS SSTTVDLKMTSLTASDTATYFCAR-SGNN--DFWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS SSTTVDLKMTSLTASDTATYFCAR-SGNN--DFWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS SSTTVDLKMTSLTASDTATYFCAR-SGNN--DFWGPGTLVTVSSGQPKAPSVTSGQAGQHHHHHHGAYPYDVPDYAS SSTTVDLKMTSLTASDTATYFCAR-SGNN--DFWGPGTLVTVSSGQPKAPSFTSGQAGQHHHHHHGAYPYDVPDYAS

Fig. 2 Sequence analysis of anti-cTnI scFv genes. Sequencing was carried out using RSC-F and RSC-B primers, and the sequences generated were aligned using CLUSTAL X. The CDRs were identified using the Kabat numbering scheme (Kabat et al. 1991)

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Fig. 3 Comparative expression analysis of scFvs with different amino acids at position 80. The wild-type scFv with Cys80 returned a titre of only 1/100, whereas the mutant scFv with Ser80 was 1/6400 while the scFv with Ala80 returned the highest titre of 1/25,600

Dissecting the effect of Cys80 on soluble functional expression levels

Effect of V domain order on functional expression and purification levels

Clone ‘MG4’ was chosen as a prototypical aberrant Cys80 model rabbit scFv. However, before proceeding with a comprehensive expression analysis, it was necessary to first confirm that the aberrant Cys80 residue was significantly compromising the expression fitness of clone ‘MG4’. Substitution with Ala or Ser at position 80 potentiated >100fold improvement in measurable functional soluble expression levels as determined by parallel titration of respective crude lysate preparations (Fig. 3).

Wild-type scFv with VL-VH domain orientation (MG4) and its domain-inverted counterpart with VH-VL domain orientation (E2) were checked for expression yield and degree of purity achievable by IMAC. The domaininverted antibody returned a much improved functional expression and purity profile (Fig. 4). A total of 0.5 mg of V L -V H domain orientation (MG4) and 2.5 mg of V H -V L domain orientation (E2) were obtained from respective 250 ml scFv cultures.

Fig. 4 Comparison of VH-VL (E2) and VL-VH (MG4) format functional expression levels. A very significant difference (1000fold) was observed in the expression levels as shown in the graph. SDS-PAGE analysis (inset) following IMAC purification clearly demonstrated that ‘E2’ yielded a much more discernible product. Lane 1: Fermentas prestained protein marker, lane 2: scFv with VL-VH domain and lane 3: scFv with VHVL domain

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Effect of incorporating C-terminal constant domain fusion on functional expression levels The effect of the presence of constant domain on MG4 scFv was studied by analysing constant domains from three different hosts (rabbit, human or chicken). Chicken Cλ and rabbit Cκ domains were amplified from chicken and rabbit cDNA and cloned into the pMoPac16 vector, substituting native human Cκ. The scFv gene sequence (both VH-VL and VLVH domain orientation) was inserted upstream of the respective constant domain within the constant domain-substituted pMoPac16 plasmid, expressing scAbs. We noted a universal increase (>1000-fold) in functional expression levels (Fig. 5) across all of the scAb variants tested, irrespective of V domain orientation.

Discussion Notwithstanding our objective in this particular study, it is probably prudent to focus on the Vκ chains, the dominant Fig. 5 Comparative expression analysis of scAbs with different constant domain fusions and variable domain orientations within the Cys80-bearing scFv composite. The cTnI-captured antibody constructs were probed with a HRP-labelled anti-His antibody since the His-tag was consistent with all formats under investigation

manifestation of the light chain affinity matured antibody response in the rabbit, when constructing a rabbit immune antibody library. Given that the major limitation with phage display is the ability to actually package and access the entirety of the natural (i.e. in vivo affinity maturation) and artificial combinatorial (i.e. non-natural chain pairs generated in vitro) cumulative diversity, diluting the accessible pool with lambda V genes (Vλ) may actually be counter-beneficial with the net result being a decrease in the percentage of accessible, potential functional binders. The isolation of clone MG4 was in itself an interesting result as it confirmed Cys80 clones could be isolated even in a population of non-Cys80-bearing target-binding clones, and indeed there are corroborating precedents in this regard with Cys80-bearing clones previously isolated by other laboratories from NZW strain rabbit immune libraries, albeit at varying degrees of frequency (Chi et al. 2002; Popkov et al. 2004). The impact of non-natural (e.g. synthetic artefacts or introduced as part of linker sequences) cysteines on general scFv expression (Schmiedl et al. 2000; Albrecht et al. 2006) and specifically in relation to rabbit scFv/Fab showing Cys80

VL VH

VL VH

VL VH

VL-VH scAb C (Human)

C (Chicken)

VH VL

VH VL

C (Rabbit)

VH V L

VH-VL scAb

2.5

C (Chicken)

C (Human)

C (Rabbit)

scFv

2

Abs @ 450 nm

VHV H-VL rabbit C scAb (B3) VHVH-VL chicken C scAb (F9)

1.5

VH-VL human C scAb (B2) VHVL-VH rabbit C scAb (B4) VLVLλ  scAb (G7) VL-VH chicken C

1

VLVL-VH human C scAb (B9) VH-VL VL-VH scFv scFv(E2) (MG4)

0.5

0 1

10

100

1,000

10,000

Reciprocal antibody dilution

100,000 1,000,000

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aberrancy (Rader et al. 2000) is acknowledged. However, in view of the numerous precedents describing successful isolation of aberrant Cys80-bearing rabbit scFv antibodies (Li et al. 2000b; Chi et al. 2002; Popkov et al. 2004), we felt it was necessary to confirm this. Our results clearly confirmed that the aberrant Cys80 imparted a refractory soluble expression bias on the scFv ‘MG4’. The role of rabbit antibodies as surrogates in preclinical therapeutic mAb validation and disease models in addition to their value as therapeutic lead mAb candidates by virtue of their phylogenetic remoteness to humans renders rabbithuman chimeric therapeutic antibodies a valuable reagent class in their own right. The need for chimerisation of therapeutic candidates will eventually be likely entirely obviated by harmonisation of transgenic or fully human synthetic platforms, which guarantee ‘humanness’ ab initio. Nonetheless, the partial (Hayhurst 2000) or full (Fab) chimerisation with human constant domains (Carter et al. 1992; Ulrich et al. 1995; Rader et al. 1998) of the standard scFv format is acknowledged to improve expression, at least in certain instances. We further speculated that by incorporating a rabbit constant light (CL) kappa domain, it might even be possible to recapitulate the natural V L Cys80-C L Cys171 bridge (McCartney-Francis et al. 1984), thereby rendering the Cys80 non-aberrant and concomitantly reducing the expression impediment. This was simply investigated by inserting the scFv gene sequence upstream of the respective constant domain (rabbit, human or chicken) within the constant domain-substituted pMoPac16 plasmid. We noted a universal increase in functional expression levels across all of the scAb variants tested. Thus, amelioration of scAb-mediated functional expression was not dependent on a restored VLCys80CLCys171 bridge and additionally, was independent of V domain orientation in the scAb context. These results might go some way to dispelling somewhat the concerns voiced by Popkov and colleagues (Popkov et al. 2004), that evidential cysteine-mediated expression recalcitrance (Schmiedl et al. 2000) might be particularly troublesome in the case of chimeric Fabs (Mage et al. 2006). However, whether our findings in this regard can be universally extrapolated across all rabbit scFvs is still debatable, given that there is at least one precedent highlighting a Fab expression campaign returning distinctly disparate functional Fab yields when the CL domain was varied from Cκ to Cλ (MacKenzie et al. 1994). It should also be noted that the pMoPac16 vector used contains a Skp chaperone-encoding element which was shown to further enhance functional expression of a difficult-to-express scFv (Hayhurst et al. 2003). These findings have particular relevance for diagnostic applications where there is less pressure to conform to compatible physiological- and immunological-defined molecular constraints. For instance, with the exception of in vivo imaging applications, serum half-life and off-target toxicity/

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immunogenicity are much less relevant in the broader in vitro diagnostic (IVD) arena. The introduction of human constant domains could potentially lead to HAHA/HACA or broader heterophilic interference if used in human blood or plasma IVD test platforms. In cases where biophysical, immobilisation or stability requirements qualify the need the scAb or Fab construct in a particular diagnostic platform, such constructs need not be chimeric in the standard sense of the term. Fully rabbit scAb or Fab antibodies can be easily constructed using natural priming from the C-terminal of the CL and CH1/hinge domain or using modular assembly in a phagemid vector cassette harbouring the desired CL and CH1 domains and receptive of independently amplified discrete VL and VH domains. This is a general observation that is worth considering not just for human diagnostics but also veterinary diagnostics where recombinant antibodies isolated from any relevant species (e.g. rabbit, mouse, chicken, cows, sheep, etc.) might benefit from this strategy. Undoubtedly, this rationale advocates using the most minimal antigen-binding entities for diagnostic applications so long as it is not to the detriment of assay performance or practicalities of antibody expression (i.e. production and downstream processes) or stability. Just as the VL to VH orientation appears to be a functionally important manifestation of antibody sequence diversity (Narayanan et al. 2009) in the natural immunoglobulin composite, it is, thus, not at all surprising that functional expression of the unnatural synthetically tethered VH/VL domains in the minimalist scFv format should be even more highly influenced by anomalous domain associations. Notwithstanding variability arising from linker length, the modular order of the V domains is indeed acknowledged to affect functional expression of discreet scFv entities (Desplancq et al. 1994; Plückthun 1994; Hamilton et al. 2001; Lu et al. 2004). Our comparison of the MG4 scFv and its domain-inverted VHVL-oriented ‘E2’ clone confirmed significantly improved expression in the latter case. Interestingly, Albrecht et al. (2006), did not report a difference on yield of a murine scFv in the VHVL or VL-VH format but reported almost complete ablation of binding activity in the latter orientation and contrary to our MG4 scFv, noted universal improved functional binding in VH-VL-Cys orientation, possibly attributable to a dimerisation effect. However, we would agree with Hu and co-workers (Hu et al. 2005) in their summation that there are no hard and fast rules in this regard. In fact, each recombinant antibody clone must be regarded as an empirical singularity requiring individual optimization of its modular structure to optimise overall functional expression fitness. Display propensity, overall expression levels, folding and localisation in the periplasm are clone-specific characteristics that vary despite the high levels of homology and framework inertia. These may be further complicated by a pronounced poorer solubility of certain clones when in the non-phage pIII-fused state (Scott et al. 2008). Although this can lead to failed screening returns and

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increased attrition of candidate scFvs, it might also contribute in some way to explaining why poorly soluble clones, such as the aberrant Cys80-bearing MG4 rabbit scFv, get enriched during sequential rounds of phage panning in spite of their intrinsic expression recalcitrance. Cumulatively, our results confirm that the presence of an aberrant Cys80 residue in a rabbit VL kappa domain scFv can render soluble expression of functional species highly intractable. However, in the case of this particular scFv, expression recalcitrance was successfully abrogated by converting it to a scAb format or simply by reversing the order of the V domains. This raises a number of technical considerations when constructing rabbit scFv libraries. It is possible to construct a dual order (i.e. comprising both VH-VL and VL-VH) library. This may have the effect of increasing the diversity of binding combinations based on domain order permutations, albeit within the context of possibly diluting fractional functional diversity if there is indeed a bona fide generic expression predilection for one orientation over the other. Notwithstanding this, it must be acknowledged that despite poor soluble expression fitness, the aberrant clone ‘MG4’ did actually get selected in a real and stringent selection campaign. This might legitimately be ascribed to pIII fusion support during the phage selection phase, only manifesting as a problem when translated in the absence of pIII read-through. The fact that the weight of Cys80 aberrancy does not preclude selection on phage (pIII-displayed) and functional soluble expression can be effected using such facile postselection domain manipulations, suggests the rabbit antibody repertoire might actually be more readily accessible than previously thought. We would temper this assertion with the caveat that in identifying the model scFv for these studies we only screened ‘soluble’ clones (i.e. non-pIII-fused scFvs expressed in non-amber-suppressing E. coli) and thus we can only speculate that this would hold true even if primary screening was conducted on monoclonal phage-displayed candidates. Nonetheless, as we are discussing an immune recombinant library from an animal returning a significant antigen-specific serum titre, we feel justified in avoiding ‘on-phage’ screening. In fact, we always consider a degree of functional soluble expression as a key criterion in our primary library screens and generally only countenance ‘onphage’ screening with naïve or synthetic libraries where there is often a much reduced functional redundancy and more pronounced anomaly between ‘on-phage’ and ‘off-phage’ functional expression. A more pertinent concern might relate to what is the likelihood of aberrant Cys80-induced dimerisation and whether this dimerisation more frequently leads to impairment of functional affinity or increased functional avidity. It is an inescapable fact that for affinity-based selection campaigns, functional expression levels are always dictated by the combination of clonal expression fitness and binding affinity/avidity. Thus, for any reasonably sized (i.e. >107)

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immune library constructed from an animal returning a high antigen-specific titre, it should be eminently possible to isolate functional binding clones. This includes rabbit allotypes with Vκ Cys80 propensity. We should also not lose sight of the fact that these antibodies can only be designated ‘aberrant’ by virtue of the library context, i.e. when they are constrained in a minimalist scFv format or paired with heterologous CH1 and CL domains in chimeric Fab constructs. Certainly, from an immunodiagnostics perspective, we are not constrained by the same restrictive format inertias that necessarily pervade the therapeutics arena. In fact, immunodiagnostics practitioners might be best served by adopting a truly ‘agnostic’ perspective when deciding on the most suitable antibody format and indeed, which species might serve as the most appropriate reservoir from which to construct any given immune library. The disparity evidenced by the paucity of comparable rabbit recombinant antibodies is an unjustified anomaly that we anticipate will dissipate as practitioners realise the value of rabbit recombinant antibodies in diagnostic and research settings. Acknowledgments The authors thank Prof. Carlos F. Barbas III (Scripps Research Institute) for kindly providing the pComb3 vector series used in this study. The authors would also like to thank Dr. Andrew Hayhurst, Department of Virology and Immunology, Texas, USA, for providing the scAb expression vector. All authors were supported by the Science Foundation Ireland under a Centres for Science Engineering and Technology (CSET) grant (05/CE3/B754).

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