Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine

Journal of Immunological Methods 280 (2003) 139 – 155 www.elsevier.com/locate/jim

Recombinant Technology

Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine Amir Moghaddam a, Tine Borgen b, John Stacy b, Louise Kausmally b, Bjørg Simonsen b, Ole J. Marvik b, Ole Henrik Brekke b, Michael Braunagel b,* a b

GeNova AS, Oslo Research Park, Guastadalle´en 21, N-0349 Oslo, Norway Affitech AS, Oslo Research Park, Guastadalle´en 21, N-0349 Oslo, Norway

Received 28 August 2002; received in revised form 15 January 2003; accepted 19 February 2003

Abstract Use of phage display of recombinant antibodies and large repertoire naı¨ve antibody libraries for identifying antibodies of high specificity has been extensively reported. Nevertheless, there have been few reported antibodies to haptens that have originated from naı¨ve antibody libraries with potential use in diagnostics. We have used chain shuffling of lead single-chain fragment variable (scFv) antibodies, isolated from a naı¨ve antibody library, to screen for antibodies that specifically recognise the major metabolite of heroin, 6-monoacetylmorphine (6MAM). The antibodies were identified by screening high-density colonies of Escherichia coli expressing soluble scFv antibody fragments without prior expression on bacteriophage (phage display). The antibodies recognise 6MAM with affinities of 1 – 3  10 7 M with no crossreactivity to morphine. These antibodies can potentially be used for developing a rapid immunoassay in drug-testing programs. To our knowledge, this is the first report of an antibody that distinguishes 6MAM from its de-acetylated form, morphine. D 2003 Elsevier B.V. All rights reserved. Keywords: scFv; Antibody fragments; 6-Monoacetylmorphine

1. Introduction Phage display of recombinant antibodies is now used widely in making antibodies with potential diagnostic and therapeutic use. This method was developed to overcome the limitations of making monoclonal

* Corresponding author. Tel.: +47-22-95-88-63; fax: +47-2295-83-58. E-mail address: [email protected] (M. Braunagel). 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-1759(03)00109-1

antibody in mice, that is, the ability to identify or to engineer antibody fragments with predefined properties. Recombinant antibodies can also originate from human lymphocytes and overcome human anti-mouse immune reactions when used as human medicine. Furthermore, selection and screening of recombinant antibody libraries is amenable to automation and has the potential to become cheaper than monoclonal antibodies, both in isolation and in production (for reviews, see Siegel, 2002; Hudson and Souriau, 2001; Soderlind et al., 2001; Schmitz et al., 2000; Hoogenboom et al., 1998a; Vaughan et al., 1998).

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Single-chain fragment variable (scFv) antibody libraries displayed on filamentous phage can be derived from non-immunized or immunized animals (Li et al., 2000; Haard et al., 1999; Little et al., 1999; Do¨rsam et al., 1997; Griffiths et al., 1994; Hoogenboom and Winter, 1992; Clackson et al., 1991). Alternatively, scFv diversity can be generated synthetically (Gargir et al., 2002; Knappik et al., 2000; Hoogenboom et al., 1998b; Pini et al., 1998; Braunagel and Little, 1997). An advantage of using scFv constructs from animals immunized against an antigen of interest is that animal immune systems are very efficient in affinity maturation of antibodies, and the resulting libraries often contain high-affinity antibodies. It is therefore feasible to work with low-diversity libraries of less than 107 clones when working with antibody repertoires originating from immunized animals or humans (Moulard et al., 2002; Tout et al., 2001; Li et al., 2000; Chowdhury et al., 1998). Naı¨ve antibody libraries overcome the use of animals altogether, and a new library need not be constructed for every new antigen. However, the antibody library has to be of high complexity to increase the chances of having specific antibodies to a wide variety of antigens. A number of high-complexity recombinant antibody libraries have been reported and shown to contain antibodies against a variety of peptide antigens, non-peptide antigens and low-molecular-weight haptens (Knappik et al., 2000; Haard et al., 1999; Little et al., 1999; Mao et al., 1999; Griffiths et al., 1994; Hoogenboom and Winter, 1992). Antibodies from naı¨ve libraries are often low affinity and they have to be affinity matured to be further engineered by chain shuffling (Klimka et al., 2000; Park et al., 2000; Schier et al., 1996; Collet et al., 1992; Marks et al., 1992), mutagenesis (Short et al., 2002; Jermutus et al., 2001; Chowdhury and Pastan, 1999; Figini et al., 1994), DNA shuffling (Huls et al., 2001; Jung et al., 1999; Proba et al., 1998) or rational design (Kusharyoto et al., 2002; Coulon et al., 2002; Salvatore et al., 2002; Leong et al., 2001; Beucken et al., 2001). There have been many reports of selecting phage antibodies from non-immunized libraries against haptens conjugated to carriers such as BSA, g-aminobutyric acid, caproic acid or biotin – streptavidin (Clackson et al., 1991; Marks et al., 1991; Hoogenboom and Winter, 1992; Griffiths et al., 1994; Do¨rsam

et al., 1997; Pini et al., 1998; Sheets et al., 1998; Haard et al., 1999; Little et al., 1999; Sblattero and Bradbury, 2000; Knappik et al., 2000). However, there have been few reports of antibodies characterized for binding to soluble unconjugated hapten (Moghaddam et al., 2001). In our experience, naı¨ve phage display antibody libraries can be used readily to identify BSA-conjugated or biotin – streptavidin-conjugated haptens, but many of these antibodies will not bind the soluble form of the hapten (Moghaddam et al., 2001). Another complication is that many of the haptens used in model studies are not readily soluble in aqueous solutions. These include haptens such as phenyl-2-oxazolin-5-one (phOx) and steroid hormones (Hoogenboom and Winter, 1992; Vaughan et al., 1996; Do¨rsam et al., 1997; Haard et al., 1999; Little et al., 1999). Similarly, reports of in vitro affinity improvement of recombinant antibodies against haptens have made use of BSA-conjugated haptens and binding to a hapten alone has not been characterized (Crameri et al., 1996; Marks et al., 1992). We have previously described the use of a highcomplexity human B-lymphocyte antibody library and a semi-synthetic antibody library for the selection of antibodies to the hapten aflatoxin-B1. We used a competitive elution strategy whereby phage antibodies binding to BSA-conjugated aflatoxin-B1 were isolated using an excess of unconjugated aflatoxin-B1 in solution. We have now extended this protocol to a number of other haptens, namely, drugs that are commonly abused in society. We found that this selection strategy readily led to the isolation of phage antibodies that recognised unconjugated metabolites of abused drugs. However, many of these scFv antibodies were relatively low affinity. Binding of scFv antibody to immobilized drug metabolite could be detected in ELISA when the antibody fragments were expressed on phage but not as soluble scFv antibody fragments. This problem has been previously reported with phage antibodies against peptide and hapten antigens (Cloutier et al., 2000; Irving et al., 1996). In this paper, we address the issues of obtaining antibodies to haptens through isolation of high-affinity scFv antibodies to the major metabolite of the abused narcotic drug, heroin. The antibodies were isolated by identifying lead antibodies from a naı¨ve

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antibody library followed by heavy chain (VH) and light chain (VL) shuffling. The chain-shuffled libraries were directly screened using high-density colony screening of Escherichia coli expressing soluble scFv antibodies without further expression on bacteriophage (phage display) and panning. 6-Monoacetylmorphine (6MAM) is a specific hydrolysed metabolite of heroin and can be detected in blood, urine and hair of heroin users (Salmon et al., 1999; O’Neal and Poklis, 1998). Heroin is one of the major target drugs in drug-testing programs because of its history of abuse, addiction and continued negative social impact. Most immunoassay screens for heroin employ antibodies to morphine that crossreact with 6MAM and are consequently prone to false-positive results. A widely cited example is the false-positive drug screen resulting from consumption of poppy seed bagels (Narcessian and Yoon, 1997). Here, we report on the isolation of scFv antibody fragments to 6MAM that do not crossreact to mor-

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phine. These antibodies have the potential to be incorporated into a high-throughput and specific immunoassay for drug detection programmes.

2. Materials 6MAM was purchased from Sigma (St. Louis, MO). Monodisperse biotinylated polyethylene glycol (biotin-PEG) was purchased from Polypure (Oslo). Biotin-PEG – 6MAM was synthesised by Ultrafine (Manchester), and its final structure (Fig. 1) was confirmed by NMR and mass spectroscopy (data not shown). Morphine was purchased from a local pharmacy in Oslo. Purchase and use of all controlled substances were performed with prior permission and according to the standards of the Norwegian Board of Medication and Drugs (Statens Legemiddeltilsynet). Streptavidin was purchased from Boehringer Mannheim (Germany) and streptavidin-coated magnetic beads were purchased from Dynal (Oslo). The

Fig. 1. Structure of 6MAM, morphine and biotin-PEG – 6MAM.

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human lymphocyte antibody library and other reagents have been described previously (Moghaddam et al., 2001; Little et al., 1999).

3. Methods 3.1. Selection of phage antibodies from non-immunized antibody library Panning of antibody libraries against antigens were carried out by using streptavidin-coated magnetic beads as means of capturing phage antibody – antigen complex. For the first round of panning, 2  1012 phage from a human lymphocyte antibody library, with a complexity of 4  109 (Little et al., 1999), was incubated in 2 ml of blocking solution (PBS/3% BSA) supplemented with 1 Ag/ml PEG 400 for 15 min at room temperature. The library was divided into two aliquots, and to one of the aliquots, morphine was added to a final concentration of 10 3 M and was incubated for an additional 30 min. The target antigen, biotin-PEG – 6MAM, was added to both aliquots to a final concentration of 10 5 M and was incubated for an additional 2 h with rotation. Phage antibody –antigen complex was captured by addition of 200 Al of streptavidincoated magnetic beads (6.7  108 beads/ml), previously blocked for non-specific binding and separated by a magnet. The beads were washed carefully 20 times with PBS/0.1% Tween with resuspension in-between each wash. Phages binding to antigen – magnetic beads were eluted by incubation with 100 Al of 100 mM triethylamine (TEA), pH 11, for 5 min followed by immediate neutralization with an equal volume of 1 M Tris, pH 7.0 (alkaline elution). Panning during the second round was performed in duplicate, and the concentration of the antigen, biotin-PEG – 6MAM, was reduced to 10 6 M. To increase the likelihood of identifying antibodies that specifically bound 6MAM and not its conjugated form, a competitive phage-antibody elution (Moghaddam et al., 2001) was performed on one of the duplicate aliquots. Basically, after washing of the phage-antibody magnetic bead complex, the magnetic beads were incubated in 10 4 M 6MAM in PBS/0.02% BSA for 2 h at RT with rotation. Eluted phages were then separated from the mag-

netic beads using a magnet. Rounds 3, 4 and 5 pannings were performed as in Round 2 keeping all four panning conditions separate (alkaline elution F excess morphine, competitive elution F excess morphine). Panning of chain-shuffled antibody clones was performed using 10 8 M antigen, biotin-PEG – 6MAM, in the presence or absence of 100 times excess morphine. Phages were eluted with 10 6 M soluble 6MAM. Phagemid DNAs from eluted phages were recovered by infection of 5 ml of exponentially growing E. coli XL-1 blue and selection on agar plates supplemented with 100 mM glucose, 50 Ag/ml ampicillin and 30 Ag/ml tetracycline. Phage expressing and displaying scFv was prepared from E. coli-harbouring phagemid DNA as described (Welschof et al., 1997; Marks et al., 1991). 3.2. Analysis of antibodies by ELISA ELISA was performed by coating microtitre plates (Nunc, Roskilde, Denmark) with 0.1 Ag/well streptavidin in a volume of 100 Al. The wells were washed 3  with PBS/0.05% BSA and were incubated for 1 h

Table 1 Isolation of phage-antibody clones to 6MAM from a naı¨ve human lymphocyte antibody library Method of recovering phage from immobilized antigen

Alkaline elution Alkaline elution Competitive elution Competitive elution

Presence of excess morphine during panning

+

+

Number of clones specifically binding to biotin-PEG – 6MAM

Number of clones that bind soluble 6MAM and morphine

Number of clones that bind soluble 6MAM but not morphine

36/96

0/36

2/36

31/96

0/31

7/31

80/96

6/80

40/80

32/96

0/32

10/32

Following five rounds of panning against immobilized biotin-PEG – 6MAM, phage was prepared from 96 individual clones and analysed for binding to biotin-PEG and biotin-PEG – 6MAM (column 3). Binding to soluble 6MAM and morphine (columns 4 and 5) was assessed by competitive ELISA using 100 times excess of each soluble hapten.

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with 100 ng/well biotin-PEG – 6MAM or biotin-PEG as control. It is estimated that this corresponds to 6 pmol of biotinylated antigen binding to streptavidin per well. The wells were washed as before and blocked with PBS/4% fat-free milk W/V for at least 2 h. The microtitre wells were incubated with phage antibody or scFv antibody preparations in a volume of 100 Al. Bound phage particles were detected with rabbit anti-Fd phage antibodies (Sigma). Binding of scFv antibodies to antigen-coated microtitre plates was detected as described (Moghaddam et al., 2001). Signals from wells were scored positive if they gave readings at least five times over background in phage-antibody ELISA and two times in soluble scFv ELISA. Competition ELISA was performed to assess binding of antibodies to soluble hapten. Phage antibody or scFv antibody fragment were incubated for 1 h in biotin-PEG – 6MAM-coated microtitre plates in the presence or absence of soluble competitor analyte in a volume of 100 Al. Unbound antibodies were

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removed by washing, and bound antibodies were detected as described above. 3.3. Chain shuffling of variable domains Unique antibody fragments identified by MvaI restriction analysis and/or sequence analysis of clones identified from five rounds of panning of the two libraries were chain shuffled to generate and identify high-affinity antibodies to 6MAM. The heavy chain (VH) and light chain (VL) of the pooled unique clones was prepared by restriction of phagemid vector DNA, pSEX-81 (Braunagel and Little, 1997), with NcoI/ HindIII and MluI/NotI, respectively, followed by electrophoresis. The DNA fragments were purified and ligated into phagemid DNA prepared from the naı¨ve antibody library restricted, in turn, with the same enzymes. Ligated DNA was used to transform electroporation competent XL-1 blue E. coli and selected on agar plates supplemented with ampicillin, tetracycline and glucose as described above. The size

Fig. 2. Dose-dependent competitive binding of unconjugated soluble 6MAM to phage antibodies selected after five rounds of panning. Binding to soluble 6MAM and morphine was assessed by competitive inhibition of binding to immobilized biotin-PEG – 6MAM. Binding to biotinPEG – 6MAM in the absence of 6MAM competition is shown in solid bars. Phage antibodies did not show non-specific binding to biotin-PEGcoated plates (background, clear bars). Concentration of soluble 6MAM corresponds to 10, 100 and 1000 times excess antigen compared to concentration of biotin-PEG – 6MAM coated on microtitre wells.

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of the chain-shuffled library was 5 –10  105 clones with less than 10% background ligation of vector without insert. This method of variable gene shuffling abrogates the potential for excess mother clones in the chain-shuffled library, even if there are background ligation colonies.

peroxidase conjugated rabbit anti-mouse antibodies (Dako, Glostrup, Denmark) and visualized using ECL (Amersham, UK). Spot positions were identified with the help of the phoretixk Array2 analysis program (Nonlinear Dynamics, Newcastle-upon-Tyne, UK).

3.4. High-density E. coli screening of soluble scFv chain-shuffled libraries

3.5. Expression and purification of soluble scFv

The unpanned chain-shuffled antibody library was cloned into the vector pHOG-21 (Moghaddam et al., 2001; Welschof et al., 1997) for the expression of soluble scFv and direct screening of E. coli clones grown in high density on nitrocellulose membranes. Approximately 12,000 clones from the primary chainshuffled library were picked and inoculated to culture medium in 384-well plates using a Q-pix robot (Genetix, New Milton, UK). Cultures were grown 16 h at 37 jC, and the plates were then used to print arrays of colonies on nitrocellulose membranes overlying nutrient agars as described (Wildt et al., 2000). Three to five replicate arrays were printed. The arrays were printed in a 4  4 pattern such that each clone is represented twice per array, causing the orientation of duplicate clones relative to one another to be unique to the source plate. Unique duplicate orientation provides a method for identifying source 384-well E. coli culture plates associated with the spots from the array, while sub-grid position within the larger grid field reveals well coordinates. The colonies were grown for 12 h at 37 jC to reach a diameter of approximately 0.5 mm each. Capture membranes were prepared by first coating with 10 Ag/ml streptavidin-NC (Spectral Diagnostics, Toronto, Canada) and then by incubating with 2 Ag/ml biotin-PEG – 6MAM or biotin-PEG. Membranes were blocked with 3% BSA for 1 h. Membranes were washed three times with PBS, 15 min each time after each of the coating steps. Antigen-coated membranes were rinsed in LB and placed on nutrient agar containing 100 AM IPTG. The membranes supporting the arrays of colonies were then positioned on the capture membranes and incubated at 30 jC for 16 h. Using this arrangement, expression is induced and candidate scFv are retained on capture membranes as described by Skerra et al. (1991). Bound scFv were detected using mouse antic-myc (Invitrogen, Carlsbad, CA) and horseradish

Expression in 96-deep-well plates (Ab-gene, Epsom, UK) or larger scale for affinity purification

Fig. 3. Screening of scFv antibody secreting E. coli clones on nitrocellulose membrane. scFv Antibody fragments secreted by IPTG induced E. coli and captured on an antigen-coated nitrocellulose membrane. Signals from unique positive clones are shown. Each clone is arrayed in duplicate to discard false-positives. The pattern of duplicates allows identification of source E. coli culture in 384-well plates.

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has been described (Moghaddam et al., 2001). Purity of soluble scFv after affinity chromatography was confirmed by denaturing electrophoresis and Coomassie blue staining. Concentration of soluble scFv after purification was assessed by Bicinchoninic Acid protein assay (Pierce).

determination of dissociation constants. Data presented are best fitted using a heterogenous ligandbinding model.

3.6. Determination of affinity

4.1. Selection of 6MAM-binding phage antibodies from non-immunized phage display antibody library

Affinities of purified scFv antibodies were determined by surface plasmon resonance (SPR) using a BIAcoreX optical biosensor (Biacore, Uppsala, Sweden), essentially as described (Moghaddam et al., 2001) and according to manufacturer’s protocols. Biotin-PEG and biotin-PEG – 6MAM was coupled to two separate flow cells of a streptavidin-coupled sensor chip SA. Coupling was performed to 250 resonance units (RU), and all samples were injected at a rate of 30 Al/min. The response sensogram curves were globally fitted to several binding models using BIAevaluation 3.0 for

4. Results

We used a human lymphocyte-derived recombinant antibody library to enrich and select for phage antibodies that bind 6MAM. Panning of non-immunized derived antibody libraries can be performed under different conditions. A major variable when using a new antigen is deciding the appropriate concentration of the antigen used in panning. As a practical rule, the lower the antigen concentration, the higher the affinity of the enriched antibodies. Equally, using too low antigen concentration may result in identifying no lead antibodies at all.

Fig. 4. Competitive binding of soluble 6MAM to scFv clones identified by screening soluble scFv antibodies on a nitrocellulose membrane. Competitive binding was assessed by incubation of 100 times excess soluble 6MAM or morphine with purified scFv antibodies in biotin-PEG – 6MAM-coated microtitre wells. In this experiment, scFv clone 6MAM-219 was tested at a different time to other clones and, hence, the chart is slightly offset. Binding to biotin-PEG – 6MAM in the absence of 6MAM competition is shown in solid bars. Background binding of scFv to biotin-PEG-coated wells is shown in clear bars.

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Table 1 shows that all four panning protocols (alkaline elution F excess morphine, competitive elution F excess morphine) led to the enrichment of phage-antibody clones that specifically bound 6MAM and not morphine in a competition binding assay. As expected, competitive elution of phage with unconjugated hapten leads to a greater number of 6MAM-binding antibodies. On the other hand, nonspecific alkaline elution leads to the enrichment of mostly clones that specifically bind immobilized biotin-PEG – 6MAM and not soluble 6MAM. Fifty-nine of the clones that specifically bound soluble 6MAM were analysed by MvaI restriction analysis and, in some cases, sequence analysis in VH sequences (data not shown). Of these, 36 had unique sequences. Phage antibodies were prepared again from 10 of these clones and reassessed for dose-dependent binding to 6MAM. Fig. 2 shows that all 10 phage antibodies tested bound 6MAM in a dose-dependent manner but not to morphine. DNA fragments from the 36 unique identified clones coding for the scFv antibody fragments were pooled and cloned into a soluble scFv expression vector. The scFv expression of unique clones was induced in E. coli in 96 wells and was assessed by ELISA. None of the soluble scFv antibody clones produced detectable signals in ELISA. However, expression of soluble scFv in culture supernatant could be confirmed by Western blot analysis (data not shown). We hypothesized that the clones identified were relatively low affinity. These antibodies produce detectable signals when expressed on phages most likely because of avidity effects when multiple anti-

Fig. 5. Dose-dependent binding of 6MAM to scFv 6MAM-219. A typical example of dose-dependent binding of scFv antibody fragment to 6MAM, identified from E. coli colony screening. Competitive binding of 6MAM to scFv 6MAM-219 can be detected at 150 – 300 nM. Binding to morphine was not detected.

In the first round of panning, we used a high concentration of the antigen, biotin-PEG – 6MAM (10 5 M). Phages were eluted from immobilized antigen non-specifically using alkaline elution (100 mM triethylamine, pH 11). This was to ensure a more complete recovery of clones in the first round. In subsequent rounds, the antigen concentration was reduced to 10 6 M. Phages binding conjugated 6MAM were eluted either by alkaline elution or competitively with unconjugated antigen. Furthermore, panning was performed in the presence or absence of excess morphine in parallel. We expected that the presence of excess morphine and elution with soluble hapten would increase the chances of enriching for specific 6MAM-binding scFv clones.

Table 2 Enrichment of phage-antibody clones from chain-shuffled libraries that specifically bind 6MAM Method of recovering phage from immobilized antigen Alkaline elution Alkaline elution Competitive elution Competitive elution

Presence of excess morphine during panning

+ +

Number of panning rounds

Number of clones specifically binding to biotin-PEG – 6MAM

Number of clones that bind soluble 6MAM and morphine

Number of clones that bind soluble 6MAM but not morphine

1 1 2 2

20/48 15/48 42/96 54/96

0/20 0/15 0/42 0/54

20/20 15/15 42/42 54/54

After one and two rounds of panning against immobilized biotin-PEG – 6MAM, phage was prepared from individual clones and analysed for binding to biotin-PEG and biotin-PEG – 6MAM (column 4). Binding to soluble 6MAM and morphine (columns 5 and 6) was assessed by competitive ELISA using 100 times excess of each soluble hapten.

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Table 3 Characterization of clones expressing soluble scFv from chain-shuffled libraries that specifically bind 6MAM Number of rounds of panning

0 (Not panned) 0 (Not panned) 1 1 2 2

Presence of excess morphine

+ + +

Number of clones specifically binding to biotin-PEG – 6MAM

Number of clones that bind soluble 6MAM and morphine

Number of clones that bind soluble 6MAM but not morphine

0/48 0/48 3/48 1/48 8/96 14/96

0/48 0/48 1/3 0/1 0/8 0/14

0/48 0/48 2/3 0/1 5/8 12/14

Names assigned to scFv clones

6MAM-120 to 6MAM-121 6MAM-101 to 6MAM-105 6MAM-106 to 6MAM-119

Polyclonal phagemid DNA from unpanned and unpanned chain-shuffled library was digested and fragments coding for scFv were cloned into soluble scFv expression vector and transformed into E. coli. Soluble scFv was prepared from individual clones and analysed for binding to biotin-PEG and biotin-PEG – 6MAM (column 3). Binding to soluble 6MAM and morphine (columns 4 and 5) was assessed by competitive ELISA using excess soluble hapten.

body fragments are present on single phage particles. Furthermore, the primary antibody used to detect phage antibodies in ELISA, anti-Fd, recognises multiple coat protein on phage particles, thus adding to the sensitivity of detection in phage-antibody ELISA.

4.2. Chain shuffling of lead antibody clones and screening soluble scFv antibody library We wanted to assess whether chain shuffling of lead hapten binding phage antibodies would lead to

Fig. 6. Dose-dependent competitive binding of unconjugated 6MAM to soluble scFv identified from panning of chain-shuffled phage-antibody library. Clones were identified from chain-shuffled phage-antibody library after one (clone 6MAM-120) or two rounds of panning (clones 102 – 119). All clones shown demonstrated dose-dependent binding to soluble 6MAM and no binding to morphine. Concentration of soluble 6MAM corresponds to 4, 20, 100 and 500 times excess competitor antigen compared to concentration of biotin-PEG – 6MAM coated on microtitre wells. Binding to biotin-PEG – 6MAM in the absence of 6MAM competition is shown in solid bars. Background binding of scFv to biotin-PEGcoated wells is shown in clear bars.

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higher affinity antibodies. Furthermore, we wanted to assess the feasibility of screening large numbers of E. coli colonies for the expression of soluble scFv antibodies to 6MAM without panning of phage antibodies. This is of interest as it may overcome the problems associated with enriching for antibody fragments that work best in multivalent phage-antibody format, or as fusion proteins to pIII protein of bacteriophage, or as a result of bias incurred due to growth cycle of E. coli. Furthermore, the time taken is drastically reduced as fewer rounds of panning need to be performed. The unique 36 lead antibodies identified from the naı¨ve antibody library were thus pooled. The DNA sequences coding for the VH and VL sequences were, in turn, cloned into the same naı¨ve lymphocyte phagemid antibody library (see Sections 2 and 3). The VH and VL shuffled libraries were pooled. DNA fragments from these two chain-shuffled phagemid libraries, coding for entire scFv sequences, VH – linker – VL, were used to clone into the soluble scFv expression vector, pHOG-21, and were transformed into E. coli. A total of 12,000 E. coli clones were picked and grown in duplicate on a nitrocellulose membrane. Each membrane was, in turn, sandwiched against a second capture membrane, coated with either biotin-PEG or biotin-PEG – 6MAM as described in Sections 2 and 3. Eighteen clones were identified in 12,000 of the unselected chain-shuffled library that showed strong binding to biotin-PEG –6MAM-coated nitrocellulose membrane compared to the control membrane. MvaI restriction analysis of purified plasmids from these clones showed seven unique clones (Fig. 3). scFv was expressed and crude bacterial culture medium from the seven unique clones was analysed by competitive ELISA. Fig. 4 shows that scFv clones 6MAM-201, -202, -205, -214 and -219 showed strong Fig. 7. Amino acid sequence analysis of scFv antibodies that specifically bind 6MAM. scFv Clones 6MAM-20 and -49 were identified from the naı¨ve human lymphocyte library and are detectable in ELISA only in phage-antibody format. Clones 6MAM-214 and 219 were identified by screening of chain-shuffled scFv library. Clones 6MAM-102 to -120 were isolated by selection of chain-shuffled phage-display library. The germ line sequences for VH and VL are shown in the top line. Differences to the germ line sequences are shown. Some amino acids are in bold for clarity. Identical sequences are shown with a dash.

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specific binding to biotin-PEG – 6MAM, and this binding was competitively inhibited by excess soluble 6MAM and not morphine. scFv clones 6MAM-203 and -217 demonstrated specific binding to biotinPEG –6MAM compared to biotin-PEG albeit with a relatively lower signal than others. However, the extent of competitive binding to excess soluble 6MAM was minimal, and these two clones were therefore not further characterized. Antibody clones 6MAM-201, -202, -205, -214 and -219 showed dose-dependent binding to soluble 6MAM in competitive ELISA assays, an example of which is shown in Fig. 5. Typically, 150 –300 nM 6MAM could be detected in competitive ELISA. Clones 214 and 219 showed the strongest competitive binding to 6MAM in repeated assays (data not shown) and were further characterized.

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rounds of panning. DNA coding for scFv fragments was prepared and cloned into the vector, pHOG-21, for the expression of soluble scFv fragments. Also, 48– 96 E. coli clones transformed with this library were picked individually in 96-deep-well plates, grown and induced for soluble scFv production. Conditioned culture medium was assessed for soluble scFv that specifically binds soluble and immobilized conjugated hapten. Table 3 shows that 22 clones from the

4.3. Comparative panning of chain-shuffled antibody library We wanted to compare direct screening of chainshuffled soluble scFv library to panning of chainshuffled phage-antibody library. The soluble scFv antibody library was derived directly from a chainshuffled library generated in a phagemid vector, pSEX-81, which could be used for phage display and panning. Hence, both libraries share the same V H – V L combination, and any antibody clones obtained could be directly compared. We thus performed panning of the phagemid chain-shuffled phage-antibody library and screened for new 6MAMbinding scFv antibodies. Two rounds of panning of the chain-shuffled library was performed using 100 times lower biotinPEG –6MAM concentration (10 8 M) than that used when panning the naı¨ve library, to increase the chances of identifying high-affinity antibodies. In all cases, phages were eluted from immobilized antigen using 100 times excess soluble 6MAM and panning was performed in the presence or absence of excess morphine. Screening of phage-antibody clones showed that after one and two rounds of panning, 30 – 50% of the clones specifically bound biotinPEG –6MAM as well as soluble 6MAM (Table 2). None of the clones demonstrated binding to morphine. To assess the selected phage antibodies in soluble scFv format, phagemid DNA was prepared from the chain-shuffled library (Round 0) and after one and two

Fig. 8. Dose-dependent binding of scFv 6MAM-219 to 6MAM by surface plasmon resonance. Purified scFv antibody fragment 6MAM-219 was injected at various concentrations over a streptavidin flow cell coupled to biotin-PEG – 6MAM (A). For assessment of binding to unconjugated hapten, 0.1 AM purified scFv 6MAM-219 was pre-incubated with various concentrations of 6MAM or morphine and injected over the streptavidin flow cell coupled to biotin-PEG – 6MAM (B). Real-time binding to the conjugated antigen was recorded and analysed for affinity determination. Values shown have been corrected for binding to biotin-PEG-coupled flow cell, which in all cases was minimal. Dissociation constant for morphine was approximated by preincubation of scFv with one concentration of morphine and comparison of binding to 6MAM. Levels of significance are indicated by standard errors. a, Below detection level.

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second round of panning produced biotin-PEG – 6MAM-binding antibodies, of which 17 bound the soluble form of 6MAM. Two clones were identified from the first round of panning that bound the soluble form of 6MAM and no clones were identified from unpanned (Round 0) chain-shuffled library. The 19 6MAM-binding clones were analysed by sequence analysis and were found to consist of 12 unique clones. The unique clones were expressed and reassessed for binding to biotin-PEG – 6MAM, 6MAM and morphine. Fig. 6 shows that all 12 scFv clones competitively bound 6MAM in a dose-dependent manner, but not morphine. Four of the scFv clones, labelled 6MAM-102, -103, -108 and -120, which seemed to give the best overall competition binding to soluble 6MAM, were chosen for analysis of kinetic binding parameters as described below.

partners obtained. Clone 6MAM-214 is a product of the lead VH clone from 6MAM-20 partnering with the lead VL clone 6MAM-49. Because both VH and VL sequences are present in the lead clones, it therefore remains ambiguous whether it is a product of VH or VL shuffling. Both VH sequence could be traced to the germ line sequence, IGHV3-9*01. All of the light chain sequences could be traced to IGLV1-40*01. 4.5. Characterization of affinity by surface plasmon resonance (SPR) To better approximate the affinity of identified clones for the hapten 6MAM, surface plasmon resonance (SPR) analysis was carried out on purified scFv from clones 6MAM-102, -103, -108 and -120 (from two rounds of selection of shuffled antibody library) and scFv 6MAM-214 and -219 (from membrane colony screening of unselected scFv chain-shuffled library). All scFv showed specific and dose-dependent binding to biotin-PEG – 6MAM conjugated chip, an example of which is shown in Fig. 8. This data was used to derive binding kinetics to the conjugated hapten (Table 4). There was no evidence of dimerisation of antibody fragments. All antibodies showed similar binding kinetics with a dissociation constant of 0.6 –3  10 7 M for biotin-PEG –6MAM. Preincubation of purified scFv with soluble 6MAM before application to the chip allows for determination of scFv antibodies for the hapten in solution. All antibodies tested showed dose-dependent binding to soluble 6MAM in solution, an example of which is shown in Fig. 8. Binding to morphine could only be

4.4. Sequence analysis of 6MAM-binding scFv antibody fragments Sequences from soluble scFv antibody clones that specifically bound 6MAM were analysed by amino acid alignment (Fig. 7). 6MAM-102, -103, -108 and 120 are from panning of the chain-shuffled phagedisplay library while 6MAM-214 and -219 are from direct screening of E. coli expressing scFv without prior panning with phage. All chain-shuffled clones identified originated from two mother clones, 6MAM20 and -49, although 36 lead phage-antibody clones were used to construct the chain-shuffled libraries. In all cases, with the exception of clone 6MAM-214, the VH of the mother clones were conserved and new VL

Table 4 Affinity and rate constants of scFv antibodies for streptavidin-chip-conjugated biotin-PEG – 6MAM and soluble 6MAM, determined by surface plasmon resonance scFv

Biotin-PEG – 6MAM kon (M

6MAM-214 6MAM-219 6MAM-102 6MAM-103 6MAM-108 6MAM-120

1

s

1

)

1  104 F 4  102 3  104 F 2  102 2  104 F 1  102 1  104 F 3  102 1.5  104 F 2  102 7  104 F 9  102

6MAM koff (s 7  10 2  10 2  10 3  10 3  10 2  10

1

)

4

F 1  10 3 F 1  10 3 F 3  10 3 F 7  10 3 F 1  10 3 F 7  10

KD (M) 4 4 5 5 4 5

6  10 6  10 1  10 3  10 2  10 3  10

Morphine

KD (M) 8 8 7 7 7 8

3  10 1.6  10 1.7  10 1  10 1.7  10 2.5  10

KD (M) 7

F 3  10 7 F 4  10 7 F 9  10 7 F 6  10 7 F 8  10 7 F 1.510

5 8 10 8 8 7

a 1  10 6  10 4  10 9  10 a

5 4 4 4

Affinities and rate constants are derived from fitting to the heterogeneous ligand binding model. Levels of significance are indicated by standard error. The scFv antibody clones 6MAM-214 and -219 are from unselected chain-shuffled scFv library screened on nitrocellulose membrane. Clones 6MAM-102 to -120 were identified from panning phage antibodies from the chain-shuffled library. a, Below detection level.

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detected when used in large excess, typically 5000 times more than that needed to detect binding to 6MAM (Fig. 8). Table 4 shows that the affinities of the antibodies for 6MAM were typically similar to biotin-PEG –6MAM, 1 – 3  10 7 M.

5. Conclusions and discussions There have been many references to the potential of using phage display of recombinant antibodies from naı¨ve libraries to obtain antibodies that have high specificity and affinity. Antibodies have been reported that distinguish heparin from heparan sulphate (Westerlo et al., 2002): estradiol – BSA from progesterone – BSA and testosterone –BSA (Little et al., 1999; Do¨rsam et al., 1997) or human luteinizing hormone from human follicle stimulating hormone and human chronic gonadotropin (Haard et al., 1999). In the experiments of Hirose et al. (1998), scFv antibodies from a non-immunized library distinguishes glutathione from its BSA-conjugated form. However, 50,000 times excess unconjugated glutathione is needed to demonstrate binding to the free hapten. To our knowledge, there are no convincing reports of phage-derived antibodies from naı¨ve antibody libraries that can distinguish closely related lowmolecular-weight haptens. Furthermore, there have been few reports of chain shuffling or other affinity maturation protocols of antibodies from naı¨ve lymphocyte libraries to increase affinity of scFv antibodies that bind unconjugated haptens. In this article, we address the problems associated with identifying recombinant antibodies from naı¨ve antibody libraries that binds acetylated morphine and not morphine. To our knowledge, this is also the first report of a monoclonal antibody that can specifically recognise the excreted heroin metabolite, 6MAM. We used a modified version of our previously published competitive elution protocol to enrich for phage antibodies that bound soluble unconjugated 6MAM. Panning was performed by preabsorbing the library with excess morphine to avoid antibodies that do not distinguish in binding between morphine and 6MAM. Using these two strategies of competitive elution and excess morphine was useful in minimising the number of unwanted biotin-PEG – 6MAM- or morphine-binding antibodies in lead candidate anti-

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bodies. Subsequently, any chain shuffling or other affinity maturation protocols can be applied polyclonally to the pooled lead clones and not necessarily to individually characterized clones. The lead phage antibodies clones obtained by panning the naı¨ve library against 10 6 M biotinPEG – 6MAM did not produce detectable signals in ELISA when used as soluble scFv antibody. These antibody clones did, on the other hand, produce secreted soluble scFv in induced E. coli culture medium as assessed by Western blot analysis (data not shown). Although one cannot exclude that these antibodies are misfolded in soluble scFv format, we hypothesized that they are simply low-affinity antibodies. Hence, binding of multimeric scFv in phage format to polyepitopic streptavidin – biotin-PEG – 6MAM can be detected in ELISA due to avidity effects. Using avidity to increase the overall binding of low-affinity scFv antibodies has been previously reported (Cloutier et al., 2000, and references therein). A second source of signal amplification in phageantibody ELISA is due to polyvalent binding of the secondary antibody against phage coat protein, antiFd. This amplification is missing in soluble scFv ELISA as the scFv antibody fragments have only one binding site for the detection antibody, anti-cmyc. Hence, in the absence of avidity and signal amplification, low-affinity scFv binding to hapten antigens, as described in these examples, falls below the standard ELISA sensitivity. There are two ways to overcome the problem of obtaining low-affinity antibodies from naı¨ve antibody libraries. Firstly, it may be feasible to only use a low concentration of antigen and hope to obtain highaffinity antibodies straight from the naı¨ve library. This is probably most feasible when screening antigens with a number of good potential epitopes, as in the case of peptide antigens. With a more ambitious project, such as that described in this paper, where we aimed to develop antibodies whose binding depends on the presence of an acetyl group on a small hapten with minimal contact surface, panning under a variety of stringent and non-stringent conditions would have to be carried out. This we find to be tedious and labour-intensive. We therefore adopted a second medium-throughput strategy to reduce the number of variables. That is, chain shuffling of lowaffinity lead antibody clones from a naı¨ve library

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followed by screening large number of individual clones for high-affinity scFv on antigen-coated membranes. It should also be feasible to perform chain shuffling on enriched libraries where a large percentage of specific binding antibodies are present without exhaustingly characterizing many individual lead clones. In our chain shuffling library construction, we chose a strategy that minimized the chances of having mother clones being present in the chain-shuffled library. We therefore inserted the VH or VL sequences of the lead antibody clones, in the absence of vector sequences, into recombinant antibody phagemid DNA libraries. This is contrary to other protocols, where the unwanted VH or VL is replaced by a library of VH or VL sequences. This strategy is to overcome having an excess of parent clones, derived from background cloning ligation, which would potentially bias downstream screenings. We screened soluble scFv expressing E. coli colonies from the pooled VH and VL chain-shuffled library, using antigen immobilized on nitrocellulose membrane. The chain-shuffled library did not undergo panning, and, hence, none of the products from the membrane colony screening are derived directly from phage display. This has the advantage of overcoming a bias inherent in E. coli and phage growth cycles as well as a bias in panning phage antibodies that favours clones that produce signals in ELISA in phage format but not as soluble scFv. A bias in panning phage-antibody libraries can be seen in the panning of chain-shuffled libraries and analysis of the clones. Two rounds of selection led to the enrichment of specific biotin-PEG – 6MAM-binding clones. Approximately, 50% of all phage antibodies tested bound the conjugated hapten in ELISA (Table 2). Yet, cloning for expression as soluble scFv led to relatively few soluble scFv that showed detectable binding in ELISA, between 8% and 15% (Table 3). Biased enrichment of phage antibodies may also be the reason why the clones 6MAM-214 and -219 (from E. coli colony screening) were not isolated by two rounds of panning. A current limitation to screening of non-enriched phage using a high density of scFv expressing E. coli on membranes is that specific antigen binding antibodies must be present at a frequency of least 1 in 104. However, recent advances in automation technology

is making screening of 105 colonies more feasible. Secondly, a competition assay has as not been developed to identify non-conjugated binding haptens. It is therefore essential to screen an enriched library or, in this case, a chain-shuffled library of pooled lead clones shown to bind soluble hapten. In this article, we report on the identification of two scFv antibodies not identified through phage panning but by direct scFv screening of a chain-shuffled library. The monomeric scFv antibodies had affinities of 1.7– 3  10 7 M for soluble 6MAM and 1 –2  10 7 M for immobilized biotin-PEG –6MAM. This is similar to affinities obtained from clones identified by classic panning (clones 102, 103, 108 and 120). Neither antibodies demonstrated binding to morphine. Additional advantages of using direct scFv on membranes screening is clearly overcoming the time-consuming panning process and subsequent need to change vectors (or E. coli strain) for the expression of soluble scFv. All clones identified from the nitrocellulose membranes do express and secrete soluble scFv at high concentration as this is a criterion for positive signals from soluble scFv screening. Hence, the clones identified can be quickly cultured for highscale expression. It is interesting that pooling of heavy and light chain-shuffled libraries led to identification of predominantly parent heavy chains with new light chain partners. Antibody clone 6MAM-214 remains ambiguous in its origin because both the VH and VL sequences were represented in two different mother clones used for chain shuffling. Conservation of VH sequences from the lead clones 6MAM-20 and 6MAM-49 after chain shuffling nevertheless implies that the majority of the specific binding to the hapten 6MAM comes from the heavy chain, while the light chain pair significantly affects the affinity of the antibody fragment for the soluble hapten. The heavy chain sequences of chain-shuffled clones can be traced to the IGVH3-9*01 germ line sequences. The chain-shuffled clones originated from one VH, 6MAM-20, except for the chain-shuffled clones 6MAM-120, which came from 6MAM-49. 6MAM-20 and 6MAM-49 VH sequences differ only by six amino acid substitutions, two of which are conservative. The light chain sequences were all of E origin and could be traced to IGLV1-40*01 germ line sequences. Majority of the sequence variation

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in VL sequences were in CDRII and to a lesser extent in FRI and CDRIII. The scFv clones 6MAM103 and 6MAM-120 have the same VL and differ from scFv 6MAM-108 VL by two amino acids. The high homology of the light chain sequences implies the non-promiscuous nature of the light chain in high-affinity binding to free 6MAM. Hence, for a predominant 6MAM-binding VH, only a narrow repertoire of VL can form a high-affinity binding pocket. The scFv clone, 6MAM-214, identified from direct E. coli colony screening, is the result of the parent clones, 6MAM-20VH and 6MAM-49VL, partnering as a result of chain shuffling. While scFv 6MAM-214 produces functional soluble scFv antibody fragment, neither parent clones give rise to functional soluble scFv as assessed by ELISA. This again implies the non-promiscuous nature of VH and VL in high-affinity binding to free 6MAM. It would be interesting to compare the 3D structure of 6MAM bound to scFvs 6MAM-214 and 6MAM-20 as the two antibody fragments differ by four non-conservative amino acid substitutions, yet, only scFv 6MAM-214 shows detectable binding as soluble scFv in ELISA. To our knowledge, this is the first set of antibodies that is reported to specifically bind 6MAM and not morphine. No doubt, there have been unsuccessful attempts at raising monoclonal or polyclonal antibodies with this specificity in animals. The obstacles could be instability of the antigen in serum or lack of flexibility in identifying antibodies that distinguish the acetylated form of morphine. This is clearly the advantage of using recombinant antibody libraries, that is, the ability to adapt screening to identify antibodies with predefined properties. Drug-testing programs, such as work place drug testing, have initial drug concentration cutoff value of 2 Ag/ml for opiates and 10 ng/ml for 6MAM. Confirmation is typically performed by GC/MS. Our ELISA assays are designed for screening of a large number of scFv candidates rather than maximising detection sensitivity. Our assays are, for example, not amplified for signal by ELAST, gold labelling or other systems. Nevertheless, we detect 6MAM at 50 – 100 ng/ml (Fig. 6). These scFv antibody fragments are therefore good candidates for further development for costeffective and rapid immunoassays for abused drugtesting programs.

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Acknowledgements We thank Erik Agner for helping with 6MAM conjugation, Kirsti Gebhardt for doing the BIAcore ˚ ge Løset for critically reading the analysis and Geir A manuscript.

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