Development of neutralising human recombinant antibodies to pertussis toxin

FEMS Immunology and Medical Microbiology 23 (1999) 313^319

Development of neutralising human recombinant antibodies to pertussis toxin Peter Williamson, Ruth Matthews * The Pertussis Reference Laboratory, University Department of Medical Microbiology, Clinical Sciences Building, Manchester Royal In¢rmary, Oxford Road, Manchester M13 9WL, UK Received 26 June 1998; received in revised form 17 November 1998 ; accepted 17 November 1998

Abstract A phage antibody display library of single chain Fv (scFv) was derived from the peripheral blood of two patients recently recovered from pertussis infection. Ten scFv, differentiated by DNA fingerprinting, were isolated by panning the library against pertussis toxin. One scFv (type 1) accounted for 33% of clones after panning. Six of the panned scFv bound to pertussis toxin. The ability of the scFv to neutralise pertussis toxin was assessed using the Chinese hamster ovary cell assay. The predominant scFv (type I) and two others (types IV and VIII) were able to neutralise the pertussis toxin. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Pertussis toxin; Phage display; Human recombinant antibody

1. Introduction Bordetella pertussis is a highly infectious respiratory pathogen which causes some 50 million cases of whooping cough (pertussis) and 350 000 deaths worldwide each year, putting it among the 10 commonest causes of fatal infectious diseases [1]. Most of these are in unvaccinated infants in developing countries. Elsewhere, the control of whooping cough is critically dependent on the continuation of an e¡ective vaccination programme, but even in countries with a high vaccination rate, such as the USA, pertussis persists and in some cases is increasing [2^4]. Antibiotics, such as erythromycin, are of limited ben* Corresponding author. Tel.: +44 (161) 276-4660; Fax: +44 (161) 276-8826.

e¢t in vivo, despite the sensitivity of the bacterium in vitro. Recently an erythromycin-resistant strain was isolated from a clinical case [5]. Pertussis toxin (PT) is one of the toxic proteins produced by B. pertussis and is the only component common to all pertussis vaccines. Its role as a protective antigen has been demonstrated in animal models of pertussis [6^8] and by a ¢eld trial with a monocomponent pertussis toxoid vaccine in Sweden [9], though multicomponent vaccines are preferred [10^12]. PT has multiple toxic e¡ects in the mouse causing lymphocytosis, hyperinsulinaemia, hypoglycaemia and histamine sensitisation and is involved in the adhesion of bacteria to epithelial cells and macrophages [13^15]. PT has a toxic e¡ect on Chinese hamster ovary (CHO) cells in culture. Under normal conditions, the cells grow as an even sheet but in the

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presence of PT they show a clustered growth pattern that 16^48 h later can be di¡erentiated from normal growth [16,17]. This toxic e¡ect is due to ADP-ribosylation of a 41-kDa cellular substrate [18]. The effect of PT on CHO cells can be inhibited by PTspeci¢c antibodies and this assay has been used both diagnostically, to detect PT-neutralising antibodies in the sera of patients with suspected whooping cough, and as a research tool to identify toxinneutralising murine monoclonal antibodies [19^22]. In one clinical trial, hyperimmune serum containing high titres of antitoxin, prepared from blood donors immunised against PT, appeared to be of some bene¢t when given early in the treatment of infants with pertussis [23]. It was argued that earlier trials, showing little or no bene¢t, may have failed due to inadequate antitoxin content [24]. To resolve this question, a standardised source of high titre antibody to PT is required. Murine monoclonal antibodies to PT have been shown to be protective in mouse models of pertussis [25], but such antibodies could not be used therapeutically in patients with pertussis because they would induce a human anti-mouse antibody (HAMA) response. Recent advances in antibody engineering have made possible the production of human recombinant antibodies to PT which should be relatively easy to bulk produce and standardise and avoid the risks of serum therapy, such as viral contamination. This technology enables the expression of human recombinant antibody fragments in phage display vectors in the form of single chain variable region fragments (scFv) [26^28]. Genes encoding the heavy (VH) and light (VL) chain variable domains are randomly assembled together and then cloned into the minor coat protein gene (g3p) of a ¢lamentous bacteriophage. The resulting library of scFv is expressed on the phage surface as g3p fusion proteins. Antibodies with high binding a¤nities for a particular antigen can be selected from the library by multiple rounds of antigen-a¤nity selection (`panning'). Here we describe the production of human scFv to PT from a phage antibody display library derived from two patients with B. pertussis infections with high titres of pertussis antibodies. These were screened for the ability to bind native PT by a dot immunobinding assay and then examined for toxinneutralising ability by the CHO assay.

2. Materials and methods 2.1. Patients Heparinised peripheral blood (20 ml) was obtained from two patients with pertussis. One was a 29-yearold unimmunised female with a 4-week history of clinically diagnosed whooping cough. She was culture-negative, but serologically positive, paired sera showing a s 4-fold rise in antibodies (to s 1:512) to agglutinogens 1 and 2 as measured by bacterial agglutination. She was diagnosed as having a B. pertussis serotype 1,2 infection. The second donor was an 8-year-old girl with a 3-week history of clinically diagnosed whooping cough. The nasopharyngeal swab was culture-positive and typing revealed she had a B. pertussis serotype 1,3 infection. 2.2. Preparation of phage antibody display library The phage antibody display library and scFv were produced essentially as described previously [26^29]. Brie£y, peripheral blood lymphocytes from each patient were obtained by separation of the heparinised blood over Ficoll and mRNA was extracted with guanidinium thiocyanate followed by puri¢cation on an oligo(dT)-cellulose column (Quick Prep mRNA; Pharmacia, St Albans, UK). First, strand cDNA was synthesised using a heavy chain constant region primer for all four subclasses of human IgG heavy chains (HuIgG1^4) [27,29] using avian myeloblastosis virus reverse transcriptase (HT Biotechnology, Cambridge, UK). The cDNA from the two patients was then combined. The VH domain genes were ampli¢ed by primary PCR with family-based forward (HuJH1^6) and backward (HuVH1a^6a, 4b) primers [26,27]. A S¢1 restriction site was introduced to the 5P-end of the products prior to assembly with a diverse pool of VL genes derived from the library of Marks et al. [27]. This also introduced a linker fragment (Gly4 Ser)3 at the 5P-end of the VL and a NotI site at the 3P-end. By use of these two restriction enzyme sites, the product was unidirectionally cloned into the phagemid vector pCANTAB 5 (Pharmacia) at unique S¢I and NotI sites. The ligated vector was introduced into Escherichia coli TG1 by electroporation and phage was rescued

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from these cells with the helper phage M13K07 (Pharmacia). 2.3. Selection of scFv against PT To enrich for PT-speci¢c scFv the phage library was panned against puri¢ed PT, kindly provided by Dr L.I. Irons, PHLS, Salisbury, prepared as previously described [19]. The PT was bound to Maxisorp tubes (Life Technologies, Paisley, UK) at a concentration of 20 Wg ml31 in phosphate-bu¡ered saline (PBS), and free protein-binding sites were blocked with blocking solution (10% foetal calf serum (FCS), 2% skimmed milk powder in phosphate-bu¡ered saline (PBS)). Phage-containing culture supernate (109 plaque forming units (pfu) ml31 ) was added to an equal volume of blocking solution and incubated in the tubes for 2 h at 30³C. Unbound phage were removed by washing ¢ve times, for 5 min, with PBS, on a rotating horizontal orbital shaker. Bound phage was eluted with log-phase E. coli TG1 and rescued with the helper phage M13K07. The phage was then re-panned against the PT a further three times. Following panning, BstNI (New England Biolabs, Hitchin, UK) DNA ¢ngerprinting was used to determine whether panning had been successful in selecting for speci¢c scFv clones. 2.4. Dot immunobinding assay Each of the dominant scFv types obtained by PTselection was grown up to give a 100 ml supernate from which the phage was precipitated using polyethylene glycol (PEG 8000) as previously described [30], and re-suspended in 1 ml of PBS. Puri¢ed PT (200 ng) was loaded onto nitrocellulose in a dot immunobinding plate (Bio-Dot, Bio-Rad Laboratories, Hemel Hempstead, UK). Incubation in blocking solution for 2 h at room temperature was followed by an overnight incubation with phage diluted 1:5 with blocking solution at 4³C. The nitrocellulose membranes were washed and incubated for 2 h at 30³C with anti-fd antibodies (Sigma, Poole, UK) at 1:1000 dilution in blocking solution. After washing they were incubated for 1 h at room temperature with alkaline-phosphatase-conjugated anti-rabbit IgG (Sigma) at 1:1000 dilution in blocking solution and stained with nitroblue tetrazolium 5-bromo-4-chloro-

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3-indolyl phosphate (Sigma). Concurrently performed negative controls included the M13K07 helper phage (in place of the recombinant phage) and a negative control scFv (against hsp90) [31]. 2.5. CHO cell toxin neutralisation test The ability of the scFv to neutralise the cytoxic activity of PT was determined using the CHO cell assay [16]. First, all PEG was removed from the PEG precipitated phage by chloroform extraction, which was repeated three times. The aqueous layer was dried down in a Speedvac (Savant Instruments, Farmingham, NY, USA) and resuspended in 0.5 ml of water. CHO cells (CHO-K1 American Culture Collection Number CCL61, BioWhittaker, Wolkingham, UK) were grown to con£uence in Ham's F-12 culture medium (Sigma) supplemented with 10% FCS at 37³C in 5% CO2 . Just prior to use they were trypsinised and diluted to 2U105 cells ml31 in F-12 medium. PT (10 ng ml31 , equal to four 100% cytotoxic doses) in 50 Wl of the F-12 culture medium was added to the each well of a 96-well tissue culture plate. To this was added 50 Wl of phage, in serial 5-fold dilutions from neat (1U1014 pfu l31 ) to a dilution of 1:10 000 (1U1010 pfu l31 ). After incubation of the phage and PT for 2 h at 37³C, 2U104 (100 Wl) of CHO cells were added to each well. After 24 and 48 h incubation at 37³C, the cells were examined with an inverted light microscope.

3. Results 3.1. Production and panning of the phage display library Primary PCR reactions directed against the seven families of heavy-chain variable-domain genes gave ampli¢ed products from ¢ve of the classes (1a, 3a, 4a, 4b, 6a) of which classes 3a, 4a and 4b gave stronger bands than those obtained from the 1a and 6a reactions. Classes 2b and 5a were negative. Approximately 2U105 colonies were recovered following transformation into E. coli TG1. A random selection of 24 colonies, following PCR ampli¢cation, each gave the expected 950-bp insert and each had a

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Fig. 1. Growth of CHO cells in tissue culture (a) in the absence of PT and (b) in the presence of PT (10 ng ml31 ).

unique BstNI ¢ngerprint, indicating a highly heterogeneous library. After four rounds of panning against PT, 30 colonies were PCR ampli¢ed and BstNI ¢ngerprinted. Two ¢ngerprint patterns predominated, constituting 33% (type I; 10 clones) and 23% (type II; 7 clones) of the 30 clones examined. A further ¢ngerprint type accounted for four (13%) of the clones (type III) and two ¢ngerprint types were each present twice (IV and V). The remaining clones were made up of ¢ve unique ¢ngerprint patterns (types VI^X).

3.2. Dot immunobinding of antigen-selected scFv against PT Each of these PT-selected scFv was screened for the ability to bind to the toxin, using a dot immunobinding assay to examine clones of ¢ngerprint types I, II and III (in duplicate, two clones of each type being examined) and one clone from each of the other less common ¢ngerprint types IV to X. Types I^V and VIII gave positive dot blots with native PT. Types VI, VII, IX, X were all negative, as were the

Fig. 2. Growth of CHO in the presence of PT which has been successfully neutralised by a scFv of (a) type I and (b) type IV.

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Fig. 3. Growth of CHO in the presence of PT which has not been successfully neutralised by a scFv of (a) type II and (b) type III.

negative controls, M13K07 helper phage, the negative control scFv and PBS (in place of scFv). Direct binding of the scFv to the nitrocellulose was used to demonstrate successful binding and visualisation by the anti-fd conjugate. With the negative control scFv, the anti-fd conjugate successfully visualised the bound scFv, but the negative control scFv did not non-speci¢cally bind to PT. 3.3. PT neutralisation test using CHO cells The clones that bound to PT in the dot immunobinding assay (clones of types I, II, III, IV, V and VIII) were functionally assessed in the CHO cell toxin neutralisation test (Fig. 1). Four of the scFv (both type I clones, the type IV clone and the type VIII clone) were able to neutralise the toxic e¡ect of PT on the CHO cells when incubated with the PT prior to addition of the cells (Fig. 2). This neutralising e¡ect was present up to dilutions of 1:1000 (1U1011 pfu l31 ) for each of these phages, but thereafter disappeared. The scFv with ¢ngerprint patterns II and III (two clones of each type) and V (one clone) showed no neutralising e¡ect even, at the highest concentrations of phage (Fig. 3).

4. Discussion A phage antibody display library was produced

from the peripheral blood lymphocyte mRNA of two patients with B. pertussis serotype 1,2 and 1,3 infections respectively, both of whom had seroconverted as a result of infection. After PT-selection, 10 di¡erent scFv were present, being di¡erentiated by BstNI ¢ngerprinting, among which type I predominated (33%), followed by type II (23%) and type III (13%). Types IV and V (7%) were commoner than types VI^X which each occurred only once out of the 30 clones examined. Ability to bind PT in the dot immunobinding assay correlated well with the results of PT-selection process (`panning') in that all of the clone types which were commonly selected by panning against PT (types I^V) also bound to PT in the dot immunobinding assay. Of the remaining types (VI^X) which were still present after panning, but relatively uncommon, only type VIII bound PT in the dot immunobinding assay. Three types (I, IV and VIII) of scFv which bound PT in the dot immunobinding assay were also found to be able to neutralise the toxic e¡ects of PT in the CHO cell assay. Presumably, these scFv recognised epitopes at sites essential to the toxicity of PT, whereas the other scFv bound epitopes on PT which were not at functionally critical sites. PT is a 105-kDa protein hexamer made up from ¢ve di¡erent subunits (S1^S5) in the ratio of 1:1:1:2:1 [14]. The S1 subunit contains the biologically active toxic region, the A-promoter, and the S2^S4 subunits together make up non-toxic B-

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oligomer involved in binding to target cells. Vaccination with PT or its subunits was protective in animal models of pertussis [6,8] and a monocomponent pertussis toxoid vaccine was 71% e¤cacious in a double-blinded, placebo-controlled trial involving 3450 children [9]. Mouse monoclonal antibodies against S1 were found to be protective against intracerebral (i.c.) and aerosol challenge in animal models, whereas antibodies against S2 and S3 were protective against aerosol challenge only [25]. In the CHO assay it has been shown that neutralisation of PT toxicity can be achieved by mouse monoclonal antibodies which block binding of the B-oligomer (S2^S4) or inactivate the toxic A-promoter (S1) [19^21]. Therefore, the three neutralising scFv described here could be binding either the B-oligomer or the A-promoter. Ohlin and Borrebaeck [33] found that the VH region gene usage among in vivo-induced, T-cell-dependent antibody responses against foreign antigens di¡ered from that found in the total B-cell population. Marks et al. [27] showed that in healthy uninfected individuals, the VH genes ampli¢ed by PCR were from each of the seven VH gene families, whereas in the current study, in patients recovering from pertussis, three classes (VH3a, 4a and 4b) predominated and two (VH2b and 5a) of the seven gene families did not amplify. Likewise we have observed previously that in patients infected with certain bacteria or fungi preferential ampli¢cation of the VH3a family occurred, while VH2b and 5a were notably absent [28]. Sequence analysis (data not shown) of the three PT neutralising scFv showed that they were all from the same VH3a gene family, while the VL domains each came from a di¡erent light chain family (VM2, VM4 and VU1) [32]. VH are believed to play a more important role in the binding of antibody fragments to antigens than VL [34]. B. pertussis infection is not mediated by a single virulence factor, but is in£uenced by a complex interaction of many cell adhesion and toxic components. The phage library we have constructed o¡ers the possibility of isolating further scFv to other virulence factors. Such human recombinant antibodies could have therapeutic potential as they avoid the problem of the HAMA response induced by mouse monoclonal antibodies. Neonates are particularly at risk from pertussis infection, because they are too

young to have been vaccinated and the mortality and morbidity is highest in this age group, often requiring hospital admission [35,36]. Erythromycin is used in the treatment and prophylaxis of pertussis because all isolates, until recently, have been susceptible to erythromycin in vitro, though it is of limited bene¢t in established infections. A case has now been reported of a 2-month-old boy with pertussis whose symptoms persisted and cultures remained positive despite 24 days of erythromycin therapy. This was found to be due to an erythromycin-resistant strain of B. pertussis with an MIC s 256 Wg ml31 [5]. Human recombinant antibodies, such as those described here, could perhaps contribute to the treatment of pertussis due to antibiotic-resistant strains or in neonates in whom mortality and morbidity is high. References [1] Matthews, R. (1997) The diagnosis of pertussis infections : a recurring challenge. PHLS Microbiol. Digest 14, 79^84. [2] Bass, J.W. and Wittler, R.R. (1994) Return of epidemic pertussis in the United States. Pediatr. Infect. Dis. J. 13, 343^345. [3] van der Zee, A., Vernooij, S., Peeters, M., van Embden, J. and Mooi, F.R. (1996) Dynamics of the population structure of Bordetella pertussis as measured by IS1002-associated RFLP: comparison of pre- and post-vaccination strains and global distribution. Microbiology 142, 3479^3485. [4] World Health Organisation (1994) Weekly Epidemiological Record 13, 95^96. [5] Lewis, K., Saubolle, M.A., Tenover, F.C., Rudinsky, M.F., Barbour, S.D. and Cherry, J.D. (1995) Pertussis caused by an erythromycin-resistant strain of Bordetella pertussis. Pediatr. Infect. Dis. J. 14, 388^391. [6] Oda, M., Cowell, J.L., Burstyn, D.G. and Manclark, C.R. (1984) Protective activities of the ¢lamentous haemagglutinin and the lymphocytosis-promoting factor of Bordetella pertussis in mice. J. Infect. Dis. 150, 823^833. [7] Schneerson, R., Robbins, J.B., Taranger, J., Lagergard, T. and Trollfors, B. (1996) A toxoid vaccine for pertussis as well as diphtheria? Lessons to be relearned. Lancet 348, 1289^1292. [8] Shahin, R.D., Witvliet, M.H. and Manclark, C.R. (1990) Mechanism of pertussis toxin B oligomer-mediated protection against Bordetella pertussis respiratory infection. Infect. Immun. 58, 4036^4068. [9] Trollfors, B., Taranger, J., Lagergard, T., Lind, L., Sundh, V., Zackrisson, G., Lowe, C.U., Blackwelder, W. and Robbins, J.B. (1995) A double-blind, placebo-controlled trial of a monocomponent pertussis toxoid vaccine. New Engl. J. Med. 333, 1045^1050. [10] Cherry, J.D. (1993) Acellular pertussis vaccines ^ a solution to the pertussis problem. J. Infect. Dis. 168, 21^24.

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