WO1998023737A9 - Production of in vivo labeled single chain synthetic antibody fragments - Google Patents

Abstract

In vivo labeled single chain antibody fragments produced by expression in a host cell of a recombinant nucleic acid which comprises a part encoding a variable domain of a heavy and/or light chain of an antibody molecule, and a part encoding a functional reporter molecule, e.g. an enzyme or a fluorescent protein, or an attachment moiety for attachment of a fluorescent chromophore. Such in vivo labeled single chain antibody fragments are useful as immunological reagent.

Description

Title: Production of in vivo labeled single chain synthetic antibody fragments

The invention relates to immunological reagents produced as fusion proteins that comprise single chain antibody fragments and reporter molecules or fragments thereof, which can for example be used in immunoassays . Diagnostic as well as other applications of antibodies are numerous. Both polyclonal as well as monoclonal antibodies (Mabs) have been used as immunological reagents to specifically bind to and detect antigens by a wide array of imunochemical techniques. Such techniques, such as enzyme- linked-immunosorbent-assays (ELISA), immunofluorescent assays (IFA), radio-immuno-assays (RIA), and others, are widely known in the field of immunological detection. These techniques require the labeling or conjugation of the antibodies used with reporter molecules such as (fluorescent) dyes, enzymes, amino acid sequences, radioactive labels, and so on.

Especially Mabs are versatile immunological reagents for diagnostic purposes and are for instance used in a large array of detection kits. Mabs owe their usefulness to their highly specific and high-affinity binding to an antigen. In an intact Mab, the V_H and V_L domain are the independently folded N- terminal variable domains of the H(eavy) and L(ight) chain of an antibody molecule and jointly they form the very specific antigen-binding site. Mabs can be generated in a well known process by fusion of a selected lymphocyte (obtained after proper immune selection) with e.g. a myeloma cell. The resulting hybridoma cell then produces the wanted Mab. The procedure to produce large amounts of these Mabs is inherently complex, because it is technically difficult to propagate the hybridoma cells and harvest sufficient amounts of the Mab. Recently, the production of immunological reagents has been strongly simplified by the design of so-called single- chain variable fragments (scFvs) or single chain synthetic antibody fragments. These can be monovalent antibody fragments which can be produced in a bacterium like Escherichia coli , or in other suitable expression systems, through the expression of the V_H and/or V_L domain of an antibody molecule, which domains may be joined covalently through a designed, flexible polypeptide linker sequence. The production of these recombinant materials in bacteria has the obvious advantage that the production is several orders of magnitude faster than when hybridoma cells are cultured, due to the high growth rate of these production hosts.

The resulting scFv is routinely expressed in such a way that it is exported to the periplasm of the prokaryotic Gram- negative producer cell, by exploiting an excretion signal sequence. However, other ways of producing scFvs in producer cells can be thought of. In E. coli the cytoplasmic expression of scFvs results in accumulation of the recombinant material in inactive form in inclusion bodies. Renaturation of these materials is possible, but high level production of active scFvs in the cytoplasm would be desirable. In a thioredoxin reductase (TrxB) mutant of E. coli functional scFv was demonstrated in the cytoplasm (Proba and Pluckthun, Gene 159: 203-207, 1995), functional expression in the cytoplasm is presumably a result of the correct formation of disulfide bridges. Functional expression in the cytoplasm is very well possible. Examples can be found with other hosts, e.g. with plants, such as tobacco, in plant or animal cell lines, yeasts, fungi, and so on. scFvs can be made by amplifying and cloning the proper region of the genes coding for a selected antibody or antibody fragment. A recent break-through in the construction of scFvs has been the application of a random selection process to identify the wanted gene from a large library of genes, each coding for a particular scFv. This can be accomplished by expressing each of these scFvs on the head of a bacteriophage, in a form such that the scFv still can bind its antigen, followed by selection of phages with a high affinity for the desired antigen. This system has become known as the phage display system.

However, for most applications as immunological reagents, scFvs must also be labeled or conjugated after production with reporter molecules before they can be used as immunological reagent. Labeling can routinely be performed in many different ways: the proteins can, e.g., be labeled chemically with (fluorescent) dyes; they can be labeled by covalent attachment of a reporter enzyme; the presence of a recognizable amino acid sequence for another antibody (a 'tag') may allow the application of the so-called sandwich technique; or a radioactive label (usually a short-living radioactive cation) can be complexed to the scFv. In the latter form scFvs have been applied in the ultra-sensitive diagnostic analyses of tumor metastasis.

The labeling of antibody molecules or scFvs with reporter molecules cannot be achieved, however, reliably in a quantitatively reproducible and consistent manner. Reaction circumstances of such conjugation procedures vary, resulting in ratios of labeled to unlabeled molecules that vary from batch to batch. Also, antibody molecules or scFv fragments may be bound to more than one reporter molecule and thus contain varying amounts of label. The end result of such batch-to- batch and within-batch variability is that batches of immunological reagents may vary widely in specificity and sensitivity.

The present invention now provides a much more reproducible and quantifiable labeling procedure of specific immunological reagents with reporter molecules by in vivo labeling of a scFv or another synthetic antibody fragment by translational fusion of an antibody gene or fragment thereof with a functional reporter gene or functional fragment of a reporter gene. Reporter genes or fragments thereof can for instance be selected from genes encoding reporter enzymes or fluorescent reporter proteins. The resulting constructs encode fusion proteins comprising an scFv fragment specifically directed against (i.e. specifically recognizing or binding) an antigen of choice and a label by which immunochemical detection can occur. After expression in a suitable expression system or host cell the in vivo labelled scFv fragments may be used, possibly after a suitable purification procedure, as immunological reagent to detect antigen by any possible immunological detection technique. The advantage of in vivo labeling of scFv fragments is that once the construct has been made via recombinant techniques, batch-to-batch or within- batch variability cannot occur; each and every time the same reactive molecule is expressed. The in vivo labeling can be carried out both before (upstream of) the N-terminal part of an scFv, or after (downstream oϊ) the C-terminal part of an scFv, and can be combined with the attachment of additional labels or tags to an scFv. The invention includes embodiments wherein the expressed product is composed of an scFv and an attachment moiety consisting of an amino acid or peptide which allows later attachment of a fluorescent chromophore.

The subject invention provides a recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding a variable domain, or a fragment thereof, of a heavy and/or light chain of an antibody molecule, and a nucleic acid sequence encoding a functional reporter molecule or functional fragment thereof, or encoding an attachment moiety for attachment of a reporter molecule.

Recently, a translational fusion of the enzyme bacterial alkaline phosphatase (PhoA) to an scFv fragment was described (Carrier, A. et al. 1995). This construct, however, misses the advantages provided by scFv fragments fused with the reporter enzymes provided by the present invention. The reporter molecule PhoA is only enzymatically active when present in a homodimeric form and thus not functional when present as a monomer. Consequently, a monomeric PhoA-scFv fragment as such is not enzymatically active and cannot be considered to be an immunological reagent. Only after a subsequent and complex dimerization process, detection of the scFv through phosphatase activity was possible, ^ t expression levels of the wanted product were very low and, iu^erently, no batch-to- batch invariability was achieved. Furthermore, the functional, dimeric, form is only present in the periplasm of the cell and can be found in the periplasm only at very low expression levels, in the cytoplasm no functional product is found. In contrast, the present invention provides a wide variety of labels or reporter molecules that do not require processes such as dimerization to render them functional. Also, the present invention provides functional immunological reagents that are produced in the cytoplasm of the host cell at high expression levels. A reporter molecule such as, e.g., the ferredoxin-NADP⁺ oxidoreductase enzyme (FNR), can be overexpressed up to levels of at least 30% of the cell weight in a soluble, active form. It is expected that the expression levels of scFv-FNR fusion proteins will roughly be equivalent to the expression levels of the un used scFvs.

A class of enzymes relevant for the uses and applications of this invention are the oxidoreductases. A ferredoxin-NADP⁺ oxidoreductase enzyme from cyanobacteria has been used in the experimental part. FNR (also designated as PetH) is the gene product of the petH gene; it may be isolated from the cyano- bacterium Anabaena variabilis ATCC 29413. Additional oxidoreductases, that can be used in a similar way, for instance the peroxidases, like haloperoxidase from Curvularia , are also discussed as well as the substrates that can be used for their subsequent detection. Also different classes of hydrolytic enzymes can translationally be coupled to the gene encoding the scFv. The best studied ferredoxin-NADP⁺ oxidoreductase is the enzyme from spinach. In its native form FNR associates with ferredoxin, a soluble Fe-S containing protein that is reduced by photosystem I during oxygenic photosynthesis. It catalyses the transfer of electrons to NADP⁺:

2Fd_red. + NADP⁺ + H⁺ -> 2Fd_0x. + NADPH FNR is a flavoprotein that performs its redox reactions with a non-covalently attached FAD as the redox-active cofactor. The visible absorption spectrum of the protein is typical for flavoproteins: it shows a maximum at 458 nm (E₄₅₈=10 mM-¹.cm^_1). Among the flavoproteins, FNR has been classified within class IV, which contains the dehydrogenases/ electron transferases. In its primary structure characteristic domains can clearly be recognised: motifs that are involved in the binding of the FAD, as well as motifs that are assigned to NADP⁺ binding. The crystal structure of the FNR from spinach has been resolved (for a review see: Karplus, P.A. and Bruns, CM., 1994). This structure reveals that FNR is composed of two independently folded domains. The N-terminal domain has a 'beta-barrel' structure and binds FAD. The C-terminal, NADP⁺- binding domain, is a structure consisting of beta sheets and alfa helices.

Many reductases have a similar domain structure: the FAD- NADP⁺ domain structure can be considered as modules with widespread occurrence. Among these homologues are also the FMN-flavoproteins and the NADH-dependent reductases. Some representative examples are: Nitric oxide synthase, Cytochrome P450 reductase, Sulfite reductase, Neutrophil NADPH oxidase, Ferric reductase, Flavohemoglobin, Cytochrome b5 reductase, Nitrate reductase, Oxygenase reductases, Phthalate dioxygenase reductase, etc. These enzymes are also applicable as reporter enzyme in translational fusion constructs with scFvs.

The activity of FNR can be straightforwardly assayed in vitro in a reverse reaction, where oxidation of NADPH is catalysed by FNR and artificial electron acceptors such as K3Fe(C )₆ or iodonitrotetrazolium violet become reduced. For the spinach enzyme K_m values towards NADPH of 10-50 μM and Vmax values of 60-150 U/mg have been reported. In our laboratory kinetic studies on the recombinant Anabaena variabilis enzyme show similar enzyme kinetics. For enzymatic labelling one may also think of: The lacZ gene encoding the β-galactosidase can be used as an alternative chromogenic tag. A well known substrate for this enzyme activity is ONPG (o-nitrophenyl-beta-D-galacto- pyranoside), which is soluble in water and can be used in ELISA assays. Also the substrate chlorophenol red-beta-D- galactopyranoside can be used for ELISA detections. Fluorescein di-beta-D-galactoside is a fluorogenic enzyme substra e for galactosidase. The substrate X-gal (5-bromo-4- chloro-3-indolyl-beta-D-galactopyranoside) is insoluble and can be used in western blot or immunohisto-/cytochemistry.

Peroxidases can be attached to scFvs. Known water soluble substrates useful for ELISA are ABTS (2,2 ' -azino-bis[3-ethyl- benzothiazoline-6-sulfonic acid], diammonium salt) and TMB ( 3 , 3 ' , 5, 5 ' -tetramethylbenzidinedichloride) and OPD (o-phenylenediaminedichloride) . Known insoluble substrates are DAB (3,3' -diaminobenzidinetetrachloride) , AEC ( 3-amino-9- ethylcarbazole) and 4-chloro-l-naphthol. Also chemiluminescent substrates are commercially available.

Bacterial luciferase (expressed in bacteria using the luxAB cassettes) fusion proteins result in bioluminescent molecules. Detection can be done in luminometers, or for convenience in a Berthold-type microplate luminometer when ELISA's are performed.

Bacterial lipases and esterases ( lipA and estA genes) are known to hydrolyze the ester bonds in the compounds p-nitrophenyl palmitate and p-nitrophenyl acetate respectively, which can be monitored in ELISA by following the extinction at 410 nm. See Kok et al . 1995.

A number of proteins display a very characteristic endo- genous type of fluorescence, that can be exploited in high- sensitivity and highly selective detection of these proteins. The best know of these is the Green Fluorescent Protein (GFP) from Aquorea victoria (Chalfie_f M. et al . , 1994), which nowadays is very often used as a reporter in gene-expression studies. GFP has a complex absorption spectrum (and thus excitation maximum, with maxima at 395 and 480 nm) . It emits intense fluorescence at 510 nm. These wavelengths nicely fit with detection systems for fluorescein-isothiocyanate (FITC), a fluorescent label that is often used to conjugate antibodies with a fluorescent tag. GFP can be produced in fluorescent form through heterologous expression, for instance in E. coli . A posttranslational maturation process gives rise to the oxidation of one of the tyrosines of the protein, resulting in a cyclization reaction that forms an imidazolin-5-one intermediate. Recently, modified forms of GFP have been selected after random mutagenesis. These (i) have a more simple and straightforward fluorescence spectrum with only a single (and stronger) absorption, and thus excitation, maximum, (ii) mature more rapidly and (iii) variants with different emission maxima are now available, so that simultaneously different variants can be detected.

Phycobiliproteins are an intrinsic part of the antenna system of cyanobacteria. They contain a linear tetrapyrrole chromophore that is posttranslationally attached to the apoprotein via a thiol-ether linkage. These proteins can be obtained with a range of absorption spectra, covering the visible part of the spectrum, depending on the particular structure of the tetrapyrrole chromophore and the amino acid sequence of the corresponding apoprotein.

Photoactive Yellow Protein is a recently discovered protein that functions as a photoreceptor in purple bacteria (Hoff, 1995). It can be heterologously produced in E. coli, be it that then only the apoprotein is formed. In vitro (and under physiological conditions) the protein can be reconstituted with a large range of chromophore analogues (A. Kroon et al . , in preparation). Among the hybrid PYP molecules thus formed are several that are highly fluorescent. The high quantum yield of this fluorescence makes these proteins also attractive tags for fluorescence detection.

Fluorescent proteins may also be translationally fused to the scFvs: PYP requires in vitro reconstitution, before it is fluorescent. This can be conveniently combined with cytoplasmic overexpression of the scFv fusion product, during renaturation. Also translational fusion constructs with phycobiliproteins can be constructed.

For the in vitro labeling of engineered scFv fragments containing an attachment moiety, use can be made of various amino acids, each with its characteristic chemical functionality. Such amino acids can be engineered at either the N-terminus or the C-terminus into the coding frame of the scFv. They can be encoded as single residue additions, or multiple additions. It should be noted that there must be a limit to the amount of added residues such as cysteine that can be introduced without interfering with either solubility or functionality. Well known chemical coupling methods can be used to introduce fluorescent chromophores . One or more cysteine residues, for example, can be used for attachment of fluorescent chromophores. The chromophores can be coupled to these residues by e.g. a maleimide reaction scheme, a pyridyl reaction scheme, an active halogen reaction scheme, or via a thioester bond linkage as in the case of reconstitution of the chromophore of the Yellow Protein.

Primary amines that are conventionally labeled via an N-hydroxy succinimide (NHS) or N-hydroxy sulfosuccinimide (Sulfo-NHS) Ester reaction scheme can be introduced when one or more lysine residues are used as attachment moiety added to the scFv coding frame.

Tyrosine and histidine residues can be used as attachment moiety and be labeled with p-diazobenzoyl compounds.

Arginine residues can be coupled selectively with phenyl glyoxal compounds. Aspartate and glutamate have carboxyl groups that can be aminated with fluorescent compounds carrying primary amine groups, via a carbodiimide-activated intermediate.

Large amounts of in vivo labeled scFv can thus, according to this invention, be produced in a very simple and robust fermentation system, for example based on Gram-positive or Gram-negative bacteria and/or yeasts. Production hosts could in principle be either prokaryotes or eukaryotes. In view of the high growth rate of bacteria, such as E. coli , however, these are preferable in industrial production processes. Depending on the particular translational fusion construct, various targeting signals can be used to produce these hybrid scFv-reporter protein constructs in (i) the cytoplasm, (ii) the periplasm or (iii) extracellularly. For all three types of localisation, the corresponding sequences are available in a form that will allow the construction of translational fusion products. It should be noted that in the case of E. coli , extracellular production of fusion protein is due to a spontaneous leakage across the outer cell wall, since this bacterium does not express the required Xcp translocational machinery necessary for active translocation to the extracellular medium.

In addition, the present invention provides in vivo labeled synthetic antibody fragments which can be used to detect antigens with even higher specificity and sensitivity. The invention provides immunological reagents comprising specifically labeled scFv fragments that utilize an intrinsic amplification mechanism, leading to a decrease in the detection limit for rare antigens. Such immunological reagents comprise labeled scFv fragments that specifically bind with the same receptor molecules as they are labeled with. scFv fragments fused with a reporter gene can be generated that specifically recognize the reporter gene product (here the PetH enzyme is used as an example but many other enzymes can be used without altering the principle disclosed by the invention), without impairing the specific activity of the reporter gene product. Thus any scFv reacting with a particular antigen, that has translationally been fused with for instance FNR or GFP, can be reacted with scFvs that specifically bind to FNR or GFP, respectively. This leads to complexes of in vivo labeled scFv fragments. For instance, it has been observed in our laboratory that the scFv that binds the Synechocystis PCC 6803 ferredoxin- NADP⁺ oxidoreductase, does not inhibit its enzyme activity when it is bound. Therefore the use of a Synechocystis FNR labeled anti-Synecnocystis FNR scFv will result in a formidable amplification of a signal, when used in combination with a specific scFv-enzyme fusion protein, to detect antigen. The resulting complexes lead to a lineair amplification of the signal upon binding of each 'next' scFv, because it binds only one antigenic determinant. The use of tags, however, leads to a complex having an expanding structure if more than one are used in the same mixture. If an antigenic determinant 'A' is bound by a scFv that is translationally fused with epitopes 'B ' and 'C ' , it could be detected with a mixture of two scFvs -anti 'B ' and -anti 'C ' that are also tagged with epitopes ' B' and 'C. In this case not a linear but an exponential amplifi- cation is achieved upon the binding of each 'next' scFv.

Since scFv fragments are recombinant molecules that can be genetically manipulated, most of these amplifications can be used to produce equivalent results as traditional methods, and allow certain improvements. The invention thus provides immunological reagents with polyclonal activity, by preparing mixtures of in vivo labeled scFv fragments that bind different antigenic determinants. In this way a polyclonal antibody can be mimicked by using such a mixture of scFvs. Further the invention provides immunological reagents that recognise a primary antibody, by making use of a mixture of in vivo labeled scFvs. The invention also provides multivalently labeled immunological reagents by integrating more copies of the gene encoding the reporter molecule in a single overexpression construct. Experimental part

Construction of a single chain Fv fragment

In order to study the expression and functionality of any scFv, fused to either the truncated version of the Anabaena variabilis petH gene encoding the ferredoxin NADP oxidoreductase or the gfp gene encoding the Aeguora Green Fluorescent Protein, it was necessary to obtain such a fragment with binding affinity for a known antigen. We selected scFv directed against ferredoxin NADP oxidoreductase from the cyanobacterium Synechocystis PCC 6803 from a library of semisynthetic human scFv fragments with designed antigen binding sites. In short, the choice of starting material for this library is based on the observation of immunologists of Cambridge University (England) that the structural repertoire of the human VH genes contains only 50 different types as far as the Complementarity Determining Regions (CDRs) CDR1 and CDR2 are concerned. This was concluded from both sequence data collected from one individual combined with the high resolution structural information that has been gathered by crystallographers . The semisynthetic library consisted of 50 representative human VH gene fragments that were fused to a synthetically randomised CDR3 (the hypervariable region) region ranging in length from 4 to 12 residues to produce a degenerate VH library with a potential size of approximately 10¹⁷ members. These were fused to a 'dummy' light chain obtained from a bovine serum albumine binding monoclonal IgG molecule. From this library functional scFvs were isolated by affinity selections by Nissim, A. et al . (1994). They can therefore be used in a model system as ' any functional scFv' . By the very nature of these synthetic binding domains combined with a human framework sequence, it was possible to isolate binders from this library directed against human antigens, resulting in anti-self antibodies. Furthermore these scFvs are of only moderate binding properties, comparable with primary- respons antibody affinities, thereby showing that no exceptio- nally strong binder is necessary tc demonstrate the use of the in vivo labeled scFv fragments as described in this text.

The sequence of the petH gene encoding the protein antigen when expressed in the cytoplasm of E. coli , is available on the internet by using the 'Cyanobase' sequence retrieval programs. The gene product is enzymatically very similar to the Anabaena petH fusion partner studied as the chromogenic tag, whose basic enzymology is described in this text as well.

The scFv 'mab4' directed against Synechocystis PCC 6803 ferredoxin-NADP⁺ oxidoreductase is of the type VH-linker-VL, with the linker sequence (Gly₄-Ser)₃, a sequence which can be cloned in frame using the 5 ' Sfil and 3 ' Ndtl restriction sites in the cytoplasmic and periplasmic expression vectors described in this text. For convenience it is noted that any scFv-coding sequence that is produced with the Pharmacia recombinant phage display kit, can be cloned in frame in the expression constructs described in this text, using the same 5' Sfil and 3' Notl restriction sites. Actually, the expression vector sold by Pharmacia, pCANTABl, is based on the pHENl vector that was constructed to clone and express the library from which (mab4) originates. Any other scFv fragment that does not have these convenient restriction sites can easily be modified for in frame cloning by a skilled worker, for example by the introduction of suitable Sfil and Wbtl restriction sites by polymerase chain reaction-based utagenesis. The nucleotide sequences of the expression constructs are given in figures 2 and 3.

Construction of the fusion vectors The cytoplasmic expression vectors that we have used are based on the Quiagens PQE30 series, which contain a strong T5 viral promoter that can be repressed by the overproduction of Lacis. The latter is achieved by co-replication of Lacl<3 expressed from E. coli plasmid pREP4. Cloning of an Open Reading Frame into this polylinker results in the translational fusion of 6 histidine residues at the N-terminus of the overexpressed protein. The promoter region contains two binding sites for the Lac repressor, and expression, when glucose is added to a culture, is additionally repressed. The polylinker of pQE30 was replaced essentially by the polylinker of pHENI. A 140 bp product generated with pHENI as the template was amplified by PCR using the oligonucleotides 'phenbam' and 'phensac'. The 5' end of this polylinker fragment now incorporates a BamHI site for in-frame cloning in the expression plasmid, and the 3' end incorporates a Sacl restriction site, also present in the polylinker of pQE30. The PCR fragment was digested with SairiHI and Sacl and cloned into the BamRl and Sacl sites of pQE30.

The resulting plasmid, pHKI was used to generate both the ferredoxin-NADP⁺ oxidoreductase-fused and the green fluorescent protein-fused expression vectors, both incorporating the N-terminally placed histidine-tag.

Only a partial fragment of the Anabaena variabilis petH gene was used in the fusion vectors: at the N-terminus of its open reading frame a domain is found that has no catalytic function, but is involved in directing the enzyme to a specific location within the cytoplasm of the cyanobacterial cell. Since it has been shown in ur laboratory that the Synechocystis enzyme in such an N-terminally truncated form can be overproduced in E. coli , yielding a fully functional reductase, we inferred that an N-terminally truncated form of the homologous Anabaena enzyme would also be functional.

Furthermore it has been known from the literature that the purified native enzyme usually looses the N-terminal domain as well, as a result of proteolysis, and still is fully active as a reductase. For use of this truncated enzyme in periplasmic expression, it was also necessary to remove the N-terminal domain, since this domain contains a number of positively charged residues that hinder the translocation of the fusion protein over the plasma membrane. The C-terminal part of the gene, encoding both the cofactor (FAD) and substrate (NADPH) binding domains was amplified by use of PCR with the oligonucleotides ' anapetkpn ' ( 5 ' ) and 'cpethind ' ( 3 ' ) . This fragment was digested with Kpnl and HindiII and cloned into the Kpnl and Hindlll sites of pHKl, to produce pHK3. This vector is shown in figure 1 and accepts ' any ' in frame scFv encoding sequence when cloned into it as a Sfil/Notl fragment. Without this cloning procedure induction of an E. coli strain harbouring this plasmid leads to the cytoplasmic over- expression of the N-terminally truncated reductase, fused at the N-terminus with respectively a histidine tag, a short nonsense peptide encoded by the polylinker, and an epitope- coding sequence called 'c-myc'. This epitope sequence could function as a 'linker' between the scFv and the reductase, but is meant to study the expression of the chimeric proteins when detected in western blot by the mouse monoclonal antibody 9E10. When an scFv coding sequence is introduced into this plasmid, it will replace the nonsense sequence in pHK3. This was done with the DNA fragment coding for the scFv directed against Synechocystis ferredoxin-NADP⁺ oxidoreductase 'mab4', resulting in construct pHK4.

The tag- and domain structure of the chimeric protein would than become (from N- to C-terminus): (His)e-VH-linker- VL-myc-FAD-NADPH. The order of these domains can also be selected in a very different way, like the reductase being placed N-terminally of the scFv fragment, or on any other side. scFvs with a reversed order of appearance of variable genes: VL-linker-VH, have been successfully produced. Also, domains can be left out at will, for instance, constructs without for example VI and/or myc domains also encode functional immmunological reagents. Also, a number of other functional linker sequences, other than the (Gly₄Ser)₃ linker, have been described. These types of nucleic acid molecules encoding in vivo labeled scFv can also be cloned into pHK3, after suitable restriction sites are introduced. The NADPH binding pocket of the reductase allows purification of the fusion protein on Red Sepharose, an affinity matrix that is known to bind NAD(P)H dependent enzymes. Therefore, the histidine tag that was introduced to facilitate purification by Metal-Chelate Affinity Chromatography, is not an essential element of the construct. The histidine tag, however, can also be placed at the C-terminus or internally at a position chosen so that it is not buried inside the protein. A vector, pHK6, was generated based on pHK3, to accept a Sfil/Notl scFv encoding fragment for the expression and trans- location of the scFv-reductase fusion protein to the periplasm. To this end three fragments were ligated to produce pHK6. The first DNA fragment contains the polylinker and the petH ' sequence and was isolated as a 1 kbp Sfil/Hindlll fragment. The second DNA fragment was generated by PCR with the vector pHENI as template, using the primers 'phenbam' and 'phensac'. This fragment was also restricted with Sfil and Hindlll and contains the leader sequence 'PelB'. The lac promoter and the translational signals were provided by pUC19, that was digested with HindiII to accept the other two fragments . In the resulting construct, pHK6, the ' mab4 ' coding sequence was cloned to produce pHK7, a high copy number plasmid (due to the use of pUC19) that overexpresses a chimeric protein with a domain structure PelB-VH-linker-VL- myc-FAD-NADPH. The PelB leader is cleaved off by the enzyme 'signal peptidase' after translocation of the construct over the cytoplasmic membrane by the secretory machinery (Sec) of E. coli , resulting in a domain structure VH-linker-VL-myc-FAD- NADPH. This protein accumulates in the periplasm and can be isolated by affinity chromatography with Red Sepharose from the periplasmic cell fraction.

For the cytoplasmic overexpression of the green fluorescent protein-labeled scFv, the gfp gene was cloned into the polylinker of pHKI as a Kpnl/Smal fragment. The gfp containing plasmid pGFP was purchased from Clonetech. An 0.7 kbp pnl/Hindlll fragment encoding gfp was first cloned into pBluescript-ll KS+, to add the suitable Smal restriction site at the 3' end of the gene, in order to clone it directionally into pHKI, which resulted in pHK2. The (mab4) coding fragment was cloned as a Sfil /Notl fragment into this construct to produce pHK5.

The resulting domain structure of this fusion protein is (His)₆-VH-linker-VL-myc-GFP. Since it has been shown that GFP can also be fused as an N-terminal fusion partner, a chimeric product with a reversed domain structure, or with the GFP on either side of the scFv fragment, could also be expressed.

A vector for the periplasmic expression of the scFv-GFP was based on pHK2. The polylinker sequence and the gfp coding region was excised from pHK2 as a 0.8 kbp Sfil/EcoRI fragment and ligated to the 3.2 kbp Sfil/EcoRI fragment of pHENI, resulting in pHK8. The 'mab4' coding fragment was cloned as a Sfil/Notl fragment into this construct to produce pHK9. This construct is a high copy number plasmid (based on pUC119) that overexpresses a chimeric protein with a domain structure PelB- VH-linker-VL-myc-GFP, that looses its N-terminal PelB leader sequence after translocation to the periplasm.

Cytoplasmic expression of scFv/reductase fusion construct

Two strains of E. coli were tested for their expression levels of fusion protein. Strains M15 and BMH71-18 were both co-transformed with pHK4, encoding the (His)₆-VH-linker-VL- myc-FAD-NADPH fusion protein, and pREP4, encoding the lac- repressor. Both strains were grown overnight in the complex

Luria-Bertani (LB) medium, containing 100 μg/ml ampicillin and 20 μg/ml kanamycin. These cultures were diluted 10-fold in LB medium containing only 100 μg/ral ampicillin. The growth was followed by reading the absorbance at 600 nm of the culture. Expression was induced at OD₆oo=0.7 by the addition of 1 mM of IPTG. induction was allowed to proceed for another 3 hours, at which point the cultures were harvested at an OD₆oo=l«4. The continuation of growth of the cultures after induction indicates that these expression levels are not toxic to the cell. Phase contrast microscopy (not shown) showed the formation of inclusion bodies inside the cytoplasm of both cultures upon induction, indicating that the overexpressed fusion protein precipates in the cytoplasm. Also, a slight elongation of the cell morphology was observed, which is not uncommon as a result of overexpression conditions.

The chimeric protein was visualised in SDS-PAGE by staining with Coomassie Blue (total protein stain) and by immunoblots (Western blots) with rabbit-anti-Anai>aena variabilis ferredoxin-NADP⁺ oxidoreductase antibody, which exclusively stains the fusion protein. The results are shown in figures 4 and 5. The arrow indicates the position of the protein in both the Coomassie stained gel and in the immunoblot. hen compared with the protein composition of plasmid-free M15 and BMH71-18, an additional band with the correct molecular mass is observed only in strain BMH71-18. The immunostaining, however, shows the overexpression in both strains. From the Coomassie stained gel it can clearly be concluded that expression levels are significantly higher in strain BMH71-18 than in M15. Immunoblot analysis of cell fractions showed that indeed in these strains all the material is present in the membrane fraction, and no signal can be observed in the soluble fraction (data not shown).

The purpose of this experiment was to show that expression levels in insoluble form in the cytoplasm reach very high levels provided that a selection for the most suitable strain is done. In our hands, the BMH71-18 strain performed best, but additional strains could be selected that reach even higher levels of expression. In this regard reference is made to non- K12 E. coli strains such as the TOPP cells (Stratagene). It should also be noted that expression technology of scFvs has been optimized by industry, and refolding protocols on an industrial scale of scFvs from inclusion bodies have been described. Periplasmic expression of reductase-fused scFv

Three E. coli strains, XLI-Blue, M15 and BMH71-18, were tested for their capacity to express the scFv-fusion construct mab4 in their periplasm. Plasmid pHK7 , encoding the PelB-VH- linker-VL-myc-FAD-NADPH chimeric protein, was originally constructed in E. coli XLI-Blue. Strains that were analysed to harbour the correct construct after ligation and tranformation had a clear phenotype, especially in liquid medium, even when additional glucose was supplied to the cultures to repress 'leaky' expression from the lac-promoter. Also an exceptional filamentous cell morphology was observed, indicative of the presence of a toxic gene construct. However, when the pHK7 construct was extracted from these cells and transformed into Ml5 and BMH71-18, this phenotype was not observed. Ml5 and BMH71-18 were co-transformed with pHK7 and pREP4 and grown in overnight cultures of LB medium containing 100 μg/ml Ampicillin and 20 μg/ml kanamycin. These cultures were diluted 10-fold in LB medium, containing only 100 μg/ml ampicillin. Their growth was followed by reading the absorbance at 600 nm of the culture. Expression was induced at OD_δoo = 0.7 by the addition of 1 mM IPTG. Induction was allowed to proceed for another 3 hours. Both strain BMH71-18 and strain M15 continued growing after induction, indicating that the phenotype of XLI-Blue (pHK7) was absent in these strains. Extracts of the periplasmic space of the induced cultures were prepared, using a conventional sucrose/EDTA treatment. SDS-PAGE analysis showed only a small amount of the fusion protein to be present in total protein extracts of both induced strains and a visible band was present in the peri- plasmic extract of strain BMH71-18 (pHK7) (pREP4), and not in M15 (pHK7) (pREP4) by Coomassie staining. The results are shown in figures 6 and 7. Western blot analysis of total E. coli proteins, using the rabbit-anti-Ana aena variabilis ferredoxin-NADP+ oxidoreductase antibody, shows both the processed fusion protein (lower arrow) and a small amount of the unprocessed protein (upper arrow) . This difference of molecular mass is due to the cleavage of the N-terminal pelB leader peptide by signal peptidase in the periplasmic space. Clearly the larger part of the expressed protein in BMH71-18 is translocated into the periplasm where it is fully soluble (lane 3). In the periplasmic extract of Ml5 (pHK7) (pREP4) the fusion protein cannot be detected by Western blot analysis. Upon further analysis of the presence of the fusion product in the supernatant, it was found that after 8 hours of induction reductase-fused scFv could be detected at a concentration of approximately 2mg/ml.

The purpose of this experiment was to show the functionality of the fusion protein by using soluble material from the periplasm. As expected the expression levels are lower than the cytoplasmic expression levels, but can be used directly to study the functionality. These expression levels of native protein are limited to the volume of the periplasm, and native expression levels could be increased by overexpressing the fusion proteins in Gram-negative bacteria such as Acinetobacter sp. or Pseudomonas sp. that are able to actively transport proteins over the outer membrane as well, resulting in the accumulation of material in the extracellular supernatant. For this purpose motifs that are recognised by the Xcp machinery should be added to the constructs.

Purification and activity of the recombinant protein

An extraction of the periplasmic fraction was performed with cells harvested from 1 liter of induced culture of BMH71- 18 (pHK7) (pREP4). Proteins were precipitated from the resulting preparation by the addition of 80% of ammonium sulfate. The pellet was taken up in a buffer containing 50 mM Tris pH 8 + 50 mM NaCl + 1 _μM FAD and desalted on a Sephadex G 50 column. Fractions that were visibly yellow, as a result of binding of FAD by the flavoprotein, were stored at -20°C after addition of 30% of glycerol. This procedure results in the preparation of a concentrated extract of the periplasmic proteins, where the FNR activity iε reconstituted by the addition of free FAD.

The presence of the fusion protein could be detected in ELISA in which the coated antigen was detected by the fusion protein, and it was shown that the fusion protein was able to detect the antigen in Western blot, by means of its diaphorase activity, employing the reaction mixture described below.

50 mM Tris.HCl pH = 8 1 mM NADPH

2 mM INT (from stock solutions in dimethyl formamide)

10 mM Glucose-6-phosphate

5 U/ml Glucose-6-phosphate dehydrogenase (from Boehringer).

Alternatively, the scFv-FNR fusion protein can be purified on a Red Sepharose column. The results are shown in figure 7. This chromatographic procedure is based on the affinity that NAD(P)H dependent enzymes have for this column material. After extraction of the periplasmic proteins they were dialysed in the presence of 0.5 mM of PMSF against a glycine buffer (50 mM glycine, 50 mM KC1). A 1 ml Red Sepharose was equilibrated with the glycine buffer. Proteins were loaded on the column, washed with glycine buffer, and eluted with a gradient of 0-lM of KC1 in glycine buffer. Fraction 3 contained the purified scFv-FNR fusion protein, as visualised by SDS-PAGE and assaying the diaphorase activity.

The diaphorase activity of the enzyme can be detected by the reduction of tetrazolium salts. Several of these salts were tested for their ability to be reduced by the reductase. Tetrazolium blue, nitrotetrazolium blue and iodotetrazolium violet all become visibly colored (blue, blue and violet, respectively) upon reduction, whereas the oxidized materials are virtually colorless or slightly yellowish. Of these three, iodotetrazolium blue (INT) was more readily reduced by the reductase than the other three. For the detection of the enzyme activity, both in ELISA and in Western blot, INT is reduced by electrons liberated from NADPH, catalysed by the reductase. Subsequently, NADP⁺ can be re-reduced by the enzyme glucose-6-phosphate dehydro- genase (G-6-P DH), which is an NADP+ oxidoreductase. If G-6-P DH and G-6-P are added to the reaction mixture the components do not become exhausted, and replace the requirement of high concentrations of NADPH in the reaction mixture.

For both ELISA and Western blots, a possible reaction mixture which allows detection of diaphorase activity is formulated as follows:

50 mM Tris.HCl pH = 8 1 mM NADPH 2 mM INT (from stock solutions in dimethyl formamide) 10 mM Glucose-6-phosphate 5 U/ml Glucose-6-phosphate dehydrogenase (from Boehringer).

Absolute activities of enzyme-labeled scFvs When scFv fragments become monovalently labeled with ferredoxin-NADP⁺ oxidoreductase a a result of a translational fusion, the resulting specific activity can be calculated to be appr. 50 U/mg. However, this unit definition is based upon the amount of substrate (NADPH) that is oxidised according to the following equation:

NADPH + H⁺ + n AQ_X -> NADP⁺ + n A_red

If tetrazolium salts can serve as the oxidant in these reactions, then it has to be taken into account that n=2 for these reactions. Therefore the rate of reduction of these compounds of 1 unit of FNR equals the rate of for instance 2 units of alkaline phosphatase.

The specific activities of commercially available chemically labeled protein G, protein A or antibody conjugates range from 300 to 1000 U/mg. Realistically this has to be compared with corresponding (apparent) value of 100 U/mg of FNR-labeled scFvs . Since scFv fragments are much smaller molecules than whole (complete) immunoglobine molecules, the specific activity as a function of the number of binding sites on tht antibody therefore will be comparable with these conjugates. For the detection of rare antigens (or detection with low affinity antibodies, Km > 10^-6) with labeled scFvs, methods are proposed for further amplification of the signal.

Fluorescent labeling

Fluorescent proteins can be translationally fused to scFvs: Good examples are green fluorescent proteins (GFP) from Aequorea victoria and Renyella and Photoactive yellow protein (PYP) from Ectothiorhodospira halophila . Recently the gene encoding the E. halophila Photoactive

Yellow Protein has been cloned and sequenced (Hoff, W. 1995). Studies of reconstitution of the chromophore on the over- expressed recombinant protein showed that fluorescent analogs of the p-coumaric acid can be linked to the apo-protein to produce a fluorescent holo-protein. These in vitro reconstitutions are specific for the unique cysteine residue in the protein. The reconstitution can be carried out by reacting the functional fragment apoform of the protein with the anhydride derivatives of these chromophores. These same couplings can therefore be performed by fusing one or more cysteine residues to the scFv coding sequence and performing the reconstitution in vitro with the desired chromophores. The colors as well as the fluorescent intensities can be varied in this way by choosing the correct coumarin analogue and the amount of cysteine residues to be coupled. The analogues studied in the photoactive yellow protein environment and their fluorescence properties are listed below. Compound Extinction λ^Max (nm) λ^Em (nm) (rcM'¹ .cm^{~ l} )

I 45.5 446 494 0.002 II 30 457 527 0.02

III 30 460 527 0.05

The above listed compounds are: (I): 4-hydroxy cinnamic acid; (II): 3,4-dihydroxy cinnamic acid and (III): 3-methoxy- 4-hydroxy cinnamic acid. Compound (I) is given mainly as a reference, since its quantum yield (Φ) is rather low. Compounds II and III, however, are of interest because of their resistance against photobleaching.

An other example of an 'activated' coumarin analogue is N-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3 ' -(2 ' - pyridyldithio)propionamide (AMCA-HPDP). This compound is sold by Pierce Chem. Comp. The resulting fluorophore absorbs at 345 nm and emits at 440-460 nm and has a high quantum yield and photostability. Also translational fusion constructs with phycobili- proteins can be constructed. Phycoerythrin (Absorption 450- 470 nm, Emission 574 nm) has already been used as a fluorescent marker by many researchers, and is sold as purified or conjugated isolate by companies such as Pierce. Depending on the source of isolation, phycoerythrin mostly contains the phycoerythrobilin chromophore, but can also contain phycourobilin. The chromophores are attached via one or two thioether linkages. This opens the possibility to covalently couple these chromophores via a thioether linkage to cysteine residues that are engineered as functional fragment into the coding frame of a scFv fragment. Figure legends

Figure 1 Physical maps of the cytoplasmic and periplasmic expression vectors of the FNR and GFP fusion constructs.

Figure 2 Sequence of the coding region of the cytoplasmic FNR fusion expression vector. Positions of motifs and restriction sites are given relative to the initiation codon.

Figure 3 Sequence of the coding region of the periplasmic FNR fusion expression vector. Positions of motifs and restriction sites are given relative to the initiation codon. Figure 4 Coomassie stained SDS-PAGE of total E. coli proteins. Lane 1: BMH 71-18 (pREP4) (pHK4), induced with IPTG. Lane 2: BMH 71-18 (contruct free). Lane 3:Ml5 (pREP4) (pHK4), induced with IPTG. Lane 4: Ml5 (construct free). The arrow indicates the position of the (visible only in lane 4) scFv- FNR fusion protein.

Figure 5 Western detection of the same SDS-PAGE gel shown in figure 4 using a rabbit anti Anabaena variabilis FNR polyclonal antiserum. The arrow indicates the position of the scFv-FNR fusion protein. Figure 6 Western detection of the periplasmic scFv-FNR fusion protein. Lane 1: Total proteins E. coli BMH 71-18 (pHK7), induced with IPTG. The upper arrow indicates the position of the PelB-scFv-FNR fusion protein not yet cleaved by the periplasmic signal peptidase. The lower arrow indicates the position of the cleaved, periplasmic, scFv-FNR fusion protein. Lane 2: Periplasmic extract from induced BMH 71-18 (pHK7). The arrow indicates the position of the cleaved, soluble, periplasmic scFv-FNR fusion protein.

Figure 7 Coomassie stained SDS-PAGE. Purification of the soluble periplasmic scFv-FNR fusion protein on Red Sepharose. Lane 1: BMH 71-18 (construct free). Lane 2: Total proteins E. coli BMH 71-18 (pHK7), induced with IPTG. Lane 3: Periplasmic extract from induced BMH 71-18 (pHK7). Lane 4: Fraction 2. Lane 3: Fraction 3. The arrow indicates the position of the purified protein. References

Carrier , A. et al . 1995.J. Immunol. Methods. 181: 177-186 Chalfie, M. et al . , 1994. Science 263: 802-805 Duenas, M. et al 1994. BioTechniques 16: 4760483

Hoff, W.D. (1995) Photoactive Yellow Protein. PhD. Thesis, University of Amsterdam.

Karplus, P.A. and Bruns, CM. 1994. J. Bioenerg. Biomembr. 26: 89-99 Kok, et al . 1995. Mol. Microbiol.15: 803-818 Nissim, A. et al . 1994. EMBO J. 13: 692-698 Whitlow, M and Filpula, D. 1991. Methods: A companion to methods in Enzymology 2: 97-105

Claims

1. A recombinant nucleic acid molecule comprising at least one nucleic acid sequence encoding a variable domain, or a fragment thereof, of a heavy and/or light chain of an antibody molecule, and a nucleic acid sequence encoding a functional reporter molecule or functional fragment thereof, or encoding an attachment moiety for attachment of a reporter molecule.

2. A recombinant nucleic acid molecule according to claim 1 wherein the reporter molecule is an enzyme.

3. A recombinant nucleic acid molecule according to claim 2 wherein the enzyme is selected from any of the following enzymes; ╬▓-galactosidase, luciferase, lipases, esterases or oxidoreductases including peroxidases and haloperoxidases

4. A recombinant nucleic acid molecule according to claim 2 wherein the enzyme is selected from reductase homologues such as the FMN-flavoproteins and NADH-dependent reductases.

5. A recombinant nucleic acid molecule according to claim 2 wherein the enzyme is PetH.

6. A recombinant nucleic acid molecule according to claim 1 wherein the reporter molecule is a fluorescent protein.

7. A recombinant nucleic acid molecule according to claim 6 wherein the fluorescent protein is the Green fluorescent protein or a phycobiliprotein or the Photoactive Yellow protein.

8. A recombinant nucleic acid molecule according to any of claims 1 to 7 wherein the antibody molecule is specifically directed against the reporter molecule.

9. A recombinant nucleic acid molecule according to claim 1 wherein the attachment moiety comprises an amino acid residue allowing attachment of a fluorescent chromophore.

10. A host cell comprising a nucleic acid molecule according to any of claims 1 to 9.

11. An immunological reagent obtained by expression of a nucleic acid molecule according to any of claims 1-8 in a host cell.

12. An immunological reagent obtained by expression of a nucleic acid molecule according to claim 9 in a host cell and attachment of a fluorescent chromophore.

13. Use of an immunological reagent according to claim 11 or claim 12 in a detection assay or kit.