A BIFUNCTIONAL OR BIVALENT ANTIBODY FRAGMENT ANALOGUE

Abstract: The invention relates to a bispecific or bivalent antibody fragment analogue comprising a binding complex containing two polypeptide chains, whereby one polypeptide chain comprises two heavy chain variable domains (VH) in series and the other polypeptide chain comprises two light chain variable domains (VL) in series, the binding complex further containing two pairs of variable domains (VH-A//VL-A and VH-B//VL-B). The two VH's and/or the two VL's are connected directly or via an intermediate peptide linker. Also a production method for such antibody fragment analogues is disclosed.

Description: Title: A bifunctional or bivalent antibody fragment analogue The invention relates to new bispecific or bivalent antibody fragment analogues, a process for preparing such antibody fragment analogues and various uses of such antibody fragment analogues. Background of the invention and prior art 1. Antibody structure Antibody molecules typically are Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions located in their V- domains (see Figure 1) . In this specification abbreviations are used having the following meaning. C-domain: Constant domain V-domain: Variable domain V L : Variable domain of the light chain V H : Variable domain of the heavy chain Fv : dual chain antibody fragment containing both a V H and a V L scFv : single-chain Fv (V H and V L genetically linked either directly or via a peptide linker) CDR : Complementarity-determining region ELISA : Enzyme Linked Immuno Sorbent Assay PCR : Polymerase Chain Reaction IPTG IsoPropyl-β-ThioGalactopyranoside PBS Phosphate Buffered Saline PBST Phosphate Buffered Saline with 0.15% Tween TMB 3,3' ,5,5' -TetraMethylBenzidine It is generally known that proteolytic digestion of an antibody with papain yields three fragments. The fragment containing the CH 2 and CH 3 domains of the two heavy chains connected by the complete hinge (see Figure 1) crystallises very easily and was therefore called Fc fragment. The two other fragments are identical and were called Fab fragments, as they contained the antigen-binding site. Digestion with pepsin is such that the two Fab's remain connected via the hinge, forming only two fragments: Fc' and Fab 2 . The Fv is the smallest unit of an antibody which still contains the complete binding site (see Figure 1) and full antigen binding activity. It consists of only the V-domains of the heavy and light chains thus forming a small, heterodimeric variable fragment or Fv. Fv's have a molecular weight of about 25 kD, which is only one sixth of the parent whole antibody (in the case of an IgG) . Previously Fv's were only available by proteolysis in a select number of cases (Givol, 1991) . The production of Fv's can now be achieved more routinely using genetic engineering methods through cloning and expressing DNA encoding only the V-domains of the antibody of interest. Smaller fragments, such as individual V-domains (Domain Antibodies or dABs, Ward et al . , 1989) , and even individual CDR's (Williams et al . , 1989; Taub et al . , 1989) were shown to retain the binding characteristics of the parent antibody. However, this is not achievable on a routine basis: most naturally occurring antibodies need both a V H and a V L to retain full immunoreactivity. For example, in the case of V H D1.3 (Ward et al . , 1989) , although it still binds hen egg lysozyme (HEL) with an affinity close to that of the parent antibody, it was shown that loss of specificity was observed in that it can no longer distinguish turkey lysozyme from HEL, whereas the Fv can (Berry and Davies, 1992) . Although murine dABs can be obtained more routinely from spleen libraries (Ward et al . , 1989) , the approach is unsustainable because of the many problems associated with their production and physical behaviour: expression is extremely poor, affinity tends to be low, stability and solubility in water is low, and non¬ specific binding is usually very high. According to the literature a possible explanation of these undesirable characteristics is the exposure of the hydrophobic residues which are normally buried in the V H -V L interface. The exposed hydrophobic patches are thought to contribute to aggregation of the protein inside the cells and/or in the culture medium, leading to poor expression and/or poor solubility (Anthony et al . , 1992; Ward et al . , 1989) . The hydrophobic patches can also explain the high non-specific binding described by Berry and Davies, 1992. These problems clearly limit the usefulness of these molecules. Most of the Camelid antibodies appear to be an exception to this rule in that they only need one V-domain, namely V H , to specifically and effectively bind an antigen (Hamers- Castermans et al . , 1993) . In addition, preliminary data indicate that they seem not to suffer from the disadvantages of mouse dABs, as these camelid antibodies or fragments thereof are soluble and have been shown to express well in yeast and Aspergillus moulds. These observations can have important consequences for the production and exploitation of antibody-based products, see patent application WO 94/25591 (UNILEVER et al . , first priority date 29.04.93) . 2. Production of antibody fragments Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli (Better et al . , 1988, Skerra and Plϋckthun, 1988, Carter et al . , 1992) , yeast (Horwitz et al . , 1988) , and the filamentous fungus Trichoderma reesei (Nyyssδnen et al . , 1993) has been described. The recombinant protein yields in these alternative systems can be relatively high (1-2 g/1 for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al . , 1992) , or at a lower level, e.g. about 0.1 mg/1 for Fab in yeast in fermenters (Horwitz et al . , 1988) , and 150 mg/1 for a fusion protein CBHI-Fab and 1 mg/1 for Fab in Trichoderma in fermenters (Nyyssδnen et al . , 1993) and such production is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO) . Although the latter can give yields of the order of 1 g/1 in high cell density fermentation, it is a time-consuming and very expensive manufacturing method resulting in a cost price of about 1000 £/gram of antibody. It was further demonstrated that plants can be used as hosts for the production of both whole antibodies (Hiatt et al . , 1989) and scFv's (Owen et al . , 1992, Firek et al. , 1993) , whereby yields of upto 0.5% of the total soluble protein content in tobacco leaves were mentioned. The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a V H and a V L can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv (Bird et al . (1988) , Huston et al . (1988) , and granted patent EP-B-0281604 (GENEX/ENZON LABS INC.; first priority date 02-09-1986) . 3. Bivalent and bispecific antibodies and antibody fragments The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the V H of an Fv (Cumber et al . , 1992) , or at the C-terminus of the V L of an scFv (Pack and Plϋckthun, 1992) , or through the hinge cysteine residues of Fab's (Carter et al . , 1992). Another approach to produce bivalent antibody fragments is described by Kostelny et al . (1992) and Pack and Plϋckthun (1992) and is based on the inclusion of a C-terminal peptide that promotes dimerization. When two different specificities are desired, one can generate bispecific antibody fragments. The traditional approach to generate bispecific whole antibodies was to fuse two hybridoma cell lines each producing an antibody having the desired specificity. Because of the random association of immunoglσbulin heavy and light chains, these hybrid hybridomas produce a mixture of up to 10 different heavy and light chain combinations, only one of which is the bispecific antibody (Milstein and Cuello, 1983) . Therefore, these bispecific antibodies have to be purified with cumbersome procedures, which considerably decrease the yield of the desired product. Alternative approaches include in-vitro linking of two antigen specificities by chemical cross-linking of cysteine residues either in the hinge or via a genetically introduced C-terminal Cys as described above. An improvement of such in vitro assembly was achieved by using recombinant fusions of Fab's with peptides that promote formation of heterodimers (Kostelny et al . , 1992). However, the yield of bispecific product in these methods is far less than 100%. A more efficient approach to produce bivalent or bispecific antibody fragments, not involving in vitro chemical assembly steps, was described by Holliger et al . (1993). This approach takes advantage of the observation that scFv's secreted from bacteria are often present as both monomers and dimers. This observation suggested that the V H and V L of different chains can pair, thus forming dimers and larger complexes. The dimeric antibody fragments, also named "diabodies" by Hollinger et al . , in fact are small bivalent antibody fragments that assembled in vivo. By linking the V H and V L of two different antibodies 1 and 2, to form "cross-over" chains V H 1V L 2 and V H 2-V L 1 (see Figure 2B) , the dimerisation process was shown to reassemble both antigen-binding sites. The affinity of the two binding sites was shown to be equal to the starting scFv's, or even to be 10-fold increased when the polypeptide linker covalently linking V H and V L was removed, thus generating two proteins each consisting of a V H directly and covalently linked to a V L not pairing with the V H (see Figure 2C) . This strategy of producing bispecific antibody fragments was also described in several patent applications. Patent application WO 94/09131 (SCOTGEN LTD; priority date 15.10.92) relates to a bispecific binding protein in which the binding domains are derived from both a V H and a V L region either present at two chains or linked in an scFv, whereas other fused antibody domains, e.g. C- terminal constant domains, are used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY / MEDICAL RESEARCH COUNCIL ; first priority date 04.12.92) relates to a polypeptide containing a V H and a V L which are incapable of associating with each other, whereby the V-domains can be connected with or without a linker. Mallender and Voss, 1994 (also described in patent application WO 94/13806; DOW CHEMICAL CO; priority date 11.12.92) reported the in vivo production of a single-chain bispecific antibody fragment in E. coli . The bispecificity of the bivalent protein was based on two previously produced monovalent scFv molecules possessing distinct specificities, being linked together at the genetic level by a flexible polypeptide linker. The thus formed V H 1- 1inker-V L 1-1inker-V H 2-1inker-V L 2 fragment (see Figure 2A) was shown to contain both antigen binding specificities 1 and 2. (1= anti-fluorescein, 2= anti-single-stranded DΝA) . Traditionally, whenever single-chain antibody fragments are referred to, a single molecule consisting of one heavy chain linked to one (corresponding) light chain in the presence or absence of a polypeptide linker is implicated. When making bivalent or bispecific antibody fragments through the 'diabody' approach (Holliger et al . , (1993) and patent application WO 94/09131) or by the 'double scFv' approach (Mallender and Voss, 1994 and patent application WO 94/13806) , again the V H is linked to a (the corresponding) V L . It is realised that claims 32 and 33 of patent application WO 93/11161 (ENZON INC.; priority date 25.11.91) and the corresponding passages in that specification on page 22, lines 1-10 may read on a polypeptide comprising two V L 's fused together via a flexible polypeptide linker, and on a polypeptide comprising two V H 's fused together via a flexible polypeptide linker, respectively. However, no examples were given to substantiate this approach, thus it was in fact a hypothetical possibility instead of an actually produced compound. A skilled person would not have expected that such approach would be viable for at least three reasons. Firstly, it is widely recognised that immunoglobulin heavy chains (excluding the above described camel immunoglobulins) have very limited solubility and spontaneously precipitate out of aqueous solution when isolated from their light chain partners. Secondly, several groups have shown (Ward et al . , 1989, Berry and Davies, 1992, and Anthony et al . , 1992) that expression of V H 's in the absence of V L 's is hampered by extremely poor yields of unstable product with many undesirable properties, e.g. non-specific binding. Thirdly in patent application WO 94/13804 it was described on page 31 lines 10-12, that in computer modelling experiments they could not model as heterodimers V H -V H and V L -V L given the constraints of short linkers. Thus the simple suggestion given in patent application WO 93/11161 is not an enabling disclosure leading a skilled person to try with a reasonable expectation of success whether such suggestion would work; therefore, that patent application should not be considered as relevant prior art for the present invention. Summary of the invention The present invention provides a bispecific or bivalent antibody fragment analogue, which comprises a binding complex containing two polypeptide chains, one of which comprises two times a variable domain of a heavy chain (V H ) in series and the other comprises two times a variable domain of a light chain (V L ) in series. In one aspect of the invention one chain of the antibody fragment analogue comprises a first V H (V H -A) connected to a second V H (V H -B) and the other chain comprises a first V L (V L -A) connected to a second V L (V L -B) . In a preferred embodiment of this aspect one chain comprises a first V H (V H -A) followed bv a second V H (V H -B) , thus [V H -A * V H -B] , and the other chain comprises a first V L (V L -A) preceded by a second V L (V L -B) , thus [V L -B * V L -A] . For some embodiments of this aspect the two V H 's are directly connected to each other, but for other embodiments of this aspect of the invention the two V L ' s are directly connected to each other._ According to another embodiment of this aspect of the invention the two V H 's are connected to each other by a linker and also the two V L 's are connected to each other by a linker. Such a linker usually comprises at least one amino acid residue. According to a special embodiment of this aspect of the invention one chain comprises a first V H (V H -A) followed by a second V H (V H -B) , thus [V H -A * V H -B] , and the other chain comprises a first V L (V L -A) followed by a second V L (V L -B) , thus [V L -A * V L -B] , and in which the two V H 's are connected to each other by a linker and also the two V L 's are connected to each other by a linker, whereas each linker comprises at least 10 amino acid residues. According to the above aspect of the invention with A being different from B there are provided bispecific antibody fragment analogues. According to another aspect of the invention the specificities A and B are the same resulting in a bivalent antibody fragment. According to a further aspect of the invention the bispecific or bivalent antibody fragment analogues can be used in a diagnostic technique or for immunoassays, in a purification method, for therapy, or in other methods in which immunoglobulins or fragments thereof are used. Such uses are well-known in the art. The invention also provides a process for producing the antibody fragments of the invention in that a host is transformed by incorporating into that host a DNA encoding the two V H 's with or without a connecting linker and a DNA encoding the two V L 's with or without a connecting linker. Preferably the two DNA's are placed in a dicistronic arrangement. It is also possible that the two linked V H 's and the two linked V L 's are produced separately by different hosts, after which the linked V H 's produced by one host can be combined with the linked V L 's produced by the other host. The hosts can be selected from the group consisting of prokaryotic bacteria of which examples are Gram-negative bacteria, e.g. E. coli , and Gram-positive bacteria, e.g. B . subtilis or lactic acid bacteria, lower eukaryotes examples of which are yeasts, e.g. belonging to the genera Sac char omyce s , Kluyveromyces , or Trichoderma, moulds, e.g. belonging to the genera Aspergillus and Neurospora, and higher eukaryotes, examples of which are plants, e.g. tobacco, and animal cells, examples of which are myeloma cells and CHO cells. The techniques to transform a host by genetic engineering methods in order to have a desirable polypeptide produced by such host are well-known to persons skilled in the art as is evident from the literature mentioned above under the heading "Background of the invention and prior art". Brief description of the drawings Figure 1 depicts in schematic form the structure of a typical antibody (immunoglobulin) molecule. Figure 2 shows a schematic representation of published arrangements of heavy and light chain V-domain gene fragments that have been proven to produce bispecific antibody fragments. Figure 3 shows in diagrammatic form the suggested arrangement of the V-domains of a double head antibody fragment according to the invention with the V-domains in the following order: V H A-V H B + V L B-V L A. Figure 4 shows the nucleotide sequence of the EcoRI- H±ndlll insert of pUR.4124 containing DNA (see SEQ ID NO: 1) encoding V L Lys-Linker-V H Lys (see SEQ ID NO: 2) . Figure 5 shows the nucleotide sequence of the Hindlll- BcoRI insert of plasmid Fv.3418 (see SEQ ID NO: 3) containing DNA encoding pelB leader- V H 3418 (see SEQ ID NO: 4) and DNA encoding pelB leader-V L 3418 (see SEQ ID NO: 5) . Figure 6 shows the nucleotide sequence of the Hindlll- BcoRI insert of plasmid Fv.4715-myc (see SEQ ID NO: 6) containing DNA encoding pelB leader- V H 4715 (see SEQ ID NO: 7) and DNA encoding pelB leader-V L 4715-Myc tag (see SEQ ID NO: 8) . Figure 7 shows the nucleotide sequence of the Hindlll- EcoRI insert of scFv.4715-myc containing DNA (see SEQ ID NO: 9) encoding pelB leader- V H 4715-Linker-V L 4715-Myc tag (see SEQ ID NO: 10) . Figure 8 a/b shows the nucleotide sequence of the Hindlll- EcoRI insert of pGOSA.E (see SEQ ID NO: 11) containing DNA encoding pelB leader-V H 4715- Linker-V L 3418 (see SEQ ID NO: 12) and DNA encoding pelB leader-V L 3418-Linker-V H 4715 (see SEQ ID NO: 13) . Figure 9 gives an overview of the oligonucleotides and their positions in pGOSA.E that can be used to replace V-domain gene fragments. Figure 10 illustrates the amino acid sequence of the V H - V H and V L -V L domain junctions in fusion polypeptides GOSA.E (see amino acids 114-145 in SEQ ID NO: 12 and amino acids 102-128 in SEQ ID NO: 13) , GOSA.V (see SEQ ID NO: 30 and amino acids 102-128 in SEQ ID NO: 13) , GOSA.S (see amino acids 114-145 in SEQ ID NO: 12 and SEQ ID NO: 31) and GOSA.T (see SEQ ID NO: 30 and SEQ ID NO: 31) . Figure 11 shows the specificity of Streptococcus binding of scFv.4715-myc. Figure 12 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.E. Figure 13 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.V. Figure 14 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.S. Figure 15 shows the specificity of glucose oxidase targeting onto the surface of various Streptococcus strains by GOSA.T. Figure 16 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.E as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity. Figure 17 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.V as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity. Figure 18 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.S as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity. Figure 19 shows the results of an ELISA. Individual fractions of a gelfiltration experiment using partially purified GOSA.T as feedstock were tested for glucose oxidase and Streptococcus sanguis bispecific binding activity, Figure 20 shows the source of fragment PCR. I BstEII/SacI Figure 21 shows the source of fragment PCR. II Sfil/EcoRI Figure 22 shows the source of fragment PCR. Ill Nhel/ SacI Figure 23 shows the source of fragment PCR. IV Xhol/EcoRI Figure 24 shows the source of fragment PCR.V SalI/EcoRI Figure 25 shows the source of fragment PCR.VI Sfil/Nhel Figure 26 shows the source of fragment PCR.VII BstEII/Nhel Figure 27 shows the source of fragment PCR.VIII Xhol/EcoRI Figure 28 shows the source of fragment PCR. IX BstEII/Nhel Figure 29 shows the source of fragment PCR.X Ps l/EcoRI Figure 30 shows the construction of plasmid pGOSA.A Figure 31 shows the construction of plasmid pGOSA.B Figure 32 shows the construction of plasmid pGOSA.C Figure 33 shows the construction of plasmid pGOSA.D Figure 34 shows the construction of plasmid pGOSA.E Figure 35 shows the construction of plasmid pGOSA.V Figure 36 shows the construction of plasmid pGOSA.S Figure 37 shows the construction of plasmid pGOSA.T Figure 38 a/b shows the construction of plasmid pGOSA.G Figure 39 shows the construction of plasmid pGOSA.J Figure 40 shows the construction of plasmid pGOSA.Z Figure 41 shows the construction of plasmid pGOSA.AA Figure 42 shows the construction of plasmid pGOSA.AB Figure 43 shows the construction of plasmid pGOSA.L Figure 44 shows the construction of plasmid pGOSA.Y Figure 45 shows the construction of plasmid pGOSA.X Figure 46 shows the construction of plasmid pGOSA.AC Figure 47 shows the construction of plasmid pGOSA.AD. Table 1 shows the nucleotide sequence of the oligonucleotides used to produce the constructs described in this specification. Restriction sites encoded by these primers are underlined. Table 2 gives an overview of all GOSA constructs described in this specification. Table 2A describes intermediate constructs that were not further tested. Table 2B describes the dicistronic constructs. Table 2C describes the monocistronic constructs. Detailed description of the invention In this specification the construction of an antibody fragment analogue consisting of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains in either order. The variable domains are linked either directly or through a polypeptide linker. Subsequent molecular modelling of this combination suggested that the protein chains could fold such that both binding sites are fully accessible, provided that the connecting linkers are kept long enough to span 30 to 35 A. Whereas in patent application WO 93/11161 it is explicitly described that for the above described bispecific complexes two flexible polypeptide linkers in the self assembling complex are required, the present invention illustrated here describes in particular the construction of a two chain protein complex containing only one linker or no linkers at all. The latter antibody fragment analogue thus consists of a two chain protein complex containing one polypeptide chain comprising heavy chain V-domains fused directly together and another polypeptide chain comprising the corresponding light chain V-domains fused together, both fusions in the absence of linkers. But also two chain protein complexes in which each chain comprises a linker between the two variable domains can be used as antibody fragment analogues according to the invention as described below with construct pGOSA.E. However, the two chain complexes containing only one linker or no linker at all are preferred. The abbreviation GOSA used in this specification relates to a combination of glucose oxidase and Streptococcus sanguis. In this specification evidence is provided that these antibody fragment analogues ("double heads") contain both antigen binding specificities of the Fv's used to generate these bispecific antibody fragments. It is exemplified that these type of constructs according to the invention can be used to target the enzyme glucose oxidase to whole bacteria, using antibody fragments derived from hybridomas expressing antibodies directed against these antigens. The present invention is now described by reference to some specific examples, which are included for purposes of illustration only and are not intended to limit the scope of the invention. EXAMPLES General experimental Strains, Plasmids and Media All cloning steps were performed in E. coli JM109 (en Al, recAl, gyrA96, thi, iιsdR17(r κ , m κ + ) , relAl, supE44, A ( lac- proAB) , [F' , traD36, proAB, lacT q ZΔM15] . E. coli cultures were grown in 2xTY medium (16 g tryptone, 10 g yeast extract, 5 g NaCl per litre H 2 0) , where indicated supplemented with 2% glucose and/or 100 μg/ml ampicillin. Transformations were plated out on SOBAG plates (20 g tryptone, 5 g yeast extract, 15 g agar, 0.5 g NaCl per litre H 2 0 plus 10 mM MgCl 2 , 2% glucose, 100 μg/ml ampicillin) The expression vectors used are derivatives of pUC19. The oligonucleotide primers used in the PCR reactions were synthesized on an Applied Biosystems 381A DNA Synthesiser by the phosphoramidite method. Expression of GOSA constructs Colonies from freshly transformed JM109 plated onto SOBAG plates were used to inoculate 2xTY medium supplemented with 100 μg/ml ampicillin, 2% glucose. Cultures were shaken at 37°C to an OD^ in the range of 0.5 to 1.0. Cells were pelleted by centrifugation and the supernatant was removed. The pelleted cells were resuspended in 2xTY medium with 100 μg/ml ampicillin, 1 mM IPTG, and grown for a further 18 hours at 25°C. Cells were pelleted by centrifugation and the supernatant, containing the secreted chains, used directly in an ELISA. The proteins in the periplasm of the pelleted cells were extracted by resuspending the cell pellet in 1/20 of the original culture volume of lysis buffer (20% sucrose, 200 mM Tris-HCl pH 7.5 , 1 mM EDTA, 500 μg/ml lysozyme) . After incubation at 25°C for 20 minutes an equal volume of H 2 0 was added and the incubation was continued for another 20 minutes. The suspension was spun at 10.000 g for 15 minutes and the supernatant containing the periplasmic proteins was used directly in an ELISA. ELISA 96 well ELISA plates (Greiner HC plates) were activated overnight at 37°C with 200 μl/well of an 1/10 dilution of an over night culture of Streptococcus cells in 0.05 M sodium carbonate buffer at pH=9.5. Following one wash with PBST, the antigen sensitised plates were pre-blocked for 1 hour at 37°C with 200 μl/well blocking buffer (2% BSA, 0.15% Tween in PBS) . Samples containing 50 μl blocking buffer plus 50 μl culture supernatants or periplasmic cell extracts (neat or diluted with PBS) were added to the Streptococcus sensitised plate and incubated for 2 hours at 37°C. Following 4 washes with PBS-T, 100 μl of blocking buffer containing glucose oxidase (50 μg/ml) was added to every well. After incubation at 37°C for 1 hour unbound glucose oxidase was removed by 4 washes with PBS-T. Bound glucose oxidase was detected by adding 100 μl substrate to each well (70 mM Na-citrate, 320 mM Na-phosphate, 27 mg/ml glucose, 0.5 μg/ml HRP, 100 μg/ml TMB) . The colour reaction was stopped after 1 hour by the addition of 35 μl 2 M HCl and the A450 was measured (compare Figures 11/15) . Affinity purification of GOSA antibody fragments GOSA.E, GOSA.V, GOSA.S and GOSA.T were partially purified by affinity chromatography. 100 ml periplasmic extract of each of these constructs was loaded onto a Glucose-oxidase- Sepharose column (CNBr-Sepharose, Pharmacia) prepared according to the manufacturer's instructions. After extensive washes with PBS the bound GOSA antibody fragments were eluted in 0. IM glycine buffer at pH=2.8. The fractions were neutralised with Tris and analysed by polyacrylamide gel electrophoresis followed by silver staining and tested for the presence of double head activity. EXAMPLE 1. Construction of the pGOS double head expression vectors In this Example the construction of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains. The variable domains are linked either directly or through a polypeptide linker. The expression vectors used are derivatives of a pUC19 derived plasmid containing a Hiπdlll-EcoRI fragment that in the case of plasmid scFv.4715-myc contains a DNA fragment encoding one pelB signal sequence fused to the N-terminus of the V H that is directly linked to the corresponding V L of the antibody through a connecting flexible peptide linker, (Gly 4 Ser) 3 (present in SEQ ID NO: 2 as amino acids 109-123 and in SEQ ID NO: 10 as amino acids 121-135) , thus generating a single-chain molecule (see Figure 7) . In the dual-chain Fv and the pGOSA expression vectors, the DNA fragments encoding both the V H and V L of the antibody are preceded by a ribosome binding site and a DNA sequence encoding the pelB signal sequence in an artificial dicistronic operon under the control of a single inducible promoter (see Figures 5, 6 and 8) . Expression of these constructs is driven by the inducible lacZ promoter. The nucleotide sequence of the Hindlll-EcoRI inserts of the plasmids pUR.4124, Fv.3418, Fv.4715-myc and scFv.4715-myc constructs used for the generation of the bispecific antibody fragments are given in Figures 4-7, respectively. Moreover, a culture of E. coli cells harbouring plasmid scFv.4715-myc and a culture of E. coli cells harbouring plasmid Fv.3418 were deposited under the Budapest Treaty at the National Collection of Type Cultures (Central Public Health Laboratory) in London (United Kingdom) with deposition numbers NCTC 12916 and NCTC 12915, respectively. In agreement with Rule 28 (4) EPC, or a similar arrangement for a State not being a Contracting State of the EPC, it is hereby requested that a sample of such deposit, when requested, will be submitted to an expert only. The construction of pGOSA.E (see Figure 8 for the Hiπdlll- EcoRI insert of pUC19) involved several cloning steps. The appropriate restriction sites in the various domains were introduced by PCR directed mutagenesis using the oligonucleotides listed in Table 1 below. The pGOSA.E derivatives pGOSA.V, pGOSA.S and pGOSA.T with only one or no linker sequence are derived from the pGOSA.E construct by removing the linker sequences by means of PCR directed mutagenesis with oligonucleotides listed in Table 1 below. Table 1 . DBL. l 5'-CAC CAT CTC CAG AGA CAA TGG CAA G-3' (=SEQ ID NO: 14) DBL.2 5 -GAG CGC GAG CTC GGC CGA ACC GGC C'GA TCC GCC ACC GCC AGA GCC-3' ( = SEQ ID NO: 15) DBL.3 5' -CAG GAT CCG GCC GGT TCG GCC' CAG GTC CAG CTG CAA CAG TCA GGA-3' ( = SEQ ID NO: 16) DBL.4 5' -CTA CAT GAA TTC 2 GCT AGC 3 TTA TTA TGA GGA GAC GGT GAC GGT GGT CCC TTG GC-3' (=SEQ ID NO: 17) DBL.5 5'-TAA TAA GCT AGC 3 GGA GCT GCA TGC AAA TTC TAT TTC-3' (= SEQ ID NO: 18) DBL.6 5'-ACC AAG CTC GAG 4 ATC AAA CGG GG-3'(= SEQ ID NO: 19) DBL.7 5'-AAT GTC GAA TTC 2 GTC GAC 5 TCC GCC ACC GCC AGA GCC-3' (= SEQ ID NO: 20) DBL.8 5' -ATT GGA GTC GAC 5 ATC GAA CTC ACT CAG TCT CCA TTC TCC-3' (= SEQ ID NO: 21) DBL.9 5' -TGA AGT GAA TTC 2 GCG_GCC_GC 6 T TAT TAC CGT TTG ATT TCG AGC TTG GTC CC-3' (= SEQ ID NO: 22) DBL.10 5'-CGA ATT CGG TCA CC 8 G TCT CCT CAC AGG TCC AGT TGC AAC AG-3' (= SEQ ID NO: 23) DBL. l l 5'-CGA ATT CTC GAG 4 ATC AAA CGG GAC ATC GAA CTC ACT CAG TCT CC-3' (= SEQ ID NO: 24) DBL.12 5'-CGA ATT CGG TCA CC g G TCT CCT CAC AGG TGC AGT TGC AGG AG-3' (= SEQ ID NO: 25) PCR.51 5'-AGG T(C/G)(A/C) AfC/A C TGC AG 7 (C/G) AGT C(A/T)G G-3' (= SEQ ID NO: 26) PCR.89 5' -TGA GGA GAC GGT GAC C 8 GT GGT CCC TTG GCC CC-3' (= SEQ ID NO: 27) PCR.90 5'-GAC ATT GAG CTC 9 ACC CAG TCT CCA-3{= SEQ ID NO: 28) PCR.116 5'-GTT AGA TCT CGA G 4 CT TGG TCC C-3' (= SEQ ID NO: 29) l =Sβl, 9 =SacI These three constructs lack some of the restriction sites at the new joining points. The V H A-V H B gene fragment without a linker lacks the 5' V H B Sfil site. The V L B-V L A gene fragment without a linker lacks the 5' V L A Sail site. The position of the oligonucleotides in the pGOSA constructs given in Table 1 are shown in Figure 9. The pGOSA expression vectors and the oligonucleotides in Table 1 have been designed to enable most specificities to be cloned into the pGOSA constructs. Figure 10 shows the amino acid sequence of the junctions between the V H A-V H B and V L B- V L A fragments encoded by DNA present in pGOSA.E, pGOSA.V, pGOSA.S and pGOSA.T. A more detailed description of the preparation of pGOSA.E, pGOSA.V, pGOSA.S and pGOSA.T is given in Example 5. EXAMPLE 2. Bifunctional binding activity of GOSA double heads In this Example we provide evidence that the above described molecules ("double heads") , i.e. the two chain protein complexes, contain both antigen binding specificities of the Fv's used to generate these multi¬ functional antibody fragment analogues. Figure 12-15 show that GOSA.E, GOSA.V, GOSA.S and GOSA.T can be used to specifically target the enzyme glucose oxidase to several Streptococcus sanguis strains using antibody fragments derived from hybridoma' s expressing antibodies directed against these antigens. Comparison of the binding specificity of the GOSA constructs (see Figures 12-15) and the binding specificity of the scFv.4715-myc (see Figure 11) shows that the fine specificity of the anti - Streptococcus sanguis scFv.4715 is preserved in the GOSA "double heads". EXAMPLE 3. FPLC analysis of GOSA double heads Partially purified GOSA.E, GOSA.V, GOSA.S and GOSA.T samples (estimated to be 50-80% pure by polyacrylamide gel electrophoresis) were analysed on a Pharmacia FPLC Superose 12 column. The analysis was performed using PBS at a flow rate of 0.3 ml/minute. Eluate was monitored at 280 nm and 0.3 ml fractions were collected and analysed by ELISA. Usually GOSA.E, GOSA.V, GOSA.S and GOSA.T samples only gave one GOSA double head activity peak as determined by ELISA (see Figures 16-19) . The position of this peak in the elution pattern indicated that the molecular weight of the GOSA double head is 40-50 kD. Since this molecular weight corresponds to the expected molecular weight of the V H 2 + V L 2 double head dimer, the conclusion is justified that GOSA.E, GOSA.V, GOSA.S and GOSA.T are primarily produced as dimeric molecules. Occasionally an activity peak with an apparent molecular weight of «=200 kD was observed (see Figure 16) . The presence of Glucose Oxidase activity in these fractions (data not shown) indicate that these fractions contain GOSA double head complexed with glucose oxidase that was eluted with the GOSA sample from the glucose oxidase-sepharose affinity matrix. EXAMPLE 4. Production of other double heads The methods described in the previous Examples were used to produce other double heads, which also appeared to be active against the antigens for which they were developed. These other double heads had the following specificities: anti-S. sanguis / anti-bet -HCG, anti-S. sanguis / anti-urease, anti-S. sanguis / anti-hen-egg-lysozyme, anti-beta-HCG / anti-hen-egg-lysozyme, anti-hen-egg-lysozyme /anti-glucose oxidase, anti-huIgG / anti-glucose oxidase, anti-urease / anti-glucose oxidase, anti-lacto-peroxidase / anti-glucose oxidase, anti-alpha-HCG / anti-glucose oxidase, and anti-reactive-Red-6 / anti-glucose oxidase. EXAMPLE 5. Detailed description of the preparation of intermediate constructs pGOSA.A, pGOSA.B pGOSA.C and pGOS .D and their use for the preparation of plasmid pGOSA.E and its derivatives pGOSA.V. pGOSA.S and pGOSA.T Oligonucleotides and PCR The primary structures of the oligonucleotide primers used in the construction of the bispecific 'pGOSA' constructs are shown in Table 1 above. Reaction mixture used for amplification of DNA fragments were 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl 2 , 50 mM KC1, 0.01% gelatin (w/v) , 0.1% Triton X- 100, 400 mM of each dNTP, 5.0 units of Vent DNA polymerase (New England Biolabs) , 100 ng of template DNA, and 500 ng of each primer (for 100 μl reactions) . Reaction conditions were: 94°C for 4 minutes, followed by 33 cycles of each 1 minute at 94°C, 1 minute at 55°C, and 1 minute 72°C. Plasmid DNA\Vector\Insert preparation and ligation\transformation. Plasmid DNA was prepared using the 'Qiagen P-100 Midi-DNA Preparation' system. Vectors and inserts were prepared by digestion of 10 μg (for vector preparation) or 20 μg (for insert preparation) with the specified restriction endonucleases under appropriate conditions (buffers and temperatures as specified by suppliers) . Modification of the DNA ends with Klenow DNA polymerase and dephosphorylation with Calf Intestine Phosphorylase were performed according to the manufacturers instructions. Vector DNAs and inserts were separated through agarose gel electrophoresis and purified with DEAE-membranes NA45 (Schleicher & Schuell) as described by Maniatis et al . Ligations were performed in 20 μl volumes containing 30 mM Tris-HCl pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 300-400 ng vector DNA, 100-200 ng insert DNA and 1 Weiss unit T 4 DNA ligase. After ligation for 2-4 h at room temperature, CaCl 2 competent E. coli JM109 (Maniatis) were transformed using 7.5 μl ligation reaction. The transformation mixtures were plated onto SOBAG plates and grown overnight at 37°C. Correct clones were identified by restriction analysis and verified by automated dideoxy sequencing (Applied Biosystems) . Restriction digestion of PCR products Following amplification each reaction was checked for the presence of a band of the appropriate size by agarose gel electrophoresis. One or two 100 μl PCR reaction mixtures of each of the PCR reactions PCR. I - PCR.X (Figure 20-29) , together containing approximately 2-4 μg DNA product were subjected to phenol-chloroform extraction, chloroform extraction and ethanol precipitation. The DNA pellets were washed twice with 70% ethanol and allowed to dry. Next, the PCR products were digested overnight (18 h) in the presence of excess restriction enzyme in the following mixes at the specified temperatures and volumes. PCR. I: 50 mM Tris-HCl pH 8.0, 10 mM MgCl 2 , 50 mM NaCl, 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (= 40 U) Sad, 4 μl (= 40 U) BstEII, in 100 μl total volume at 37°C. PCR. II: 10 mM Tris-Acetate pH 7.5, 10 mM MgAc 2 , 50 mM KAc (lx "One-Phor-All" buffer ex Pharmacia) , 4 μl (= 48 U) Sfil, in 50 μl total volume at 50°C under mineral oil. After overnight digestion, PCR. II-Sfil was digested with EcoRI (overnight at 37°C) by the addition of 16 μl H 2 0, 30 μl lOx "One-Phor-All" buffer (Pharmacia) (100 mM Tris-Acetate pH 7.5, 100 mM MgAc 2 , 500 mM KAc) and 4 μl (= 40 U) EcoRI. PCR . I l l : 10 mM Tris-Acetate pH 7.5, 10 mM MgAc 2 , 50 mM KAc (lx "One-Phor-All" buffer {Pharmacia}) , 4 μl (= 40 U) Nhel , 4 μl (= 40 U) Sad, in 100 μl total volume at 37°C. PCR. IV: 20 mM Tris-Acetate pH 7.5, 20 mM MgAc 2 , 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}) , 4μl (= 40 U) Xhol, 4 μl (= 40 U) EcoRI, in 100 μl total volume at 37°C. PCR.V: 20 mM Tris-Acetate pH 7.5, 20 mM MgAc 2 , 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}) , 4 μl (= 40 U) Sail, 4 μl (= 40 U) EcoRI, in 100 μl total volume at 37°C. PCR.VI: 10 mM Tris-Acetate pH 7.5, 10 mM MgAc 2 , 50 mM KAc (lx "One-Phor-All" buffer {Pharmacia}) , 4 μl (= 48 U) Sfil, in 50 μl total volume at 50°C under mineral oil. After overnight digestion, PCR.VI-Sfil was digested with Nhel (overnight at 37°C) by the addition of 41 μl H 2 0, 5 μl lOx "One-Phor-All" buffer (Pharmacia) (100 mM Tris-Acetate pH 7.5, 100 mM MgAc 2 , 500 mM KAc) and 4 μl (= 40 U) Nhel . PCR.VII: 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 50 mM ΝaCl, 4 mM sper idine, 0.4 μg/ml BSA, 4 μl (= 40 U) Nhel , 4 μl (= 40 U) BstEII, in 100 μl total volume at 37°C. PCR.VIII: 20 mM Tris-Acetate pH 7.5, 20 mM MgAc 2 , 100 mM KAc (2x "One-Phor-All" buffer {Pharmacia}) , 4 μl (» 40 U) EcoRI, in 50 μl total volume at 37°C. After overnight digestion, PCR.VIII-EcoRI was digested with Xhol (overnight at 37°) by the addition of 46 μl H 2 0 and 4 μl (= 40 U) Xhol. PCR.IX: 25 mM Tris-Acetate, pH 7.8, 100 mM KAc, 10 mM MgAc, ImM DTT (lx "Multi-Core" buffer {Promega}) , 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (= 40 U) Nhel , 4 μl (= 40 U) BstEII, in 100 μl total volume at 37°C. PCR . X : 50 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 50 mM NaCl, 4 mM spermidine, 0.4 μg/ml BSA, 4 μl (= 40 U) PstI, 4 μl (= 40 U) EcoRI, in 100 μl total volume at 37°C. The digested PCR fragments PCR. I-Sad/Bs EII, PCR.II- SfiI/EcoRI, PCR . II I - Nhel /SacT , PCR. IV-Xhol/EcoRI , PCR.V-Sall/EcoRI, PCR.VI - SfiI /Nhel , PCR.VII - BstEII/Nhel , PCR.VIII-XhoI/EcoRI, PCR. IX- BstEII/Nhel , and PCR.X-PstI/EcoRI were purified on an 1.2% agarose gel using DEAE-membranes ΝA45 (Schleicher & Schuell) as described by Maniatis et al . The purified fragments were dissolved in H 2 0 at a concentration of 100-150 ng/μl . Construction of the pGOSA double head expression vectors. The construction of pGOSA.E (see Figure 8) involved several cloning steps that produced 4 intermediate constructs pGOSA.A to pGOSA.D (see Figure 30-34) . The final expression vector pGOSA.E and the oligonucleotides in Table 1 above have been designed to enable most specificities to be cloned into the final pGOSA.E construct (Figure 9) . The upstream V H domain can be replaced by any PstI-BstEII V H gene fragment obtained with oligonucleotides PCR.51 and PCR.89 (see Table 1 above) . The oligonucleotides DBL.3 and DBL.4 (see Table 1 above) were designed to introduce Sfil and Nhel restriction sites in the V H gene fragments thus allowing cloning of those V H gene fragments into the Sfil- Nhel sites as the downstream V H domain. All V L gene fragments obtained with oligonucleotides PCR.116 and PCR.90 (see Table 1 above) can be cloned into the position of the V L .3418 gene fragment as a Sacl-Xhol fragment. A complication here however is the presence of an internal SacI site in the V H .3418 gene fragment. Oligonucleotides DBL.8 and DBL.9 (see Table 1 above) are designed to allow cloning of V L gene fragments into the position of the V L .4715 gene fragment as a Sall -NotI fragment. The pGOSA.E derivatives pGOSA.V, pGOSA.S and pGOSA.T with only one or no linker sequences contain some aberrant restriction sites at the new joining points. The V H A-V H B construct without a linker lacks the 5' V H B Sfil site. The V H B fragment is cloned into these constructs as a BstEII/Nhel fragment using oligonucleotides DBL.10 or DBL.ll and DBL.4 (see Table 1 above) . The V L B-V L A construct without a linker lacks the 5' V L A Sail site. The V L A fragment is cloned into these constructs as a XhoI/EcoRI fragment using oligonucleotides DBL.ll and DBL.9 (see Table l above) . In the following part of the description the following linkers are mentioned which are also present in the sequence listing: the (Gly 4 Ser) 3 linker, present in SEQ ID NO: 2 as amino acids 109-123 and SEQ ID NO: 10 as amino acids 121-135, the (Gly 4 Ser) 3 AlaGlySerAla linker (= linkerA) , present in SEQ ID NO: 12 as amino acids 121-139, and the (Gly 4 Ser) 2 Gly 4 Val linker (= linkerV) , present in SEQ ID NO: 13 as amino acids 108-122. pGOSA.A This plasmid is derived from both the Fv.4715-myc construct and the scFv.4715-myc construct. An Sfil restriction site was introduced between the DNA sequence encoding the (Gly 4 Ser) 3 linker and the gene fragment encoding the V L of the scFv.4715-myc construct (see Figure 30) . This was achieved by replacing the BstEII-Sad fragment of the latter construct by the fragment PCR-I BstEII/SacI (Figure 20) that contains an Sfil site between the DNA encoding the (Gly 4 Ser) 3 linker and the V L .4715 gene fragment. The introduction of the Sfil site also introduced 4 additional amino acids (AlaGlySerAla) between the (Gly 4 Ser) 3 linker and V L .4715 resulting in a (Gly 4 Ser) 3 AlaGlySerAla linker (linkerA) . The oligonucleotides used to produce PCR-I (DBL.l and DBL.2, see Table 1 above) were designed to match the sequence of the framework-3 region of V H .4715 and to prime at the junction of the DNA encoding the (Gly 4 Ser) 3 linker and the V L .4715 gene fragment, respectively. Thus pGOSA.A can be indicated as: pelB-V H 4715-linkerA- (Sfil) -V L 4715-myc. pGOS .B This plasmid is derived from plasmid Fv.3418 (see Figure 31) . The XhoI-EcoRI fragment of plasmid Fv.3418 comprising the 3' end of DNA encoding framework-4 of the V L including the stop codon was removed and replaced by the fragment PCR-IV Xhol/EcoRI (Figure 23) . The oligonucleotides used to produce PCR-IV (DBL.6 and DBL.7, see Table 1 above) were designed to match the sequence at the junction of the V L and the (Gly 4 Ser) 3 linker perfectly (DBL.6) , and to be able to prime at the junction of the (Gly 4 Ser) 3 linker and the V H in pUR.4124 (DBL.7) . DBL.7 removed the PstI site in the V H (silent mutation) and introduced a Sail restriction site at the junction of the (Gly 4 Ser) 3 linker and the V H , thereby replacing the last Ser of the linker by a Val residue resulting in a (Gly 4 Ser) 2 Gly 4 Val linker (linkerV) . Thus pGOSA.B can be indicated as: pelB-V H 3418 + pelB-V L 3418-linkerV- (Sall-EcoRI) pGOSA.C This plasmid contains DNA encoding V H .4715 linked by the (Gly 4 Ser) 3 AlaGlySerAla linker to V H .3418 (see Figure 32) , thus: pelB-V H 4715-linkerA-V„3418. This construct was obtained by replacing the Sfil-EcoRI fragment from pGOSA.A encoding V L .4715 by the fragment PCR¬ II Sfil/EcoRI containing the V H .3418 gene (see Figure 21) . The oligonucleotides used to produce PCR-II (DBL.3 and DBL.4, see Table 1 above) hybridize in the framework-1 and framework-4 region of the gene encoding V H .3418, respectively. DBL.3 was designed to remove the PstI restriction site (silent mutation) and to introduce an Sfil restriction site upstream of the V H gene. DBL.4 destroys the BstEII restriction site in the framework-4 region and introduces an Nhel restriction site downstream of the stopcodon. pGOSA.D This plasmid contains a dicistronic operon comprising the V H .3418 gene and DΝA encoding V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715 (see Figure 33) , thus: pelB-V H 3418 + pelB-V L 3418-linkerV-V L 4715. This construct was obtained by digesting plasmid pGOSA.B with Sall-EcoRI and inserting the fragment PCR-V Sall/EcoRI (Figure 24) containing the V L .4715 gene. The oligonucleotides used to obtain PCR-V (DBL.8 and DBL.9, see Table 1 above) were designed to match the nucleotide sequence of the framework-1 and framework-4 regions of the V L .4715 gene, respectively. DBL.8 removed the SacI site from the framework-1 region (silent mutation) and introduced a Sail restriction site upstream of the V L .4715 gene. DBL.9 destroyed the Xhol restriction site in the framework-4 region of the V L .4715 gene (silent mutation) and introduced a NotI and an EcoRI restriction site downstream of the stop codon. pGOSA.E This plasmid contains a dicistronic operon comprising DΝA encoding V H .4715 linked by the (Gly 4 Ser) 3 AlaGlySerAla linker to V H .3418 plus DΝA encoding V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715 (see Figure 34) , thus: peIB-V H 4715-linkerA-V H 3418 + peIB-V L 3418-linkerV-V L 4715. Both translational units are preceded by a ribosome binding site and DΝA encoding a pelB leader sequence. This plasmid was obtained by a three-point ligation by mixing the vector resulting from pGOSA.D after removal of the V H 3418-encoding PstI-SacI insert with the PstI -Nhel pGOSA.C insert containing V H .4715 linked to V H .3418 and the PCR-III Nhel/ SacI fragment (see Figure 22) . The remaining PstI-SacI pGOSA.D vector contains the 5' end of the framework-1 region of V H .3418 upto the PstI restriction site and V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715 starting from the SacI restriction site in V L .3418. The Pstl - Nhel pGOSA.C insert contains V H .4715 linked by the (Gly 4 Ser) 3 - AlaGlySerAla linker to V H .3418, starting from the PstI restriction site in the framework-1 region in V H .4715. The Nhel-SacI PCR-III fragment provides the ribosome binding site and DΝA encoding the pelB leader sequence for the V L .3418- (Gly 4 Ser) 2 Gly 4 Val-V L .4715 construct. The oligo- nucleotides DBL.5 and PCR.116 (see Table 1 above) used to generate PCR-III were designed to match the sequence upstream of the ribosome binding site of V L .4715 in Fv.4715 and to introduce an Nhel restriction site (DBL.5) , and to match the framework-4 region of V L .3418 (PCR.116) . pGOSA.V This plasmid is derived from pGOSA.E (see Figure 35) from which the BstEII/Nhel fragment containing DΝA encoding linkerA-V H .3418 was excised and replaced by the fragment PCR-VII BstEII/Nhel containing the V H .3418 gene (see Figure 26) . The resulting plasmid pGOSA.V contains V H .3418 linked directly to the framework-4 region of V H .4715, plus V L .4715 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to the framework-4 region of V L .3418, thus: pelB-V H 4715*V H 3418 + pelB-V L 3418-linkerV-V L 4715. . pGOSA.S This plasmid is derived from pGOSA.E (see Figure 36) from which the (Gly 4 Ser) 2 Gly 4 Val-V L 4715 Xhol/ EcoRI fragment was excised and replaced by the fragment PCR-VIII Xhol/ EcoRI which contains V L .4715 (see Figure 27) . The resulting plasmid pGOSA.S contains V H .4715 linked by the (Gly 4 Ser) 3 - AlaGlySerAla linker to V H .3418 plus V L .3418 linked directly to the 5' end of the framework-1 region of V L -4715, thus: pelB-V H .4715-linkerA-V H .3418 + pelB-V L .3418*V L .4715. pGOSA. T This plasmid contains a dicistronic operon consisting of V H .3418 directly to the framework-4 region of V H .4715 plus V L .3418 linked directly to the 5' end of the framework-1 region of V L .4715 (see Figure 37) . Both transcriptional units are preceded by a ribosome binding site and a pelB leader sequence, thus: pelB-V H .4715*V H .3418 + pelB-V, .3418*V L .4715. This construct was obtained by inserting the Nhel/EcoRl fragment of pGOSA.S which contains V L .3418 linked directly to the 5' end of the framework-1 region of V L .4715, into the vector pGOSA.V from which the Nhel/EcoRl fragment containing V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715 was removed. EXAMPLE 6. Detailed description of the preparation of other dicistronic constructs pGOSA.G, and pGOSA.J. pGOSA.Z, pGOSA.AA and pGOSA.AB pGOSA.G This plasmid is an intermediate for the synthesis of pGOSA.J. It is derived from pGOSA.E from which the V H 4715 Pstl/BstEII fragment has been excised and replaced by the V H 3418 Pstl/BstEII fragment (excised from Fv.3418) . The resulting plasmid pGOSA.G (see Figure 38) contains two copies of V H .3418 linked by the (Gly 4 Ser) 3 AlaGlySerAla linker, plus V L .4715 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to the framework-4 region of V L .3418, thus: pe!B-V H .3418-linkerA-V H .3418 + pelB-V L .3418-linkerV-V L .4715. pGOSA.J This plasmid contains a dicistronic operon consisting of V H .3418 linked by the (Gly 4 Ser) 3 AlaGlySerAla linker to V H .4715 plus V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715. Both transcriptional units are preceded by a ribosome binding site and a pelB leader sequence (see Figure 39) , thus: pelB-V H .3418-linkerA-V H .4715 + pelB-V, .3418-linkerV-V L .4715. This construct was obtained by inserting the fragment PCR- VI Sfil /Nhel which contains V H 4715 (Figure 25) , into the vector pGOSA.G from which the Sfil /Nhel V H 3418 fragment was removed. pGOSA.Z This plasmid is derived from pGOSA.G from which the (Gly 4 Ser) 3 AlaGlySerAla Iinker-V H 3418 BstEII/Nhel fragment was excised and replaced by the fragment PCR-IX BstEII/Nhel which contains V H .4715 (Figure 28) . The resulting plasmid pGOSA.Z (see Figure 40) contains V H .3418 linked directly to the framework-1 region of V H .4715, plus V L .4715 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to the framework-4 region of V L .3418, thus: pelB-V H .3418*V H .4715 + pelB-V L .3418-linkerV-V L .4715. pGOSA.AA This plasmid contains a dicistronic operon consisting of the V H .3418 linked directly to the 5' end of the framework- 1 region of V H .4715 plus V L .3418 linked directly to the 5' end of the framework-1 region of V L .4715. Both transcriptional units are preceded by a ribosome binding site and a pelB leader sequence (see Figure 41) . This construct was obtained by inserting the Nhel/EcoRl fragment of pGOSA.T which contains V L .3418 linked directly to the 5' end of the framework-1 region of V L .4715, into the vector pGOSA.Z from which the Nhel/EcoRl fragment containing V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to V L .4715 was removed, thus: pelB-V H .3418*V H .4715 + pelB-V, .3418*V L .4715. pGOSA. AB This plasmid is derived from pGOSA.J by a three point ligation reaction (see Figure 42) . The SacI/ EcoRI insert, containing part of V H .3418 and the full (Gly 4 Ser) 3 AlaGlySerAla linker-V H .4715 and the V L .3418- (Gly 4 Ser) 2 Gly 4 Val-V L .4715 encoding sequences, was removed and replaced by the SacI/ SacI pGOSA.J fragment containing the same part of V H .3418 and the full (Gly 4 Ser) 3 AlaGlySerAla linker-V H .4715 and the SacI/ EcoRI pGOSA.T fragment containing V L .3418 linked directly to the framework-1 region of V L .4715 (see Figure 37) . The resulting plasmid contains V H .3418 linked by the (Gly 4 Ser) 3 AlaGlySerAla linker to the 5' end of the framework-1 region of V H .4715 plus V L .3418 linked directly to the 5' end of the framework-1 region of V L .4715, thus: pelB-V H .3418-linkerA-V H .4715 + pelB-V L .3418*V L .4715. EXAMPLE 7. Detailed description of the preparation of monocistronic constructs pGOSA.L and pGOSA.Y, . and pGOSA.C. pGOSA.X, pGOSA.AC and pGOSA.AD pGOSA.L This plasmid is derived from pGOSA.E from which the Hindlll/Nhel fragment containing DΝA encoding V H .4715.- (Gly 4 Ser) 3 AlaGlySerAla-V H .3418 was removed (see Figure 43) . The DΝA ends of the vector were made blunt-end using Klenow DΝA polymerase and ligated. The resulting plasmid pGOSA.L contains V L .3418 linked by the (Gly 4 Ser) 2 Gly 4 Val linker to the 5' end of the framework-1 region of V L .4715, thus: pelB-V L .3418-linkerV-V L .4715. pGOSA.Y This plasmid is derived from pGOSA.T from which the Hindlll/Nhel fragment containing DΝA encoding V H .4715- V H .3418 was removed (see Figure 44) . The DΝA ends of the vector were made blunt-end using Klenow DΝA polymerase and ligated. The resulting plasmid pGOSA.Y contains V L .3418 linked directly to 5' end of the framework-1 region of V L .4715, thus: pelB-V L .3418*V L .4715. The preparation of pGOSA.C was given in Example 5 above; it can be indicated with: pelB-V H .4715-linkerA-V H .3418. pGOSA.X This plasmid is derived from pGOSA.T from which the Nhel/EcoRl fragment containing DNA encoding V L .3418-V L .4715 was removed. The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.X (see Figure 45) contains V H .4715 linked directly to 5' end of the framework-1 region of V H .3418, thus: peIB-V H .4715*V H .3418. pGOSA.AC This plasmid is derived from pGOSA.Z from which the Nhel/EcoRl fragment containing DNA encoding V L .3418- (Gly 4 Ser) 2 Gly 4 Val-V L .4715 was removed (see Figure 46) . The DNA ends of the vector were made blunt-end using Klenow DNA polymerase and ligated. The resulting plasmid pGOSA.AC contains V H .3418 linked directly to 5' end of the framework-1 region of V H .4715, thus: pelB-V H .3418*V H .4715. pGOSA.AD This plasmid was obtained by inserting the PstI/EcoRI PCR.X fragment containing DNA encoding V H .3418- (Gly 4 Ser) 3 AlaGly- SerAla-V H .4715 (see Figure 29) into the Fv.4715-myc vector from which the PstI/ EcoRI Fv.4715-myc insert was removed (see Figure 47) , thus: pelB-V H .3418-linkerA-V H .4715. These monocistronic constructs can be used to transform the same host with two different plasmids or to transform two different hosts, so that the two V H 's in series can be produced separately from the two V L 's in series. Evaluation of the results obtained Bifunctional binding activity of GOSA double heads In this specification the construction of a two chain protein complex is described, in which one of the chains consists of two heavy chain V-domains and the other chain consists of the two corresponding light chain V-domains. The variable domains are linked either directly or through a polypeptide linker. In this specification evidence is provided that these type of molecules ("double heads") contain both antigen binding specificities of the Fv's used to generate these multi-functional antibody fragments. Figure 12 shows that GOSA.E can be used to specifically target the enzyme glucose oxidase to several Streptococcus sanguis strains, using antibody fragments derived from hybridomas expressing antibodies directed against these antigens. Figure 12 further shows that the fine specificity of the anti - Streptococcus sanguis scFv 4715 is preserved in the GOSA.E double head. Effect of linkers and relative position of V-domains on double head activity After it was shown that the "cross-over double-head" approach (V H A-V H B + V L B-V L A) yields active bispecific molecules, the importance of the relative position of the V-domains in these constructs was investigated. Both possible positional orientations (GOSA.E = V H A-LinkerA-V H B + V L B-LinkerV-V L A and GOSA.J = V H B-LinkerA-V H A + V L B-LinkerV- V L A) were constructed and tested for bispecific activity, despite the suggestion obtained by molecular modelling that the binding site formed by the second (downstream/C- terminal) V-domains in the configuration V H B-V H A + V L B-V L A (GOSA.J) was in an unfavourable position for binding to large protein antigens on the surface of cells. Surprisingly however, it was found experimentally that the downstream binding site is in fact accessible. Although the relative position of the heavy chains and the light chains was found to have an effect on the observed reactivity both tested combinations show bispecific activity with the "cross-over" combination (GOSA.E = V H A-V H B + V L B-V L A) exhibiting a higher level of reactivity compared to the combination V H B-V H A + V L B-V L A (= GOSA.J) as demonstrated for A=anti-Strep and B=anti-Gox. Molecular modelling of the V H B-V H A + V t B-V L A (= GOSA.J) configuration further suggested that, only when the connecting linkers are kept long enough (to span 30 to 35 A) , the protein chains could fold such that both binding sites are fully accessible. The "cross-over" configuration: V H A-V H B + V L B-V L A (GOSA.E) wherein linker length was not critical, was predicted to result in a complex with both binding sites facing in opposite directions, without the restraints suggested for the configuration V H B-V H A + V L B-V L A (GOSA.J) . Removing the flexible polypeptide linker from the V H A-V H B chain only had a minimal effect on the ability of the double head in the "cross-over" configuration (GOSA.V = V H A*V H B + V L B-V L A) to bind both S . sangui s and Glucose oxidase. However, removing the flexible polypeptide linker from the V H B-V H A chain from the molecule in the V H B-V H A + V L B-V L A configuration (GOSA.Z = V H B*V H A + V L B-V L A ) resulted in a dramatic reduction of its ability to bind both S. sanguis and Glucose oxidase. In contrast with the double head in the "cross-over" configuration without the flexible polypeptide linker between the two heavy chain domains (GOSA.V) , where molecular modelling predicted the resulting molecule to be active, removal of the flexible linker from the V L B-V L A chain could not be modelled such that both binding sites were fully accessible. ELISA results confirm that the double head in the V H B-V H A + V L B-V L A configuration without a linker between the two light chain domains (GOSA.AB) exhibits only minimal S. sanguis and glucose oxidase binding activity. Surprisingly, deletion of the flexible linker from the V L B-V L A chain from the double head in the "cross-over" configuration (GOSA.S) only had a small effect on the bispecific activity of the resulting molecule. As expected from the molecular modelling results from the double heads without a flexible linker between the two light chain domains, removal of both the flexible polypeptide linkers from the double head molecules, could not be modelled such that both binding sites were fully accessible. In agreement with the ELISA results obtained with the GOSA.AB construct, the double head in the V H B-V H A + V L B-V L A configuration without any linkers (GOSA.AA) only exhibits minimal if any S. sanguis and glucose oxidase binding activity. Surprisingly, the double head in the "cross-over" configuration without any linkers (GOSA.T = V H A*V H B + V L B*V L A) still exhibited 25-50% of S. sanguis and glucose oxidase bispecific binding activity when compared to the activity of the double head in the "cross-over" configuration with two linkers (GOSA.E) . Thus the conclusion of this work is that modelling can give some indications, but that the computer programmes cannot predict what is possible and what not. Several deviations from the modelling expectations were found. With a paraphrase on an old saying: theories are nice but the experiment is the ultimate proof. Sensitivity of GOSA double heads Using an ELISA format it was shown that the sensitivity of the GOSA.E double head is as least as a sensitive as an IgG-glucose oxidase conjugate, as determined by the lowest concentration of Streptococcus sanguis antigen immobilised on a solid phase that is still detectable. GOSA double heads are produced as dimers FPLC analysis of partially affinity-purified GOSA.E, GOSA.V, GOSA.S and GOSA.T samples usually gave only one GOSA double head activity peak as determined by ELISA (Figures 16-19) . The position of this peak in the elution pattern indicated that the molecular weight of the GOSA double head is 40-50 kD. Since this molecular weight corresponds to the expected molecular weight of the V H 2 + V L 2 double head dimer, it was concluded that GOSA.E, GOSA.V, GOSA.S and GOSA.T are primarily produced as dimeric molecules. Occasionally an activity peak with an apparent molecular weight of «=200 kD was observed (Figure 16) . The presence of glucose oxidase activity in these fractions indicate that these fractions contain GOSA double head complexed with glucose oxidase. In vitro assembly of GOSA double heads It was shown that bifunctionally active dimeric GOSA molecules together in one cell can be produced by transla¬ tion from one dicistronic messenger (GOSA.E, GOSA.S, GOSA.T, GOSA.V, GOSA.J, GOSA.AB, GOSA.AA and GOSA.Z) . In addition high levels of S. sanguis and glucose oxidase bispecific binding activity is formed when supernatants of cultures producing the separate GOSA subunits are mixed (see Example 7) . The effects of linkers and the relative position of the individual V H -domains on the S. sanguis and glucose oxidase bispecific binding activity observed in these mixing experiments are comparable to the dicistronic constructs. The constructs described above are summarised in Table 2 below. Table 2A describes intermediate constructs that were not further tested. Table 2B describes the dicistronic constructs. Table 2C describes the monocistronic constructs. (LiA) stands for the V H -V H linker (Gly 4 Ser) 3 AlaGlySerAla (= linkerA) (LiV) stands for the V L -V L linker (Gly 4 Ser),Gly 4 Val (= linkerV) (*) indicates that the two heavy chain domains or the two light chain domains are fused together without a connecting linker. Table 2 Table 2A GOSA.A : V H .4715-LiA- (Sfil) -V L .4715-myc GOSA.B : V H .3418-LiV-V L .3418- (Sall/EcoRI) GOSA.D : V H .3418 + V L .3418-LiV-V L .4715 GOSA.G : V H .3418-LiA-V H .3418 + V L .3418-LiV-V L .4715 Table 2B GOSA.E : V H .4715-LiA-V H .3418 + V L .3418-LiV-V L .4715 GOSA.S V H .4715-LiA-V H .3418 + V L .3418*V L .4715 GOSA.T V H .4715*V H .3418 + V L .3418*V L .4715 GOSA.V V H .4715*V H .3418 + V L .3418-LiV-V L .4715 GOSA.J V H .3418-LiA-V H .4715 + V L .3418-LiV-V L .4715 GOSA.AB V H .3418-LiA-V H .4715 + V L .3418*V L .4715 GOSA.AA V H .3418*V H .4715 + V L .3418*V L .4715 GOSA.Z V H .3418*V H .4715 + V L .3418-LiV-V L .4715 Table 2C GOSA. L : V L . 3418 - LiV-V L . 4715 GOSA. Y : V L . 3418*V L . 4715 GOSA.AD V H .3418-LiA-V H .4715 GOSA.AC V H .3418*V H .4715 GOSA.C V H .4715-LiA-V H .3418 GOSA.X V H .4715*V H .3418 REFERENCES Patent literature - EP-0281604; GENEX/ENZON; SINGLE POLYPEPTIDE CHAIN BINDING MOLECULES; priority date 02.09.86. - WO 93/11161; ENZON, INC; MULTIVALENT ANTIGEN-BINDING PROTEINS; priority date 25.11.91. - WO 94/09131; SCOTGEN LIMITED; RECOMBINANT SPECIFIC BINDING PROTEIN; priority date 15.10.92) . - WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY / MEDICAL RESEARCH COUNCIL; MULTIVALENT AND MULTISPECIFIC BINDING PROTEINS, THEIR MANUFACTURE AND USE; first priority date 04.12.92 - WO 94/13806; THE DOW CHEMICAL COMPANY; MULTIVALENT SINGLE CHAIN ANTIBODIES; priority date 11.12.92 - WO 94/25591; UNILEVER N.V., UNILEVER PLC, Hamers, R. , Hamers-Casterman, C. , & Muyldermans, S; PRODUCTION OF ANTIBODIES OR (FUNCTIONALIZED) FRAGMENTS THEREOF DERIVED FROM HEAVY CHAIN IMMUNOGLOBULINS OF CAMELIDAE; first priority date 29.04.93. Non-patent literature Anthony, J. , Near, R. , Wong, S.L. , Iida, E., Ernst, E., Wittekind, M. , Haber, E. and Ng, S-C; Molec. Immunol. 29. (1992) 1237-1247, PRODUCTION OF STABLE ANTI -DIGOXIN Fv IN ESCHERICHIA COLI . Berry, M.J. and Davies, J. ; J. Chromatography 597 (1992] 239-245; Use of antibody fragments in immunoaffinity chromatography: comparison of Fv fragments, V H fragments and paralog peptides. - Better, M. , Chang, C.P., Robinson, R.R. and Horwitz A.H.; Science 240 (1988) 1041-1043; Escheri chia coli Secretion of an Active Chimeric Antibody Fragment. Bird, R.E., Hardman, K.D. , Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.M., Lee, T. , Pope, S.H., Riordan, G.S. and Whitlow, M. ; Science 242 (1988) 423-426; Single- Chain Antigen-Binding Proteins. Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias, M. , Velligan, M.D., Wong, W.L.T., Rowland, A.M., Kotts, C.E., Carver, M.E., Yang, M. , Bourell, J.H., Shepard, H.M. and Henner, D. ; BIO/TECHNOLOGY 0 (1992) 163- 167; High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Cumber, A.J., Ward, E.S., Winter, G. , Parnell, G.D. and Wawrzynczak. E.J.; J. Immunol. 149 (1992) 120-126; COMPA¬ RATIVE STABILITIES IN VITRO AND IN VIVO OF A RECOMBINANT MOUSE ANTIBODY FvCys FRAGMENT AND A bisFvCys CONJUGATE. Firek, S., Draper, J. , Owen. M.R.L., Gandecha, A., Cock- burn, B., and Whitelam, G.C.; Plant Mol. Biol. 23 (1993) 861-870; Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures. - Givol, D.; Molec. Immunol. 2_8 (1991) 1379; THE MINIMAL ANTIGEN BINDING FRAGMENT OF ANTIBODIES - Fv FRAGMENT. Hamers-Casterman, C. , Atarhouch, T. , Muyldermans, S., Robinson, G. , Hamers, C. , Bajyana Songa, E., Bendahman, N. and Hamers, R.; Nature 363 (1993) 446-448; Naturally occurring antibodies devoid of light chains. Hiatt, A., Cafferkey, R. and Bowdish, K. ; Nature 342 (1989) 76-78; Production of antibodies in transgenic plants. Holliger, P., Prospero, T. , and Winter, G.; Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448; "Diabodies": Small bivalent and bispecific antibody fragments. Horwitz, A.H., Chang, C.P., Better, M. , Hellstrom, K.E. and Robinson, R.R.; Proc. Natl. Acad. Sci. USA 85 (1988) 8678-8682; Secretion of functional antibody and Fab fragment from yeast cells. Huston, J.S., Levinson, D., Mudgett-Hunter, M. , Tai, M- S., Novotny, J. , Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. ; Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escheri chia coli . Kostelny, S.A., Cole, M.S. and Tso, J.Y.; J. Immunol. 148 (1992) 1547-1553; FORMATION OF A BISPECIFIC ANTIBODY BY THE USE OF LEUCINE ZIPPERS. Mallender, W.D., and Voss, Jr., E.W.; J. Biol. Chem. 269 (1994) 199-206; Construction, Expression, and Activity of a Bivalent Bispecific Single-chain Antibody. - Milstein, C. and Cuello, A.C.; Nature, 305 (1983) 537- 540; Hybrid hybridomas and their use in immunohisto- chemistry. - Nyyssδnen, E., Penttilla., M. , Harkki, A., Saloheimo, A., Knowles, J.K.C. and Keranen, S.; BIO/TECHNOLOGY Bio/tech. 11 (1993) 591-595; Efficient Production of Antibody Fragments in Filamentous Fungus Tri choderma reesei . Owen, M. , Gandecha, A., Cockburn, B. and Whitelam G. ; BIO/TECHNOLOGY ) (1992) 790-794; Synthesis of a functional an i-phytochrome single-chain Fv protein in transgenic tobacco. Pack, P. and Plϋckthun, A.; Biochemistry 31 (1992) 1579- 1584; Miniantibodies : Use of Amphiphatic Helices to Produce Functional, Flexibly Linked Dimeric Fv Fragments with High Avidity in Escheri chia coli . Skerra, A. and Plϋckthun, A.; Science, 240 (1988) 1038- 1041; Assembly of a Functional Immunoglobulin Fv Fragment in Escherichia coli . - Taub, R. , Gould, R.J., Ciccarone, T.M. , Hoxie, J., Friedman, P.A., Shattil, S.J. and Garsky, V.M. ; J. Biol. Chem. 264 (1989) 259-265; A Monoclonal Antibody against the Platelet Fibrinogen Receptor Contains a Sequence That Mimics a Receptor Recognition Domain in Fibrinogen. - Ward, E.S., Gussow, D., Griffiths, A.D. , Jones, P.T. and Winter G.; Nature 341 (1989) 544-546; Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli . Williams, W,V., Moss, D.A. , Kieber-Emmons, T. , Cohen, J.A., Myers, J.N. , Weiner, D.B. and Greene, M.I.; Proc. Natl. Acad. Sci. USA. . 86 (1989) 5537-5541; Development of biologically active peptides based on antibody structure. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A NAME: Unilever PLC (B STREET: Unilever House, Blac friars (C CITY: London (E COUNTRY: United Kingdom (F POSTAL CODE (ZIP) : EC4P 4BQ (GB) (A NAME: Unilever N.V. (B STREET: Weena 455 (c; CITY: Rotterdam (E COUNTRY: The Netherlands (F POSTAL CODE (ZIP) : NL-3013 AL (A NAME: Paul James DAVIS (B STREET: The Hawthorns, Pavenham Road (C CITY: Felmersham (Bedfordshire) (E COUNTRY: United Kingdom (F POSTAL CODE (ZIP) : MK43 7EX (GB) (A NAME: Cornelis Paul Erik van der LOGT (B STREET: 1 Bluebell Rise (Peverel Manor Estate) (c: CITY: Rushden (Northamptonshire) (E COUNTRY: United Kingdom (F POSTAL CODE (ZIP) : NN10 OTU (GB) (A NAME: Martine Elisa VERHOEIJEN (B STREET: 1 Tintagel Close (Manor Farm Estate) (C CITY: Rushden (Northamptonshire) (E COUNTRY: United Kingdom (F POSTAL CODE (ZIP) : NN10 ONP (GB) (A NAME: Steve Wilson (B STREET: 3 Aldenham Close (Goldington ) (C CITY: Bedford, (E COUNTRY: United Kingdom (F POSTAL CODE (ZIP) : MK41 OFQ (GB) (ii) TITLE OF INVENTION: A bifunctional or bivalent antibody fragment analogue (iii) NUMBER OF SEQUENCES: 31 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: EP 95307332. (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 737 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "cDNA domains with synthetic linker (s) " (vii) IMMEDIATE SOURCE: (B) CLONE: EcoRI-Hindlll insert of pUR4124 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:11..730 (D) OTHER INFORMATION: /product= "VLlys-GS-VHlys" (ix) FEATURE: (A) NAME/KEY: at peptide (B) LOCATION:11..334 (D) OTHER INFORMATION: /product= "VLlys" (ix) FEATURE: (A) NAME/KEY: misc_RNA (B) LOCATION:335..379 (D) OTHER INFORMATION: /product= " (Gly4Ser) 3 linker" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:380..727 (D) OTHER INFORMATION: /product= "VHlys" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGGCC GAC ATC GAG CTC ACC CAG TCT CCA GCC TCC CTT TCT GCG 49 Asp lie Glu Leu Thr Gin Ser Pro Ala Ser Leu Ser Ala 1 5 10 TCT GTG GGA GAA ACT GTC ACC ATC ACA TGT CGA GCA AGT GGG AAT ATT 97 Sex Val Gly Glu Thr Val Thr lie Thr Cys Arg Ala Ser Gly Asn lie 15 20 25 CAC AAT TAT TTA GCA TGG TAT CAG CAG AAA CAG GGA AAA TCT CCT CAG 145 His Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin 30 35 40 45 CTC CTG GTC TAT TAT ACA ACA ACC TTA GCA GAT GGT GTG CCA TCA AGG 193 Leu Leu Val Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val Pro Ser Arg 50 55 60 TTC AGT GGC AGT GGA TCA GGA ACA CAA TAT TCT CTC AAG ATC AAC AGC 241 Phe Ser Gly Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys lie Asn Ser 65 70 75 CTG CAA CCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGG AGT 289 Leu Gin Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gin His Phe Trp Ser 80 85 90 ACT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTC GAG ATC AAA CGG GGT 337 Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Gly 95 100 105 GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG CAG GTG 385 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val 110 115 120 125 CAG CTG CAG GAG TCA GGA CCT GGC CTG GTG GCG CCC TCA CAG AGC CTG 433 Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu 130 135 140 TCC ATC ACA TGC ACC GTC TCA GGG TTC TCA TTA ACC GGC TAT GGT GTA 481 Ser He Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val 145 150 155 AAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG GAG TGG CTG GGA ATG 529 Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met 160 165 170 ATT TGG GGT GAT GGA AAC ACA GAC TAT AAT TCA GCT CTC AAA TCC AGA 577 He Trp Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg 175 180 185 CTG AGC ATC AGC AAG GAC AAC TCC AAG AGC CAA GTT TTC TTA AAA ATG 625 Leu Ser He Ser Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met 190 195 200 205 AAC AGT CTG CAC ACT GAT GAC ACA GCC AGG TAC TAC TGT GCC AGA GAG 673 Asn Ser Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Glu 210 215 220 AGA GAT TAT AGG CTT GAC TAC TGG GGC CAA GGG ACC ACG GTC ACC GTC 721 Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val 225 230 235 TCC TCA TGA TAAGCTT 737 Ser Ser * 240 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 240 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Asp He Glu Leu Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr He Thr Cys Arg Ala Ser Gly Asn He His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val 35 40 45 Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gin Tyr Ser Leu Lys He Asn Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gin His Phe Trp Ser Thr Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Gin 115 120 125 Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser He Thr 130 135 140 Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val 145 150 155 160 Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly 165 170 175 Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He 180 185 190 Ser Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu 195 200 205 His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Glu Arg Asp Tyr 210 215 220 Arg Leu Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser * 225 230 235 240 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 920 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /deεc = "cDNA domains with synthetic linker(ε) " (vii) IMMEDIATE SOURCE: (B) CLONE: Hindlll-EcoRI insert Fv.3418 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 6..443 (D) OTHER INFORMATION: /product= "pelB-VH3418" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 36..101 (D) OTHER INFORMATION: /product= "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 102..440 (D) OTHER INFORMATION: /product= "VH3418" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:495..884 (D) OTHER INFORMATION: /product= "pelB-VL4318" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION:495..560 (D) OTHER INFORMATION: /product= "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat__peptide (B) LOCATION:561..881 (D) OTHER INFORMATION: /product= "VL3418" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AAGCTTGCAA ATTCTATTTC AAGGAGACAG TCATA ATG AAA TAC CTA TTG CCT 53 Met Lys Tyr Leu Leu Pro -22 -20 ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG GCC 101 Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met Ala -15 -10 -5 CAG GTG CAG CTG CAG CAG TCA GGA CCT GAG CTG GTA AAG CCT GGG GCT 149 Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 TCA GTG AAG ATG TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT AGC TAT 197 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 GTT ATG CAC TGG GTG AAA CAG AAG CCT GGG CAG GGC CTT GAG TGG ATT 245 Val Met His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp He 35 40 45 GGA TAT ATT TAT CCT TAC AAT GAT GGT ACT AAG TAC AAT GAG AAG TTC 293 Gly Tyr He Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 AAA GGC AAG GCC ACA CTG ACT TCA GAC AAA TCC TCC AGC ACA GCC TAC 341 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 ATG GAG CTC AGC AGC CTG ACC TCT GAG GAC TCT GCG GTC TAT TAC TGT 389 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 TCA AGA CGC TTT GAC TAC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCC 437 Ser Arg Arg Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser 100 105 110 TCA TAA TAAGAGCTAT GGGAGCTTGC ATGCAAATTC TATTTCAAGG AGACAGTCAT 493 Ser * A ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC 539 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu -22 -20 -15 -10 GCT GCC CAA CCA GCG ATG GCC GAC ATC GAG CTC ACC CAG TCT CCA TCT 587 Ala Ala Gin Pro Ala Met Ala Asp He Glu Leu Thr Gin Ser Pro Ser -5 1 5 TCC ATG TAT GCA TCT CTA GGA GAG AGA ATC ACT ATC ACT TGC AAG GCG 635 Ser Met Tyr Ala Ser Leu Gly Glu Arg He Thr He Thr Cys Lys Ala 10 15 20 25 AGT CAG GAC ATT AAT ACC TAT TTA ACC TGG TTC CAG CAG AAA CCA GGG 683 Ser Gin Asp He Asn Thr Tyr Leu Thr Trp Phe Gin Gin Lys Pro Gly 30 35 40 AAA TCT CCC AAG ACC CTG ATC TAT CGT GCA AAC AGA TTG CTA GAT GGG 731 Lys Ser Pro Lys Thr Leu He Tyr Arg Ala Asn Arg Leu Leu Asp Gly 45 50 55 GTC CCA TCA AGG TTC AGT GGC AGT GGA TCT GGG CAA GAT TAT TCT CTC 779 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gin Asp Tyr Ser Leu 60 65 70 ACC ATC AGC AGC CTG GAC TAT GAA GAT ATG GGA ATT TAT TAT TGT CTA 827 Thr He Ser Ser Leu Asp Tyr Glu Asp Met Gly He Tyr Tyr Cys Leu 75 80 85 CAA TAT GAT GAG TTG TAC ACG TTC GGA GGG GGG ACC AAG CTC GAG ATC 875 Gin Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He 90 95 100 105 AAA CGG TAA TAATGATCAA ACGGTATAAG GATCCAGCTC GAATTC 920 Lys Arg * (2) INFORMATION FOR SEQ ID NO: : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 136 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4- Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin Ser Gly Pro Glu -5 1 5 10 Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly 15 20 25 Tyr Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gin Lys Pro Gly 30 35 40 Gin Gly Leu Glu Trp He Gly Tyr He Tyr Pro Tyr Asn Asp Gly Thr 45 50 55 Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys 60 65 70 Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp 75 80 85 90 Ser Ala Val Tyr Tyr Cys Ser Arg Arg Phe Asp Tyr Trp Gly Gin Gly 95 100 105 Thr Thr Val Thr Val Ser Ser * 110 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 130 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Asp He Glu Leu Thr Gin Ser Pro Ser Ser -5 1 5 10 Met Tyr Ala Ser Leu Gly Glu Arg He Thr He Thr Cys Lys Ala Ser 15 20 25 Gin Asp He Asn Thr Tyr Leu Thr Trp Phe Gin Gin Lys Pro Gly Lys 30 35 40 Ser Pro Lys Thr Leu He Tyr Arg Ala Asn Arg Leu Leu Asp Gly Val 45 50 55 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gin Asp Tyr Ser Leu Thr 60 65 70 He Ser Ser Leu Asp Tyr Glu Asp Met Gly He Tyr Tyr Cys Leu Gin 75 80 85 90 Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 95 100 105 Arg * (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 999 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "cDNA domains with synthetic linker (s) " (vii) IMMEDIATE SOURCE: (B) CLONE: Hindlll-EcoRI insert of Fv.4715-myc (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:40..468 (D) OTHER INFORMATION: /product= "pelB-VH4715" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION:40..105 (D) OTHER INFORMATION: /product= "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:106..465 (D) OTHER INFORMATION: /product= "VH4715" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:520..963 (D) OTHER INFORMATION: /product= "pelB-VL4715-myc" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION:520..585 (D) OTHER INFORMATION: /product= "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_j?eptide (B) LOCATION:586..927 (D) OTHER INFORMATION: /produc = "VL4715" (ix) FEATURE: (A) NAME/KEY: misc_RNA (B) LOCATION:928..960 (D) OTHER INFORMATION: /product= "myc-tag" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC CTA TTG 54 Met Lys Tyr Leu Leu -22 -20 CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG 102 Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met -15 -10 -5 GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GAC TTA GTG AAG CCT GGA 150 Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGT AGT 198 Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser 20 25 30 TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GAC AAG AGT CTG GAG TGG 246 Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp Lys Ser Leu Glu Trp 35 40 45 GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT ACC TAT TAT TCA GAC AAT 294 Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr Tyr Tyr Ser Asp Asn 50 55 60 GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AAT GGC AAG AAC ACC CTG 342 Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Gly Lys Asn Thr Leu 65 70 75 TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GAC ACA GCC GTG TAT TAC 390 Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 80 85 90 95 TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TAT TTT GAC TAC TGG GGC 438 Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr Phe Asp Tyr Trp Gly 100 105 110 CAA GGG ACC ACG GTC ACC GTC TCC TCA TAA TAAGAGCTAT GGGAGCTTGC 488 Gin Gly Thr Thr Val Thr Val Ser Ser * 115 120 ATGCAAATTC TATTTCAAGG AGACAGTCAT A ATG AAA TAC CTA TTG CCT ACG 540 Met Lys Tyr Leu Leu Pro Thr -22 -20 GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG GCC GAC 588 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met Ala Asp -15 -10 -5 1 ATC GAG CTC ACT CAG TCT CCA TTC TCC CTG ACT GTG ACA GCA GGA GAG 636 He Glu Leu Thr Gin Ser Pro Phe Ser Leu Thr Val Thr Ala Gly Glu 5 10 15 AAG GTC ACT ATG AAT TGC AAG TCC GGT CAG AGT CTG TTA AAC AGT GTA 684 Lys Val Thr Met Asn Cys Lys Ser Gly Gin Ser Leu Leu Asn Ser Val 20 25 30 AAT CAG AGG AAC TAC TTG ACC TGG TAC CAG CAG AAG CCA GGG CAG CCT 732 Asn Gin Arg Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin Pro 35 40 45 CCT AAA CTG TTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGA GTC CCT 780 Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro 50 55 60 65 GAT CGC TTC ACA GCC AGT GGA TCT GGA ACA GAT TTC ACT CTC ACC ATC 828 Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He 70 75 80 AGC AGT GTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG AAT GAT 876 Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp 85 90 95 TAT ACT TAT CCG TTC ACG TTC GGA GGG GGG ACC AAG CTC GAG ATC AAA 924 Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 100 105 110 CGG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT TAA TAAGATCAAA 973 Arg Glu Gin Lys Leu He Ser Glu Glu Asp Leu Asn * 115 120 125 CGGTAATAAG GATCCAGCTC GAATTC 999 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 143 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp -5 1 5 10 Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly 15 20 25 Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp 30 35 40 Lys Ser Leu Glu Trp Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr 45 50 55 Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn 60 65 70 Gly Lys Asn Thr Leu Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp 75 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr 95 100 105 Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser * 110 115 120 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 148 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Asp He Glu Leu Thr Gin Ser Pro Phe Ser -5 1 5 10 Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Asn Cys Lys Ser Gly 15 20 25 Gin Ser Leu Leu Asn Ser Val Asn Gin Arg Asn Tyr Leu Thr Trp Tyr 30 35 40 Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Tyr Trp Ala Ser 45 50 55 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly 60 65 70 Thr Asp Phe Thr Leu Thr He Ser Ser Val Gin Ala Glu Asp Leu Ala 75 80 85 90 Val Tyr Tyr Cys Gin Asn Asp Tyr Thr Tyr Pro Phe Thr Phe Gly Gly 95 100 105 Gly Thr Lys Leu Glu He Lys Arg Glu Gin Lys Leu He Ser Glu Glu 110 115 120 Asp Leu Asn * 125 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 924 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "cDNA domains with synthetic linker (s) " (vii) IMMEDIATE SOURCE: (B) CLONE: Hindlll-EcoRI insert of scF .4715-myc (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION:40..105 (D) OTHER INFORMATION: /product= "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION-.106..465 (D) OTHER INFORMATION: /products "VH4715" (ix) FEATURE: (A) NAME/KEY: misc_RNA (B) LOCATION:466..510 (D) OTHER INFORMATION: /products " (Gly4Ser) 3-linker" (ix) FEATURE : (A) NAME/KEY: mat_peptide (B) LOCATION:511..852 (D) OTHER INFORMATION: /product= "VL4715" ( ix) FEATURE : (A) NAME/KEY: misc_RNA (B) LOCATION: 85 ..885 (D) OTHER INFORMATION: /products "myc-tag" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:40..888 (D) OTHER INFORMATION: /products "pelB-VH4715- (Gly4Ser) 3-VL4715-myc" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC CTA TTG 54 Met Lys Tyr Leu Leu -22 -20 CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG 102 Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met -15 -10 -5 GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GAC TTA GTG AAG CCT GGA 150 Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGT AGT 198 Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser 20 25 30 TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GAC AAG AGT CTG GAG TGG 246 Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp Lys Ser Leu Glu Trp 35 40 45 GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT ACC TAT TAT TCA GAC AAT 294 Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr Tyr Tyr Ser Asp Asn 50 55 60 GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AAT GGC AAG AAC ACC CTG 342 Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Gly Lys Asn Thr Leu 65 70 75 TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GAC ACA GCC GTG TAT TAC 390 Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 80 85 90 95 TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TAT TTT GAC TAC TGG GGC 438 Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr Phe Asp Tyr Trp Gly 100 105 110 CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA 486 Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC GAG CTC ACT CAG TCT CCA 534 Gly Gly Ser Gly Gly Gly Gly Ser Asp He Glu Leu Thr Gin Ser Pro 130 135 140 TTC TCC CTG ACT GTG ACA GCA GGA GAG AAG GTC ACT ATG AAT TGC AAG 582 Phe Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Asn Cys Lys 145 150 155 TCC GGT CAG AGT CTG TTA AAC AGT GTA AAT CAG AGG AAC TAC TTG ACC 630 Ser Gly Gin Ser Leu Leu Asn Ser Val Asn Gin Arg Asn Tyr Leu Thr 160 165 170 175 TGG TAC CAG CAG AAG CCA GGG CAG CCT CCT AAA CTG TTG ATC TAC TGG 678 Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Tyr Trp 180 185 190 GCA TCC ACT AGG GAA TCT GGA GTC CCT GAT CGC TTC ACA GCC AGT GGA 726 Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Ala Ser Gly 195 200 205 TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AGT GTG CAG GCT GAA GAC 774 Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Val Gin Ala Glu Asp 210 215 220 CTG GCA GTT TAT TAC TGT CAG AAT GAT TAT ACT TAT CCG TTC ACG TTC 822 Leu Ala Val Tyr Tyr Cys Gin Asn Asp Tyr Thr Tyr Pro Phe Thr Phe 225 230 235 GGA GGG GGG ACC AAG CTC GAG ATC AAA CGG GAA CAA AAA CTC ATC TCA 870 Gly Gly Gly Thr Lys Leu Glu He Lys Arg Glu Gin Lys Leu He Ser 240 245 250 255 GAA GAG GAT CTG AAT TAA TAAGATCAAA CGGTAATAAG GATCCAGCTC GAATTC 924 Glu Glu Asp Leu Asn * 260 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 283 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp -5 1 5 10 Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly 15 20 25 Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp 30 35 40 Lys Ser Leu Glu Trp Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr 45 50 55 Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn 60 65 70 Gly Lys Asn Thr Leu Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp 75 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr 95 100 105 Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly 110 115 120 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Glu 125 130 135 Leu Thr Gin Ser Pro Phe Ser Leu Thr Val Thr Ala Gly Glu Lys Val 140 145 150 Thr Met Asn Cys Lys Ser Gly Gin Ser Leu Leu Asn Ser Val Asn Gin 155 160 165 170 Arg Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys 175 180 185 Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 190 195 200 Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser 205 210 215 Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp Tyr Thr 220 225 230 Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Glu 235 240 245 250 Gin Lys Leu He Ser Glu Glu Asp Leu Asn * 255 260 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1706 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "cDNA domains with synthetic linker(s)" (vii) IMMEDIATE SOURCE: (B) CLONE: Hindlll-EcoRI insert of pGOSA.E (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:40..864 (D) OTHER INFORMATION: /products "pelB-VH4715-LiA-VH3418" (ix) FEATURE: (A) NAME/KEY: sigjpeptide (B) LOCATION:40..105 (D) OTHER INFORMATION: /products "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:106..465 (D) OTHER INFORMATION: /products "VH4715" (ix) FEATURE : (A) NAME/KEY: misc_RNA (B) LOCATION:466..522 (D) OTHER INFORMATION: /products "linkerA (Gly4Ser) 3AlaGlySerAla" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:523..861 (D) OTHER INFORMATION: /products "VH3418" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:913..1689 (D) OTHER INFORMATION: /products "pelB-VL3418-LiV-VL4715" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION:913..978 (D) OTHER INFORMATION: /products "pectate lyase" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:979..1299 (D) OTHER INFORMATION:/products "VL3418" (ix) FEATURE: (A) NAME/KEY: misc_RNA (B) LOCATION: 1300..1344 (D) OTHER INFORMATION:/products "linker V (Gly4Ser) 2Gly4Val" (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION:1345..1686 (D) OTHER INFORMATION: /products "VL4715" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 AAGCTTGCAT GGAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC CTA TTG 54 Met Lys Tyr Leu Leu -22 -20 CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG 102 Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met -15 -10 -5 GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GAC TTA GTG AAG CCT GGA 150 Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGT AGT 198 Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser 20 25 30 TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GAC AAG AGT CTG GAG TGG 246 Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp Lys Ser Leu Glu Trp 35 40 45 GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT ACC TAT TAT TCA GAC AAT 294 Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr Tyr Tyr Ser Asp Asn 50 55 60 GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AAT GGC AAG AAC ACC CTG 342 Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn Gly Lys Asn Thr Leu 65 70 75 TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GAC ACA GCC GTG TAT TAC 390 Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 80 85 90 95 TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TAT TTT GAC TAC TGG GGC 438 Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr Phe Asp Tyr Trp Gly 100 105 110 CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA 486 Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 GGT GGC TCT GGC GGT GGC GGA TCG GCC GGT TCG GCC CAG GTC CAG CTG 534 Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Ser Ala Gin Val Gin Leu 130 135 140 CAA CAG TCA GGA CCT GAG CTG GTA AAG CCT GGG GCT TCA GTG AAG ATG 582 Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met 145 150 155 TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT AGC TAT GTT ATG CAC TGG 630 Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Met His Trp 160 165 170 175 GTG AAA CAG AAG CCT GGG CAG GGC CTT GAG TGG ATT GGA TAT ATT TAT 678 Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp He Gly Tyr He Tyr 180 185 190 CCT TAC AAT GAT GGT ACT AAG TAC AAT GAG AAG TTC AAA GGC AAG GCC 726 Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala 195 200 205 ACA CTG ACT TCA GAC AAA TCC TCC AGC ACA GCC TAC ATG GAG CTC AGC 774 Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser 210 215 220 AGC CTG ACC TCT GAG GAC TCT GCG GTC TAT TAC TGT TCA AGA CGC TTT 822 Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser Arg Arg Phe 225 230 235 GAC TAC TGG GGC CAA GGG ACC ACC GTC ACC GTC TCC TCA TAA 864 Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser * 240 245 250 TAAGCTAGCG GAGCTGCATG CAAATTCTAT TTCAAGGAGA CAGTCATA ATG AAA TAC 921 Met Lys Tyr -22 -20 CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA 969 Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro -15 -10 -5 GCG ATG GCC GAC ATC GAG CTC ACC CAG TCT CCA TCT TCC ATG TAT GCA 1017 Ala Met Ala Asp He Glu Leu Thr Gin Ser Pro Ser Ser Met Tyr Ala 1 5 10 TCT CTA GGA GAG AGA ATC ACT ATC ACT TGC AAG GCG AGT CAG GAC ATT 1065 Ser Leu Gly Glu Arg He Thr He Thr Cys Lys Ala Ser Gin Asp He 15 20 25 AAT ACC TAT TTA ACC TGG TTC CAG CAG AAA CCA GGG AAA TCT CCC AAG 1113 Asn Thr Tyr Leu Thr Trp Phe Gin Gin Lys Pro Gly Lys Ser Pro Lys 30 35 40 45 ACC CTG ATC TAT CGT GCA AAC AGA TTG CTA GAT GGG GTC CCA TCA AGG 1161 Thr Leu He Tyr Arg Ala Asn Arg Leu Leu Asp Gly Val Pro Ser Arg 50 55 60 TTC AGT GGC AGT GGA TCT GGG CAA GAT TAT TCT CTC ACC ATC AGC AGC 1209 Phe Ser Gly Ser Gly Ser Gly Gin Asp Tyr Ser Leu Thr He Ser Ser 65 70 75 CTG GAC TAT GAA GAT ATG GGA ATT TAT TAT TGT CTA CAA TAT GAT GAG 1257 Leu Asp Tyr Glu Asp Met Gly He Tyr Tyr Cys Leu Gin Tyr Asp Glu 80 85 90 TTG TAC ACG TTC GGA GGG GGG ACC AAG CTC GAG ATC AAA CGG GGT GGA 1305 Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Gly Gly 95 100 105 GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA GTC GAC ATC GAA 1353 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val Asp He Glu 110 115 120 125 CTC ACT CAG TCT CCA TTC TCC CTG ACT GTG ACA GCA GGA GAG AAG GTC 1401 Leu Thr Gin Ser Pro Phe Ser Leu Thr Val Thr Ala Gly Glu Lys Val 130 135 140 ACT ATG AAT TGC AAG TCC GGT CAG AGT CTG TTA AAC AGT GTA AAT CAG 1449 Thr Met Asn Cys Lys Ser Gly Gin Ser Leu Leu Asn Ser Val Asn Gin 145 150 155 AGG AAC TAC TTG ACC TGG TAC CAG CAG AAG CCA GGG CAG CCT CCT AAA 1497 Arg Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys 160 165 170 CTG TTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGA GTC CCT GAT CGC 1545 Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg 175 180 185 TTC ACA GCC AGT GGA TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AGT 1593 Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser 190 195 200 205 GTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG AAT GAT TAT ACT 1641 Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp Tyr Thr 210 215 220 TAT CCG TTC ACG TTC GGA GGG GGG ACC AAG CTC GAA ATC AAA CGG TAA 1689 Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg * 225 230 235 TAAGCGGCCG CGAATTC 1706 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 275 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Glu Ser Gly Gly Asp -5 1 5 10 Leu Val Lys Pro Gly Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly 15 20 25 Phe Thr Phe Ser Ser Tyr Ala Phe Ser Trp Val Arg Gin Thr Ser Asp 30 35 40 Lys Ser Leu Glu Trp Val Ala Thr He Ser Ser Thr Asp Thr Tyr Thr 45 50 55 Tyr Tyr Ser Asp Asn Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn 60 65 70 Gly Lys Asn Thr Leu Tyr Leu Gin Met Ser Ser Leu Lys Ser Glu Asp 75 80 85 90 Thr Ala Val Tyr Tyr Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr 95 100 105 Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly 110 115 120 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Ser 125 130 135 Ala Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly 140 145 150 Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 155 160 165 170 Tyr Val Met His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp 175 180 185 He Gly Tyr He Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys 190 195 200 Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala 205 210 215 Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 220 225 230 Cys Ser Arg Arg Phe Asp Tyr Trp Gly Gin Gly Thr Thr Val Thr Val 235 240 245 250 Ser Ser * (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 259 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -22 -20 -15 -10 Ala Gin Pro Ala Met Ala Asp He Glu Leu Thr Gin Ser Pro Ser Ser -5 1 5 10 Met Tyr Ala Ser Leu Gly Glu Arg He Thr He Thr Cys Lys Ala Ser 15 20 25 Gin Asp He Asn Thr Tyr Leu Thr Trp Phe Gin Gin Lys Pro Gly Lys 30 35 40 Ser Pro Lys Thr Leu He Tyr Arg Ala Asn Arg Leu Leu Asp Gly Val 45 50 55 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gin Asp Tyr Ser Leu Thr 60 65 70 He Ser Ser Leu Asp Tyr Glu Asp Met Gly He Tyr Tyr Cys Leu Gin 75 80 85 90 Tyr Asp Glu Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys 95 100 105 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Val 110 115 120 Asp He Glu Leu Thr Gin Ser Pro Phe Ser Leu Thr Val Thr Ala Gly 125 130 135 Glu Lys Val Thr Met Asn Cys Lys Ser Gly Gin Ser Leu Leu Asn Ser 140 145 150 Val Asn Gin Arg Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin 155 160 165 170 Pro Pro Lys Leu Leu He Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 175 180 185 Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 190 195 200 He Ser Ser Val Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn 205 210 215 Asp Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu He 220 225 230 Lys Arg * 235 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.l (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CACCATCTCC AGAGACAATG GCAAG 25 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: GAGCGCGAGC TCGGCCGAAC CGGCCGATCC GCCACCGCCA GAGCC 45 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CAGGATCCGG CCGGTTCGGC CCAGGTCCAG CTGCAACAGT CAGGA 45 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTACATGAAT TCGCTAGCTT ATTATGAGGA GACGGTGACG GTGGTCCCTT GGC 53 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TAATAAGCTA GCGGAGCTGC ATGCAAATTC TATTTC 36 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ACCAAGCTCG AGATCAAACG GGG 23 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: AATGTCGAAT TCGTCGACTC CGCCACCGCC AGAGCC 36 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.8 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: ATTGGAGTCG ACATCGAACT CACTCAGTCT CCATTCTCC 39 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: TGAAGTGAAT TCGCGGCCGC TTATTACCGT TTGATTTCGA GCTTGGTCCC 50 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: CGAATTCGGT CACCGTCTCC TCACAGGTCC AGTTGCAACA G 41 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.ll (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: CβAATTCTCG AGATCAAACG GGACATCGAA CTCACTCAGT CTCC 44 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer DBL.12 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: CGAATTCGGT CACCGTCTCC TCACAGGTGC AGTTGCAGGA G 41 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer PCR.51 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: AGGTSMAMCT GCAGSAGTCW GG 22 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer PCR.89 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27- TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer PCR.90 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GACATTGAGC TCACCCAGTC TCCA 24 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "synthetic DNA" (vii) IMMEDIATE SOURCE: (B) CLONE: primer PCR.116 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GTTAGATCTC GAGCTTGGTC CC 22 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Thr Thr Val Thr Val Ser Ser Gin Val Gin Leu Gin Gin 1 5 10 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Lys Leu Glu He Lys Arg Asp He Glu Leu Thr Gin 1 5 10 DtVΛ/ I I /12 pogo 11 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION Or THE DEPOSIT OF MICROORGANISMS ron THE PURPOSES OF PATE T PROCEDURE : NTEHNΛTIOMΛ FORM [ TO Dr. P van der Logc | Unilever Research ' RECEI PT I H THE CASE OF AN ORIGINAL DEPOS IT Colworth Laboratory 1-iEuod pursuant to Rule 7. 1 by the INTERNATIONAL DEPOSITARY AUTHORITY Biosciences Division identi f ied a t the bottom of this page Colworth House, Sharnbroo Bedford MK44 1LQ L UΛfis AND ΛDURΠSS i OF DEPOSITOR I . IDENTIFICATION OF Tllϋ n- lOORCAIUS.S Identi f ication reference gi ven by the Access ion nu ber given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY : Escherichia coli NCTC 12916 ScFy 4715.myc II . SCIENTIFIC DESCRIPTION ΛCD/OR PROPOSED TΛXONOMΪC DESIGNATION The microorganism identi f ied under I above was ccompanied by : a scientific description I 1 I a proposed tsxonomic des i gna tion {Ht.tr. wi th a cross where app l icable ) HI . RECEIPT AJJD ACCEPTANCE This Internatioonnaal Depositaryv Authority accepts the microorganism identified under I above. which was reccive! by it on 14th October 1 * 995 (date of original deposit) 1 IV. INTERNATIONAL DEPOSITARY AUTHORITY National Collection of Type Cultures Signature(s) of person(β) having the power to represent the International Depositary Authority or of authorised official (β): dress: Central Public Health Laboratory 61 Colindale Avenue Date: 4/12/95 London NW9 5HT B Holmes Cl inirnl Sn nffsf Where Rule β.lld ) applies, such date is the dato on which the status of international de osit.- authority was acquired-, where a deposit mode outside tho Dudapest Treaty after the aequl β ltic oi tho status of international depositary authority is converted Into a deposit under the Dudαpest Treaty, such dato is the date on which the 'microorganism was received by tne international depositary authority. Forn DP/-I (solo rJSC) BUDAPEST TREATY OH HE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM Dr P van der Logt : L lever Research jlworth Laboratory VIABILITY STATEMENT Iss ued pursuant to Rule 10 . 2 by the . osciences Division INTERNATIONAL DEPOSITARY AUTHORITY ■ Lworth House, Sharnbrook Identified on the following page iford K4 1LQ Λ::S ADDRESS OF THE PARTY .-SC-K THE VIA3I.MTY STATEMENT IS ISSUED .ate. tho dato of the original deposit or, where a^new deposit or a t""'" J" b#βn . the cast recent relevant date (date of the new deposit or date of the transfer ) . : referred to in Rule 10.2(a) (li) and (ill), refer to the most recent viability cracs the applicable box. ', 'i.za page) BP/Λ/II/12 page 25 CO::O:TIO!:S VI:OSP. WHICH THE VIABILITY TEST ILΛS DEEH PERΓΌΠMED 4 Nutrient Agar without additions (a) Λ β ar (17R Bacto-Tryptone, lOg Bacto-Yeast Extract, 5R NaCl, per litre) with lOOug/ml ampicillin and LZ glucose (b) Aerobic incubation, 37°C, 24 hrs (a) 8 x IO 5 cfu/ml (b) 6 x IO 5 cfu/ml :TεKMΛTZOt.'AI. DEPOSITARY AUTHORITY Slenneurα Co) of pezaon(a) having tlie power ofTyps to represent tha International Depositary Authority or of authorised official (β) i iitlie::-:: Trispbe-Mi OlSI40O4-U> Teleat B 93MZ(DβR-«Dα> Date: 4/12/95 B Hθl-0βa Pa»01Si^C37a74 Clinical Scientist rill in if the inioraation has been requested and if tha results of the test were negative. DI>/Λ/ I I/12 pago 14 UUDΛPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOS IT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE I HTLHNΛTIONΛL IΌKM Fro Dr. P van der Logt 1 Unilever Research ' RECEI PT I N TilE CASE OF AN ORIGINAL DEPOS IT Colworth Laboratory l :: suc(l |> rn u.-ιιι L Lo Rule 7 . 1 by tlir: Biosciences Division I NTEKNATIONΛL DCPOSITΛRΪ AUTHORITY identi fied at the bottom of this page Colworth House, Sharnbrook Bedford MK44 1LQ IIΛΠC AND ΛDUHCSS L OF DEPOSITOR J I DENTIFICATION OF THE MICROORGANISM Identi ication reference gi ven by the Accession number given by the L l'O lTOlt: IIITEIlNΛTIONΛIx'Dl.POSITΛHY AUTHORITY : Escherichia coli NCTC 12915 Fv 3418 .1 . SCIENTIFIC DESCRIPTION Λ1.D/0R PROPOSED TΛXONOMIC DESIGNATION The rclcroorganlSKt identified under I above was accompanied by : I I a scienti fic description j j^ l α proposed laxonoinic designation (Mark with a cross v.- crc applicable ) III . RECEIPT A2ID ACCEPTANCE Tl'.ls International Depositary Authority accepts the microorganism identified under I above, which was received by it on j^ th October 1995 (date of ori ginal Hp ns r ) 1 IV. INTERNATIONAL DEPOSITARY AUTHORITY :; MKS :National Collection of Type Cultures Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorised o icial(s) t Ad ress: Central Public Health Laboratory Sft T rM He* *"*. 61 Colindale Avenue Date: 4/12/95 London NW9 5hT B Holmes Clinical Scientis _ Where Ru l i]e 6 4 Id ) applies, such dote Is the date on which tho status of international deposita authorit:yy was a_c_q..uired; where a deposit πade outside the Dudαpest Treaty after the acquisitio ot the status of international depositary authority is converted into a deposit under the Budapest Treaty, such date is the date on which the microorganism was received by the international depositary authority. torn 9P/4 (sole page) U / A/II/ 12 page 24 1IUDΛPE5T TREATY ON THE INTERNATIONAL HECOCillTION OF THE DEPOSIT OF MICROORGANISMS TOR THE URPOSES OF PATENT ROCEDURE - INTERNATIONAL FORM . Dr P van der Logt ~) illever Research jlworth Laboratory VIABILITY STATEMENT issued pursuant to Rule 10 .2 by the iosciences Division INTERNATIONAL DEPOSITARY AUTHORITY olworth House, Sharnbrook iden tified on the following page edford M 44 1LQ ;.:-i ;.:.Ό ΛS.OSSSS OF THE PARTY KJ:::Λ • .:• • VIΛ3∑LΣTY STATEMENT l≤ ' SSUED II. IDENTIFICATION OF THE MICROORGANISM Dr 1' van der Logt Accession number given by the INTERNATIONAL DEPOSITARY AUTJIOIUTY: As above NCTC U9I5 Dato of tlie deposit-or of the transfer: 14th October 199S STΛTEME::? the Kiel'saI-?.: .".;..τι i cntl ied under II above was tested 2 On that date, the said microorganism was ir viable date o: t>u v:.i iιιal deposit or, where a new deposit or a transfer has bean :t ι-3 5 uιιt .cii.-.-.iήt ϋaue (date of tho new deposit or date of tha transfer). rc-ic: '.ule 10.2(a) (ii) and (iii) , refer to the nost recant viability zcosz sac sc licaiile box. ,-- • . • :;- β.;a; "m t cc:oιτιo::5 uimsn HHICII TΠE VIABILITY TEST HAS ΛEEII PEIUΌIU-IED 4 Nutrient Λgar without additions (a) ' ΛRΠΓ (17β Hncto-Tryp.tone, IOR Bacto-Yeast Extrnς.^.--5B NoCl, per I i.rc) wj.Uι lOOug ml ampicillin and IZ lύcϋfaj^(b) Aerobic incubation, 37&Q; 24 hrs. (a) 2 x IO 6 cfu/ml (b) 1 x IO 6 cfu/ml' ZVΛZPJ's.SτiOUΛ OEPOSITAnv AUTHORITY .-Signature, a) of pax*on(s) having..tha powαr NaiMtnal CaHeedoa cit Type QUt—a "Cβ; represent the International Depositary Cenuar Puelle Health saenter/ .Authority or of authorised ό fieial(a)ι 61 CuUndale Awaa, Lsarlsa N 93MT Telaphew OtSl<∞O 40O Telex.* 8933M2 BHIMD 0> ; pate.- 4/12/95 B Holαiβ Pax: 0181-3007*74 Clinical Scientist i l in if the information has been requested and lx the rasuits of the text ware ne a ive . -.•;:_. iϋ>/5- 'aeco-i-- ΛII-J last page)

Claims: C A I M S 1. A bispecific or bivalent antibody fragment analogue, which comprises a binding complex containing two polypeptide chains, whereby one polypeptide chain comprises two times a variable domain of a heavy chain (V H ) in series and the other polypeptide chain comprises two times a variable domain of a light chain (V L ) in series, and the binding complex contains two pairs of variable domains (V H - A//V L -A and V H -B//V L -B) . 2. An antibody fragment analogue according to claim 1, in which one polypeptide chain comprises a first V H connected to a second V H and the other polypeptide chain comprises a first V L connected to a second V L . 3. An antibody fragment analogue according to claim 2, in which the two V H 's are directly connected to each other without an intermediate peptide linker. 4. An antibody fragment analogue according to claim 2, in which the two V L ' s are directly connected to each other without an intermediate peptide linker. 5. An antibody fragment analogue according to claim 3 or claim 4, in which one polypeptide chain comprises a first V H directly connected to a second V H , and the other polypeptide chain comprises a first V L directly connected to a second V L . 6. An antibody fragment analogue according to claim 2, in which the two V H 's are connected to each other by a peptide linker and also the two V L 's are connected to each other by a peptide linker, each peptide linker comprising at least one amino acid residue. 7. An antibody fragment analogue according to claim 6, in which one polypeptide chain comprises a first V H (V H -A) followed by a second V H (V H -B) and the other polypeptide chain comprises a first V L (V L -A) followed by a second V L (V L -B) , and in which the two V H 's are connected to each other by a peptide linker (Li H ) , thus [V H -A * Li H * V H -B] , and also the two V L ' s are connected to each other by a peptide linker (Li L ) , thus [V L -A * Li L * V L -B] , each peptide linker comprising at least 10 amino acid residues. 8. An antibody fragment analogue according to claim 2, in which one polypeptide chain comprises a first V H (V H -A) followed by a second V H (V H -B) with or without a connecting peptide linker (Li H ) , thus [V H -A * (Li H ) * V H -B] , and the other polypeptide chain comprises a first V L (V L -A) preceded by a second V L (V L -B) with or without a connecting peptide linker (Li L ) , thus [V L -B * (Li L ) * V L -A] . 9. An antibody fragment analogue according to claim 1, in which the two variable domains are different resulting in a bispecific antibody fragment analogue. 10. An antibody fragment analogue according to claim 1, in which the specificities A and B are the same resulting in a bivalent antibody fragment analogue. 11. Use of an antibody fragment analogue according to claim 1, in immunoassays including diagnostic techniques, in agglutination assays, in a purification method, for compositions suitable for therapy, or in other methods in which immunoglobulins or fragments thereof can be used. 12. A process for producing an antibody fragment analogue according to any one of claims 1-10, which comprises (1) transforming a host by incorporating into that host a DNA encoding the two V H 's in series with or without a connecting peptide linker and a DNA encoding the two V L ' s in series with or without a connecting peptide linker, (2) culturing such transformed host under conditions whereby the connected V H 's and the connected V L 's are formed, and (3) allowing the two connected V H 's and the two connected V L 's to combine to each other under formation of a double head antibody fragment analogue, and (4) optionally collecting the double head antibody fragment analogue. 13. A process for producing an antibody fragment analogue according to any one of claims 1-10, which comprises (1) transforming a first host by incorporating into that first host a DNA encoding the two V H 's in series with or without a connecting peptide linker, (2) transforming a second host by incorporating into that second host a DNA encoding the two V L ' s in series with or without a connecting peptide linker, (3) culturing the first and second transformed host under conditions whereby the connected V H 's and the connected V L 's, respectively, are formed, (4) optionally collecting separately the two connected V H 's and the two connected V L 's, and (5) combining the connected V H 's and the connected V L 's under conditions that they can form a double head antibody fragment analogue. 14. A process according to claim 12 or 13, in which the host is selected from the group consisting of prokaryotic micro-organisms comprising Gram-negative bacteria (e.g. E. coli ) and Gram-positive bacteria (e.g. B . subtilis or lactic acid bacteria) , lower eukaryotic microorganisms comprising yeasts (e.g. belonging to the genera Saccharomyces , Kluyveromyces , Pichia , and Hansenula) and moulds (e.g. belonging to the genera Aspergillus, Neurospora or Trichoderma) , and higher eukaryotic organisms (e.g. plants) or cell cultures thereof (e.g. hybridoma's) .

Inventors: DAVIS PAUL JAMES, VAN DER LOGT CORNELIS PAUL ERI, VERHOEIJEN MARTINE ELISA, WILSON STEVE, DAVIS, PAUL, JAMES, VAN DER LOGT, CORNELIS, PAUL, ERIK, VERHOEIJEN, MARTINE, ELISA, WILSON, STEVE
Applicants: DAVIS PAUL JAMES, LOGT CORNELIS PAUL ERIK V D, UNILEVER NV, UNILEVER PLC, VERHOEIJEN MARTINE ELISA, WILSON STEVE, DAVIS, PAUL, JAMES, UNILEVER N.V., VAN DER LOGT, CORNELIS, PAUL, ERIK, VERHOEIJEN, MARTINE, ELISA, WILSON, STEVE
Application year: 1996
Main classification: C07K16
IPC codes: C07K16, C12P21