Abstract:
A process is provided for the production of an antibody or a fragment or functionalized fragment thereof using a transformed lower eukaryotic host containing an expressible DNA sequence encoding the antibody or (functionalized) fragment thereof, wherein the antibody or (functionalized) fragment thereof is derived from a heavy chain immunoglobulin of  Camelidae  and is devoid of light chains, and wherein the lower eukaryotic host is a mould, preferably belonging to the genera  Aspergillus  or  Trichoderma,  or a yeast, preferably belonging to the yeast genera  Saccharomyces, Kluyveromyces, Hansenula, or Pichia . The heavy chain fragment can contain at least the whole variable domain. A complementary determining region (CDR) different from the CDR belonging to the natural antibody ex  Camelidae  can be grafted on the framework of the variable domain of the heavy chain immunoglobulin. The catalytic antibodies can be raised in  Camelidae  against transition state molecules. The functionalized antibody or fragment thereof can comprise a fusion protein of both heavy chain immunoglobulin from  Camelidae  or a fragment thereof and another polypeptide, e.g., an enzyme, preferably an oxido-reductase. Also provided are new products obtainable by a process as described, and compositions containing a product produced by a process as described, which composition may contain a new product as provided.

Description:
The present invention relates to a process for the production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of  Camelidae  and is partly based on research investigations carried out at the Free University of Brussels. A draft publication thereon already submitted to the periodical Nature and communicated to the present applicants by Prof. R. Hamers reads as follows. 
     FUNCTIONAL HEAVY CHAIN IMMUNOGLOBULINS IN THE CAMELIDS 
     Random association of V L  and V H  repertoires contributes considerably to antibody diversity (1). The diversity and the affinity are then increased by hypermutation in B-cells located in germinal centres (2). Except in the heavy chain disease (3), naturally occurring heavy chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains (4) or cloned V H  domains (5). The presence of considerable amounts IgG like material of 100 Kd in the serum of the camel ( Camelus dromedarius ) (6) was confirmed. These molecules are composed of heavy chain dimers and are devoid of light chains. Nevertheless they bear an extensive antigen binding repertoire, a finding which questions the role of the light chains in the camel. Camel heavy chain IgGs lack the C H 1, which in one IgG class might be structurally replaced by an extended hinge. Heavy chain IgGs are a feature of all camelids. These findings open perspectives in engineering of antibodies. 
     By a combination of affinity chromatography on Protein A and Protein G, three quantitatively important fractions corresponding to subclasses of IgG can be isolated from the serum of camels ( Camelus dromedarius ) (FIG. 1A, lanes c-f). One fraction (IgG 1 ) contains molecules of 170 Kd (FIG. 1B, lane 2) which upon reduction yield 50 Kd heavy chains and large 30 kD light chains (FIG. 1C, lane 2). The two other immunoglobulin fractions contain molecules of approximately 100 Kd (FIG. 1B, lanes 1 and 3) which upon reduction yield only heavy chains of respectively 46 Kd (IgG 2  fraction binding only to Protein A) (FIG. 1C, lane 3) and 43 Kd (IgG 3  fraction binding to Protein A and Protein G) (FIG. 1C, lane 1). These two IgG classes appear to lack the light chain completely. 
     To exclude the possibility that the light chains were only weakly associated with the heavy chains and lost during the selective purification, whole serum was size fractionated by gel filtration. Coomassie blue staining of unreduced fractions revealed the sequential elution of the 170 Kd IgG 1  followed by the incompletely resolved isotypes IgG 2  and IgG 3  (90 Kd) (FIG. 1D, upper inset). Immunostaining of the same fractions after reduction confirmed that the light chains were present solely in the 50 Kd heavy chain containing fractions (FIG. 1D, lower inset). 
     A comparative study of old world camelids ( Camelus bactrianus  and  Camelus dromedarius ) and new world camelids ( Lama pacos, Lama glama  and  Lama vicugna ) showed that heavy chain immunoglobulins are abundant in the sera of all species examined (data not shown) and total up to 75% of the molecules binding to protein A. 
     The abundance of the heavy chain immunoglobulins in the serum of camelids raises the question as to whether they bear an extensive antigen binding repertoire. This question could be answered by examining the IgG 1 , IgG 2  and IgG 3  fractions from the serum of camels ( Camelus dromedarius ) with a high antitrypanosome titer (7). In radio-immunoprecipitation, purified fractions of IgG 1 , IgG 2  and IgG 3  derived from infected camels were shown to bind a large number of antigens present in a  35 S methionine labelled trypanosome lysate (FIG. 2A), indicating an extensive repertoire complexity for the three IgG classes. Conversely, in blotting experiments, 35S methionine labelled trypanosome lysate hinds to SDS-PAGE separated IgG 1 , IgG 2  and IgG 3  obtained from infected animals (FIG. 2B). These findings indicate that the heavy chains alone can generate an extensive repertoire and question the obligatory contribution of the light chain to the useful antibody repertoire in the camelids. 
     The camelid γ2 and γ3 chains are considerably shorter than the normal mammalian γ or camel γ1 chains. This would suggest that, as in the case of heavy chain disease (3), deletions have occurred in the C H 1 protein domain (8,9). To address this question, cDNA was synthesized from camel spleen mRNA and the sequences between the 5′ end of the V H  and the C H 2 were amplified by a Polymerase Chain Reaction (PCR), and cloned. Seventeen clones presenting a different V H  sequence were isolated and sequenced. Their most striking feature was the complete lack of the C H 1 domain, the last framework (FR4) residues of the V H  region being immediately followed by the hinge (FIG. 3, lower part). The absence of the C H 1 domain clarifies two important dilemmas. 
     First, immunoglobulin heavy chains are normally not secreted unless the heavy chain chaperoning protein or BIP (10) has been replaced by the L chain (11), or alternatively the C H 1 domain has been deleted (3,8,9). Secondly, isolated heavy chains from mammalian immunoglobulins tend to aggregate, but are only solubilized by light chains (8,12) which bind to the C H 1 and the V H  domains (13). 
     14 of the 17 clones were characterized by a short hinge sequence with a length equal to that of human IgG 2  and IgG 4  (14) (FIG. 3). The other 3 had a long hinge sequence containing the ‘EPK’ hinge motif found in human IgG 1  and IgG 3  (14). They possess the C H   2  ‘APELL/P’ motif also found in human IgG 1  and IgG 3  (see SEQ. ID. NO: 1-2), and which is associated with mammary transport of bovine IgG 1  (15). On basis of molecular weight, we expect the “short hinge” clones to correspond to IgG 3  and the “long hinge” clones to IgG 2 . 
     In the short hinge containing antibody, the extreme distance between the extremities of the V H  regions will be of the order of 80 Å corresponding to twice the size of a single domain of 40 Å (2×V H ) (16). This could be a severe limitation for agglutinating, cross linking or complement fixation (17,18). In the long hinge containing immunoglobulin the absence of C H 1 might be compensated by the extremely long hinge itself, composed of a 12 fold repeat of the sequence Pro-X (X=Gln, Glu, Lys) (FIG. 3 &amp; 4). NMR (19) and molecular modelling (20) of Pro-X repeats present in the TonB protein of  E. coil  (X=Glu, Lys) and the membrane procyclin of trypanosomes (X=Asp, Glu) indicate that these repeated sequences function as rigid rodlike spacers with a diameter of 8 Å and a rise of 2.9 Å per residue. Assuming the same geometry, the long hinge would be 70 Å which compensates for the absence of the C H 1 domain. 
     The binding site of heavy chain antibodies cannot form the pocket resulting from adjoining light and heavy chain V regions and the residues of the V H  which normally interact with V L  will be exposed to solvent (3,5,13). It was found that leucine at position 45 conserved in 98% of human and murine V H  sequences (14), and crucial in the V H -V L  association (13), can be replaced by an arginine (FIG. 3, upper part). This substitution is in accordance with both the lost contact with a V L  domain and an increased solubility. 
     Unlike myeloma heavy chains which result mainly from C H 1 deletion in a single antibody producing cell (21) the camelid heavy chain antibodies have emerged in a normal immunological environment and it is expected that they will have undergone the selective refinement in specificity and affinity accompanying B cell maturation (1, 2). The obtention of camelid heavy chain antibodies could therefore be an invaluable asset in the development and engineering of soluble V H  domains (5) or of new immunologicals for diagnostic, therapeutic or biochemical purposes. 
     References 
     
         
         1. Tonegawa, S.  Nature  302, 575-581 (1983). 
         2. Jacob, J., Kelsoe, G., Rajewski, K., &amp; Weiss, U.  Nature  354, 389-392 (1991). 
         3. Fleischman J. B., Pain R. H. &amp; Porter R. R.  Arch. Biochem. Biophys  Suppl. 1, 174-180 (1962). 
         4. Utsumi, S. &amp; Karush, F.  Biochemistry  3, 1329-1338 (1964). 
         5. Ward, E. S., Güssow, D., Griffiths, A. d., Jones, P. T. &amp; Winter G.  Nature  341, 544-546 (1989). 
         6. Ungar-Waron H., Eliase E., Gluckman A. and Trainin Z.  Isr. J. Vet. Med . 43, 198-203 (1987). 
         7. Bajyana Songa, E., &amp; Hamers R.  Ann. Soc. Belge Méd. Trop . 68, 233-240 (1988). 
         8. Seligmann M., Mihaesco E., Preud&#39;homme J.-L., Danon F. &amp; Brouet J.-C.  Immun. Rev . 48, 145-167 (1979). 
         9. Traunecker, A., Schneider, J., Kiefer, H., Karjalaien, K.,  Nature  339, 68-70 (1989). 
         10. Henderschot L. M., Bole D., Köhler, G. &amp; Kearney, J. F.  J. Cell Biol . 104, 761-767 (1987). 
         11. Henderschot L. M.  J. Cell Biol . 111, 829-837 (1990). 
         12. Roholt O., Onoue K. &amp; Pressman D.  Proc. Natn. Acad. Sci. USA  51, 173-178 (1964). 
         13. Chothia, C., Novotny, J., Bruccoleri, R., Karplus, M.  J. Mol. Biol . 186, 651-663 (1985). 
         14. Kabat E. A., Wu, T. T., Reid-Miller, M., Perry H. M. &amp; Gottesman, K. S.  Sequences of Proteins of Immunological Interest  511 (U.S. Dept of Health and Human Services, US Public Health Service, National Institutes of Health, Bethesda, 1987). 
         15. Jackson, T., Morris, B. A, Sanders, P. G.  Molec. Immun . 29, 667-676 (1992). 
         16. Poljak R. J. et al.  Proc. Natn. Acad. Sci. USA  70, 3305-3310 (1973). 
         17. Dangl J. L., et al.  EMBO J . 7, 1989-1994 (1988). 
         18. Schneider W. P. et al.  Proc. Natn. Acad. Sci. USA  85, 2509-2513 (1988). 
         19. Evans, J. S. et al.  FEBS Lett . 208, 211-216 (1986). 
         20. Roditi, I. et al.  J. Cell Biol . 108, 737-746 (1989). 
         21. Dunnick, W., Rabbits, T. H., Milstein, C.  Nucl. Acids Res ., 8, 1475-1484 (1980). 
         22. Bülow, R., Nonnengässer, C., Overath, P.  Mol. Biochem. Parasitol . 32, 85-92 (1989). 
         23. Sambrook, J., Fritsch, E. F. &amp; Maniatis, T.  Molecular Cloning: A Laboratory Manual  2nd Edn (Cold Spring Harbor Laboratory Press, New York, 1989). 
         24. Sastry, L et al.  Proc. Natn. Acad. Sci. USA  86, 5728-5732 (1989). 
         25. Sanger, F., Nicklen, S. &amp; Coulson, A. R.  Proc. Natn. Acad. Sci. USA  74, 5463-5467 (1977). 
         26. Klein, J.  Immunology  (Blackwell Scientific Publications, London, 1990). 
       
    
     FIG. 1 Characterisation and purification or camel IgG classes on Protein A, Protein G and gel filtration. 
     (A) The fraction of  C. dromedarius  serum adsorbed on Protein A shows upon reduction on SDS-PAGE three heavy chain components of respectively 50, 46, and 43 Kd (bands between dots), absent in the non adsorbed fraction (lane d), and light chain components of around 30 Kd (lane c) considerably larger than rabbit light chain (lane a, rabbit IgG). The fractions adsorbed on Protein G (lane e) lack the 46 Kd heavy chain which remains in the non adsorbed fraction (lane f). Lane b contains a size marker. 
     (B and C) By differential adsorption and elution on Protein G and Protein A, the IgG fractions containing 43 Kd (lane 1), 46 Kd (lane 3) and 50 Kd (lanes 2) heavy chains were purified and analysed on SDS-PAGE in absence (B) or presence (C) of DTT. 
     (D) Whole camel serum (0.1 ml) was fractionated by gel filtration on a Superdex 200 column using 150 mM NaCl, 50 mM sodium phosphate buffer pH 7.0 as eluent. Affinity purified IgG 2  and IgG 3  elute at the positions indicated by arrows. The fractions of interest were further analysed by SDS-PAGE with or without prior reduction. The protein contents as visualized by Coomassie blue (without reduction, upper inset) are compared with the immunoglobulins from the same fractions (after reduction with DTT, lower inset) as revealed by Western blotting with a rabbit anti-camel-IgG (lower inset). 
     METHODS. 5 ml of  C. dromedarius  serum is adsorbed onto a 5 ml Protein G SEPHAROSE (Pharmacia) column, and washed with 20 mM phosphate buffer, pH 7.0. Upon elution with 0.15 M NaCl, 0.58% acetic acid (pH 3.5), IgG 3  of 100 Kd is eluted which upon reduction yields heavy chains of 43 Kd (lane 1, B and C). IgG 1  of 170 Kd can subsequently be eluted with a pH 2.7 buffer (0.1 M Gly-HCl). This fraction, upon reduction, yields a 50 Kd heavy chain and a broad light chain band (lane 2, C). The fraction not adsorbed on Protein G is brought on a 5 ml Protein A SEPHAROSE column. After washing and elution with 0.15 M NaCl, 0.58% acetic acid (pH 4.5) IgG 2  of 100 Kd is obtained which consists solely of 46 Kd heavy chains (lane 3, C). 
     FIG. 2 Repertoire complexity and antigen binding capacity or camel IgG 1 , IgG 2  and IgG 3  analysed by radioimmunoprecipitation (A) or Western blotting (B &amp; C). 
     (A) Serum or purified IgG fractions from healthy or  Trypanoma evansi  infected  C. dromedarius  (CATT titer 1/160 (7)) were incubated with labelled trypanosome lysate, recovered with Protein A SEPHAROSE and analysed by SDS-PAGE. The relative counts recovered are inscribed below each lane. No trypanosome proteins bind to the Protein A or to the healthy camel immunoglobulins. 
     (b) 20 μg of IgG 1 , IgG 2  and IgG 3  from healthy and trypanosome infected animals were separated by SDS-PAGE without prior reduction or heating. The electroblotted proteins were incubated with the labelled trypanosome lysate. The IgG 2  shows a single antigen binding component corresponding to the heavy chain immunoglobulin whereas the IgG 3  fraction appears to contain in addition two larger antigen binding components barely detectable by PONCEAU RED staining (C). These are possibly Ig classes copurified as immunocomplexes present in the serum of the infected animals. 
     METHODS. ( 35 S)-methionine labelled  Trypanosoma evansi  lysate (500,000 counts) (22) was incubated (4° C., 1 hour) with 10 μl of serum or 20 μg of IgG 1 , IgG 2  or IgG 3  in 200 μl of 0.4 M NaCl, 10 mM EDTA, 10 mM Tris (pH 8.3), containing 0.1 M TLCK. 10 mg of Protein A SEPHAROSE suspended in 200 μl of the same buffer was added (4° C., 1 hour). After washing and centrifugation, each pellet was resuspended in 75 μl SDS PAGE sample solution containing DTT, and heated for 3 min. at 100° C. After centrifugation, 5 μl of the supernatant was saved for radioactivity counting and the remainder analysed by SDS PAGE and fluorography. The nitrocellullose filter of the Western blot of purified fractions IgG 1 , IgG 2  and IgG 3  was stained with Ponceau Red (C) or incubated with 1% ovalbumin in TST buffer (Tris 10 mM, NaCl 150 mM, Tween 0.05%) (B). The membrane was extensively washed with TST buffer and incubated for 2 house with ( 35 S)-labelled trypanosome antigen. To avoid unspecific binding, the labelled trypanosome antigen lysate was filtered (45μ) and incubated with healthy camel immunoglobulin and ovalbumin adsorbed on a nitrocellulose membrane. 
     FIG. 3 Amino acid sequences of the V H  framework, and hinge/C H 2 of  Camelus dromedarius  heavy chain immunoglobulins, compared to human (italic) V H  framework (subgroup III) and hinges of human IgG (14). 
     METHODS. Total RNA was isolated from a dromedary spleen (23). mRNA was purified with oligo T-paramagnetic beads (PolyATract-Promega). 1 μg mRNA was used for preparing double-strand cDNA (23) after an oligo-dt priming using enzymes provided by Boehringer Mannheim. 5 μg of cDNA was amplified by PCR in a 100 μl reaction mixture (10 mM Tris-HCl pH 8.3, 50 mM KCl, 15 mM MgCl 2 , 0.01% (w/v) gelatine, 200 μM of each dNTP). 25 pmoles of each oligonucleotide of the mouse V H  (24), containing a XhoI site, and 5′-CGCCATCAAGGTACCAGT-TGA-3′ (see SEQ. ID. NO: 3) were used as primers. The 3′ end primer was deduced from partial sequences corresponding to γ chain amino acid 296 to 288 (T. Atarhouch, C. Hamers-Casterman, G. Robinson, private communication) in which one mismatch was introduced to create a KDnI restriction site. After a round of denaturing annealing (94° C. for 5 min. and 54° C. for 5 min.), 2 U of Taq DNA polymerase were added, to the reaction mixture before subjecting it to 35 cycles of amplification (5). The PCR products were purified by phenol-chloroform extraction followed by HPLC (Genpak-fax column, Waters) and finally by MERMAID (BIO 101, Inc.). After these purification steps, the amplified cDNA was digested with XhoI and KpnI, and ligated into pBluescript. 
     The clones were sequenced by the dideoxy chain termination method (25). The sequences were translated into amino acids which allowed their assignment to well defined domains of the Ig molecule (14); see SEQ. ID. NO: 4-12 and 63-71. 
     FIG. 4 Schematic representation of the structural organisation of the camel immunoglobulins (adapted from 26). 
     On the basis of size consideration, the IgG 1  fraction possess probably the normal antibody assembly of two light and two heavy chains. IgG 3  would have a hinge comparable in size to the human IgG 1 , IgG 2  and IgG 3 . The two antigen binding sites are much closer to each other is this camel IgG lacks the C H 1 domain. In the camel IgG 2  the long hinge, being formed of Pro-X repeats (X=Glu, Gln or Lys), most likely adopt a rigid structure (19,20). This long hinge could therefore substitute the C H 1 domain and bring the two antigen binding sites of IgG 2  to normal positions. 
     BACKGROUND OF THE INVENTION 
     Already at a very early stage during evolution antibodies have been developed to protect the host organisms against invading molecules or organisms. Most likely one of the earliest forms of antibodies must have been developed in Agnatha. In these primitive fishes antibodies of the IgM type consisting of heavy and lights chains have been detected. Also in many other forms of life ranging from amphibians to mammals antibodies are characterized by the feature that they consist of two heavy and two light chains, although the heavy chains of the various classes of immunoglobulins are quite different. These heavy and light chains interact with each other by a number of different physical forces, but interactions between hydrophobic patches present on both the heavy and light chain are always important The interaction between heavy and light chains exposes the complementarity determining regions (CDRs) of both chains in such a way that the immunoglobulin can bind the antigen optimally. Although individual heavy or light chains have also the capability to bind antigens (Ward et al., Nature 341 (1989) 544-546=ref. 5 of the above given draft publication) this binding is in general much less strong than that of combined heavy and light chains. 
     Heavy and light chains are composed of constant and variable domains. In the organisms producing immunoglobulins in their natural state the constant domains are very important for a number of functions, but for many applications of antibodies in industrial processes and products their variable domains are sufficient. Consequently many methods have been described to produce antibody fragments. 
     One of these methods is characterized by cleavage of the antibodies with proteolytic enzymes like papain and pepsin resulting in (a) antibody fragment comprising a light chain bound via an S-S bridge to part of a corresponding heavy chain formed by proteolytic cleavage of the heavy chain (Fab), or (b) a larger fragment of the antibody comprising two of these Fabs still connected to each other via an S-S bridge in enlargements of the heavy chain parts, indicated with F(ab) 2 , respectively (see patent applications EP-A-0125023 (GENENTECH/Cabilly et al., 1984) and WO-A-93/02198 (TECH. RES. CENT. FINLAND/Teeri et al., 1993) for definitions of these abbreviations). The disadvantage of the enzymatic route is that the production of whole antibodies is expensive and the enzymatic processing increases the costs of these fragments even more. The high costs of antibody fragments block the application of these fragments in processes and products outside the pharmaceutical industry. 
     Another method is based on linkage on DNA level of the genes encoding (parts of) the heavy chain and the light chain. This linkage and the subsequent production of these chimeric immunoglobulins in microorganisms have been described (for Fab fragments see e.g. Better et al., Science 240 (1988) 1041-1043, for F v  fragments (combination of variable fragments of the heavy chain (V H ) and light chain (V L ) still connected to each other by non-covalent binding interactions) see e.g. Skerra et al., Science 240 (1988) 1938, and for single chain F v  fragments (ScF v ; an F v  fragment in which the two variable fragments are linked to each other by a linker peptide) see e.g. Bird et al., Science 242 (1988) 423-426. Provided that an appropriate signal sequence has been placed in front of the single chain V H  and V L  antibody fragment (ScF v ), these products are translocated in  E. coli  into the periplasmic space and can be isolated and activated using quite elaborate and costly procedures. Moreover the application of antibody fragments produced by  E. coli  in consumer products requires extensive purification processes to remove pyrogenic factors originating from  E. coli . For this and other reasons the production of ScF v  in microorganisms that are normally used in the fermentation industry, like prokaryotes as Streptomyces or Bacillus (see e.g. Wu et al. Bio/Technology 11 (1993) 71) or yeasts belonging to the genera  Saccharomyces  (Teeri et al., 1993, supra),  Kluyveromyces, Hansenula,  or  Pichia  or moulds belonging to the genera  Aspergillus  or  Trichoderma  is preferred. However with a very few exceptions the production of ScF v  antibodies using these systems proved to be impossible or quite poor. Although the exact reasons for the poor production are not well known, the use of linkers between the V H  and V L  chains not designed for secretion (Teeri et al., 1993, supra) may be a reason. 
     Another reason may be incorrect folding of ScF v . The frameworks and to a limited extend the CDRs of variable domains of light and heavy chains interact with each other. It has been described by Chothia et al. (J. Mol. Biol. 186 (1985) 651-663=ref. 13 of the above given drift publication) that this interaction involves amino acids at the following positions of the variable region of the heavy chain: 35, 37, 39, 44-45, 47, 100-103 and 105 (numbering according to Kabat et al., In “ Sequences of Proteins of Immunological Interest , Public Health Service, NIH, Washington D.C., 1983=ref. 14 of the above given draft publication). Especially leucine at position 45 is strongly conserved and the whole apolar side chain of this amino acid seems to be involved in the interaction with the light chain. These strong interactions may fold the ScF v  into a structure that can not be translocated in certain types of lower eukaryotes. 
     Thus the use of a linker in the production of ScF v  for connecting a V H  chain to a V L  chain, might negatively influence either the translocation, or the folding of such ScF v  or both. 
     Not prior-published European patent application 92402326.0 filed 21 Aug. 1992 (C. Casterman &amp; R. Hamers) discloses the isolation of new animal-derived immunoglobulins devoid of light chains (also indicated as heavy chain immunoglobulins), which can especially originate from animals of the camelid family ( Camelidae ). This European patent specification, now publicly available as EP-A1-0 584 421, is incorporated herein by reference. These heavy chain immunoglobulins are characterized in that they comprise two heavy polypeptide chains sufficient for the formation of one or more complete antigen binding sites, whereby a complete antigen binding site means a site which will alone allow the recognition and complete binding of an antigen, which can be verified by any known method regarding the testing of the binding affinity. The European patent specification further discloses methods for isolating these heavy chain immunoglobulins from the serum of  Camelidae  and details of the chemical structure of these heavy chain immunoglobulins. It also indicates that these heavy chain immunoglobulins and derivatives thereof can be made by using recombinant DNA technology in both prokaryotes and eukaryotes. The present invention relates to a further development of the work disclosed in that prior-filed but not prior-published European specification. 
     Due to the absence of light chains in most of the immunoglobulins of  Camelidae  such linkers are not necessary, thereby avoiding the above-mentioned potential problems. 
     As described above in the draft publication for Nature, now publicly available as Nature 363 (3 Jun. 1993) 446-448, and in the not prior-published European patent application 92402326.0 (supra) it was surprisingly found that the majority of the protein A-binding immunoglobulins of  Camelidae  consists just of two heavy chains and that these heavy chains are quite different from common forms of heavy chains, as the C H 1 domain is replaced by a long or short hinge (indicated for IgG 2  and IgG 3 , respectively, in FIG. 4 of the above given draft publication for Nature). 
     Moreover these heavy chains have a number of other features that make them remarkably different from the heavy chains of common immunoglobulins. 
     One of the most significant features is that they contain quite different amino acid residues at those positions involved in binding to the light chain, which amino acids are highly conserved in common immunoglobulins consisting of two heavy and two light chains (see Table 1 and SEQ. ID. NO: 13-31). 
     
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Comparison al amino acid sequences of various immunoglobulins 
               
               
                 Alignment of a number of V H  regions of Camel heavy 
               
               
                 chain antibodies compared with those of mouse (M, top line) 
               
               
                 and human (H, second line). Framework fragments are indicated 
               
               
                 in capitals, CDR fragments in small print; see SEQ. ID. NO: 
               
               
                 13-31 for sequences indicated by M, H, 1, 2, 3, 7, 9, 11, 
               
               
                 13, 16, 17, 18, 19, 20, 21, 24, 25, 27, 29, respectively. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1                                                   50 
               
               
                 m 
                 EVKLVESGGG LVQPGGSLRL SCATSGFTFS dfyme..WVR QPPGKRLEWI 
               
               
                 h 
                 EVQLVESGGG LVQPGGSLRL SCAASGFTFS syams..WVR QAPGKGLEWV 
               
               
                 cam1 
                 ........GG SVQAGGSLRL SCAASGYSNC pltws..WYR QFPGTEREFV 
               
               
                 cam2 
                 DVQLVASGGG SVQAGGSLRL SCTASGDSFS rfams..WFR QAPGKECELV 
               
               
                 cam3 
                 ........GG SVQTGGSLRL SCAVSGFSFS tscma..WFR QASGKQREGV 
               
               
                 cam7 
                 ........GG SVQGGGSLRL SCAISGYTYG sfcmg..WFR EGPGKEREGI 
               
               
                 cam9 
                 ........GG SVQAGGSLTL SCVYTNDTGT ...mg..WFR QAPGKECERV 
               
               
                 cam11 
                 ........GG SVQAGGSLRL SCNVSGSPSS tyclg..WFR QAPGREREGV 
               
               
                 cam13 
                 ........GG SVEAGGSLRL SCTASGYVSS ...ma..WFR QVPGQEREGV 
               
               
                 cam16 
                 ........GG SAQAGGSLRL SCAAHGIPLN gyyia..WFR QAPGKGREGV 
               
               
                 cam17 
                 ........GG SVQPGGSLTL SCTVSGATYS dysig..WIR QAPGKDREVV 
               
               
                 cam18 
                 ........GG SVQAGGSLRL SCTGSGFPYS tfclg..WFR QAPGKEREGV 
               
               
                 cam19 
                 ........GG SVQAGGSLRL SCAASDYTIT dycma..WFR QAPGKERELV 
               
               
                 cam20 
                 ........GG SVQVGGSLRL SCVASTHTDS stcig..WFR QAPGKEREGV 
               
               
                 cam21 
                 ........GG SVQVGGSLKL SCKISGGTPD rvpkslaWFR QAPEKEREGI 
               
               
                 cam24 
                 ........GG SVQAGGSLRL SCNVSGSPSS tyclg..WFR QAPGKEREGV 
               
               
                 cam25 
                 ........GG SVQTGGSLRL SCEISGLTFD dsdvg..WYR QAPGDECKLV 
               
               
                 cam27 
                 ........GG SVQAGGSLRL SCASSSKYMP ctydmt.WYR QAPGKEREFV 
               
               
                 cam29 
                 .....exxGG SVQAGGSLRL SCVASGFNFE tsrma..WYR QTPGNVCELV 
               
               
                   
                 51=================================================100 
               
               
                 m 
                 A..asrnkan dytteysasv kgRFIVSRDT SQSILYLQMN ALRAEDTAIY 
               
               
                 h 
                 S..xisxktd ggxtyyadsv kgRFTISRDN SKNTLYLQMN SLRAEDTAVY 
               
               
                 cam1 
                 S..smd...p dgntkytysv kgRFTMSRGS TEYTVFLQMD NLKPEDTAMY 
               
               
                 cam2 
                 S..siq...s ngrtteadsv qgRFTISRDN SRNTVYLQMN SLKPEDTAVY 
               
               
                 cam3 
                 Aainsgggrt yyntyvaesv kgRFAISQDN AKTTVYLDMN NLTPEDTATY 
               
               
                 cam7 
                 A..tiln..g gtntyyadsv kgRFTISQDS TLKTMYLLMN NLKPEDTGTY 
               
               
                 cam9 
                 A..hit...p dgmtfidepv kgRFTISRDN AQKTLSLRMN SLRPEDTAVY 
               
               
                 cam11 
                 T..aint..d gsiiyaadsv kgRFTISQDT AKETVHLQMN NLQPEDTATY 
               
               
                 cam13 
                 A..fvqt..a dnsalygdsv kgRFTISHDN AKNTLYLQMR NLQPDDTGVY 
               
               
                 cam16 
                 A..ting..g rdvtyyadsv tgRFTISRDS PKNTVYLQMN SLKPEDTAIY 
               
               
                 cam17 
                 A..aant..g atskfyvdfv kgRFTISQDN AKNTVYLQMS FLKPEDTAIY 
               
               
                 cam18 
                 A..gins..a ggntyyadav kgRFTISQGN AKNTVFLQMD NLKPEDTAIY 
               
               
                 cam19 
                 A.aiqvvrsd trltdyadsv kgRFTISQGN TKNTVNLQMN SLTPEDTAIY 
               
               
                 cam20 
                 A..siyf..g dggtnyrdsv kgRFTISQLN AQNTVYLQMN SLKPEDSAMY 
               
               
                 cam21 
                 A..vlst..k dgktfyadsv kgRFTIFLDN DKTTFSLQLD RLNPEDTADY 
               
               
                 cam24 
                 T..aint..d gsviyaadsv kgRFTISQDT AKKTVYLQMN NLQPEDTATY 
               
               
                 cam25 
                 Sgilsdgtpy tksgdyaesv rgRVTISRDN AKNMIYLQMN DLKPEDTAMY 
               
               
                 cam27 
                 S..sin...i dgkttyadsv kgRFTISQDS AKNTVYLQMN SLKPEDTAMY 
               
               
                 cam29 
                 S..siy...s dgktyyvdrm kgRFTISREN AKNTLYLQLS GLKPEDTAMY 
               
               
                   
                 101=====================================139 
               
               
                 m 
                 YCARdyygss .......y.. f.....dvWG AGTTVTVSS 
               
               
                 h 
                 YCARxxxxxx xxxxxyyyyh x....fdywG QGTLVTVSS 
               
               
                 cam1 
                 YCKTalqpgg ycgygx.... ......clWG QGTQVTVSS 
               
               
                 cam2 
                 YCGAvslmdr isqh...... ......gcRG QGTQVTVSL 
               
               
                 cam3 
                 YCAAvpahlg pgaildlkky ......kyWG QGTQVTVSS 
               
               
                 cam7 
                 YCAAelsggs celpllf... ......dyWG QGTQVTVSS 
               
               
                 cam9 
                 YCAAdwkywt cgaqtggyf. ......gqWG QGAQVTVSS 
               
               
                 cam11 
                 YCAArltemg acdarwatla trtfaynywG QGTQVTVSS 
               
               
                 cam13 
                 YCAAqkkdrt rwaeprew.. ......nnWG QGTQVTASS 
               
               
                 cam16 
                 FCAAgsrfss pvgstsrles .sdy..nyWG QGIQVTASS 
               
               
                 cam17 
                 YCAAadpsiy ysilxiey.. ......kyWG QGTQVTVSS 
               
               
                 cam18 
                 YCAAdspcym ptmpappird sfgw..ddFG QGTQVTVSS 
               
               
                 cam19 
                 SCAAtssfyw ycttapy... ......nvWG QGTQVTVSS 
               
               
                 cam20 
                 YCAIteiewy gcnlrttf.. ......trWG QGTQVTVSS 
               
               
                 cam21 
                 YCAAnqlagg wyldpnywls vgay..aiWG QGTHVTVSS 
               
               
                 cam24 
                 YCAArltemg acdarwatla trtfaynyWG RGTQVTVSS 
               
               
                 cam25 
                 YCAVdgwtrk eggiglpwsv qcedgynywG QGTQVTVSS 
               
               
                 cam27 
                 YCKIdsypch ll........ ......dvWG QGTQVTVSS 
               
               
                 cam29 
                 YCAPveypia dmcs...... ......ryGD PGTQVTVSS 
               
               
                   
               
             
          
         
       
     
     For example, according to Pessi et al. (1993) a subdomain portion of a V H  region of common antibodies (containing both heavy chains and light chains) is sufficient to direct its folding, provided that a cognate V L  moiety is present. Thus it might be expected from literature on the common antibodies that without V L  chains proper folding of heavy chains cannot be achieved. A striking difference between the common antibodies and the  Camelidae -derived heavy chain antibodies is, that the highly conserved apolar amino acid leucine (L) at place 45 present in common antibodies is replaced in most of the  Camelidae -derived heavy chain antibodies by the charged amino acid arginine (R), thereby preventing binding of the variable region of the heavy chain to that of the light chains. 
     Another remarkable feature is that one of the CDRs of the heavy chains of this type of immunoglobulins from  Camelidae , CDR3, is often much longer than the corresponding CDR3 of common heavy chains. Besides the two conserved cysteines forming a disulphide bridge in common V H  fragments, the  Camelidae  V H  fragments often contain two additional cysteine residues, one of which often is present in CDR3. 
     According to the present inventors these features indicate that CDR3 may play an important role in the binding of antigens by these heavy chain antibodies and can compensate for the absence of light chains (also containing CDRs) in binding of antigens by immunoglobulins in  Camelidae . 
     Thus, as the heavy chains of  Camelidae  do not have special features for interacting with corresponding light chains (which are absent), these heavy chains are very different from common heavy chains of immunoglobulins and seem intrinsically more suitable for secretion by prokaryotic and lower eukaryotic cells. 
     The present inventors realized that these features make both intact heavy chain immunoglobulins of  Camelidae  and fragments thereof very attractive for their production by microorganisms. The same holds for derivatives thereof including functionalized fragments. In this specification the term “functionalized fragment” is used for indicating an antibody or fragment thereof to which one or more functional groups, including enzymes and other binding polypeptides, are attached resulting in fusion products of such antibody fragment with another biofunctional molecule. 
     SUMMARY OF THE INVENTION 
     In a broad sense the invention provides a process for the production of an antibody or a fragment or functionalized fragment thereof using a transformed lower eukaryotic host containing an expressible DNA sequence encoding the antibody or (functionalized) fragment thereof, wherein the antibody or (functionalized) fragment thereof is derived from a heavy chain immunoglobulin of  Camelidae  and is devoid of light chains, and wherein the lower eukaryotic host is a mould or a yeast. Thus the lower eukaryotic host can be a mould e.g. belonging to the genera  Aspergillus  or  Trichoderma , or a yeast, preferably belonging to the yeast genera  Saccharomyces ,  Kluyveromyces, Hansenula,  or  Pichia . Preferably the fragments still contain the whole variable domain of these heavy chains. 
     The invention also provides methods to produce such heavy chain immunoglobulins or (functionalized) fragments thereof in which methods the framework or the CDRs of these heavy chains are modified by random or directed mutagenesis in such a way that the mutated heavy chain is optimized for secretion by the host microorganism into the fermentation medium. 
     Another embodiment of the invention is that CDRs can be grafted on these optimized frameworks (compare grafting of CDRs on human immunoglobulins as described by e.g. Jones et al., Nature 321 (1986) 522). These CDRs can be obtained from common antibodies or they may originate from heavy chain immunoglobulins of  Camelidae.  The binding properties may be optimized by random or directed mutagenesis. Thus in a process according to the invention an antibody or (functionalized) fragment thereof derived from a heavy chain immunoglobulin of  Camelidae  can be produced which comprises a CDR different from the CDR belonging to the natural antibody ex  Camelidae  which is grafted on the framework of the variable domain of the heavy chain immunoglobulin ex  Camelidae.    
     The invention also provides a method for the microbiological production of catalytic antibodies. These antibodies are preferably raised in  Camelidae  against transition state molecules following procedures similar to the one described by Lerner et al., Science 252 (1991) 659-667. Using random or site-directed mutagenesis such catalytic antibodies or fragments thereof can be modified in such a way that the catalytic activity of these (functionalized) antibodies or fragments can be further improved. 
     For preparing modified heavy chain antibodies a process according to the invention is provided, in which the DNA sequence encodes a modified heavy chain immunoglobulin or a (functionalized) fragment thereof derived from  Camelidae  and being devoid of light chains, and is made by random or directed mutagenesis or both. Thus the resulting immunoglobulin or (functionalized) fragment thereof is modified such that
         it is better adapted for production by the host cell, or   it is optimized for secretion by the lower eukaryotic host into the fermentation medium, or   its binding properties (k on  and k off ) are optimized, or   its catalytic activity is improved, or   it has acquired a metal chelating activity, or   its physical stability is improved.       

     Another particular embodiment of the present invention relates to genes encoding fusion proteins consisting of both a heavy chain immunoglobulin from  Camelidae  or part thereof and a second protein or another polypeptide, e.g. an enzyme, in particular an oxido-reductase, and to expression products of such genes. By means of the heavy chain immunoglobulin (fragment) the protein or enzyme can be guided to a target thereby increasing the local efficiency of the protein or enzyme significantly. Thus according to this embodiment of the invention a process is provided, in which the functionalized antibody or fragment thereof comprises a fusion protein of both a heavy chain immunoglobulin from  Camelidae  or a fragment thereof and another polypeptide, e.g. an enzyme, preferably an oxido-reductase. 
     As a result of a process according to the invention known products may be produced, e.g. antibodies also produced by  Camelidae,  but many of the possible products will be new products, thus the invention also provides new products obtainable by a process according to the invention. 
     The products so produced can be used in compositions for various applications. Therefore, the invention also relates to compositions containing a product produced by a process according to the invention. This holds for both old products and new products. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       FIGS. 1A-1D Characterisation and purification of camel IgG classes on Protein A, Protein G and gel filtration. 
       FIGS. 2A-2C Repertoire complexity and antigen binding capacity of camel IgG 1 , IgG 2  and IgG 3  analysed by radioimmunoprecipitation (A) or Western blotting (B &amp; C). 
       FIGS. 3A-3B Amino acid sequences of the V H  framework (see A) and hinge C H 2 (see B) of  Camelus dromedarius  heavy chain immunoglobulins, compared to human (italic) V H  framework (subgroup III) and hinges of human IgG (14); see SEQ ID NO: 4-12. 
       FIGS. 4A-4C Schematic representation of the structural organization of the camel immunoglobulins (adapted from 26). 
       FIG. 5 DNA and amino acid sequences of the Camel V H  fragments followed by the Flag sequence as present in pB03 (FIG. 5B), pB09 (FIG. 5A) and pB24 (FIG. 5C); see SEQ ID NOs. 34 and 35, 32 and 33, and 36 and 37, respectively. 
       FIG. 6 Nucleotide sequence of synthetic DNA fragment cloned into pEMBL9 (Example 1) (see SEQ. ID. NO: 38-39) and encoded protein sequences (SEQ ID NO: 40-41). The bottom strand of the DNA restriction fragrant, SEQ ID NO: 39, is shown 3′ to 5′. 
       FIG. 7 Schematic drawing of plasmid pUR4423 
       FIG. 8 Schematic drawing of plasmid pUR4426 
       FIG. 9 Schematic drawing of plasmid pUR2778 
       FIG. 10 Schematic drawing of plasmid pUR4429 
       FIG. 11 Schematic drawing of plasmid pUR4430 
       FIG. 12 Schematic drawing of plasmid pUR4445 
       FIG. 13 Schematic drawing of plasmid pUR4446 
       FIG. 14 Schematic drawing of plasmid pUR4447 
       FIG. 15 Schematic drawing of plasmid pUR4451 
       FIG. 16 Schematic drawing of plasmid pUR4453 
       FIG. 17 Schematic drawings of plasmids pUR4437 and pUR4438 
       FIGS. 18A-B Schematic drawings of plasmids pUR4439 (see A) and pUR4440 (see B). 
       FIG. 19 Nucleotide sequence of synthetic DNA fragment cloned into pEMBL9 (Example 6) (see SEQ. ID. NO: 42-43) and encoded protein sequences (SEQ ID NO: 44-45). The bottom strand of the DNA restriction fragment, SEQ ID NO: 43, is shown 3′ to 5′. 
       FIG. 20 Schematic drawing of plasmid pAW14B. 
       FIGS.  21A-B Western blot analysis of culture medium of    S. cerevisiae  transformants containing pUR4423M (see A) or pUR4425M (see B). Samples were taken after 24 (see 1) or 48 hours (see 2). For pUR4425M, two bands were found due to glycosylation of the antibody fragment. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention relates to the production of antibodies or (functionalized) fragments thereof derived from heavy chain immunoglobulins of  Camelidae  by eukaryotes, more in particular by lower eukaryotes such as yeasts and fungi. Therefore, mRNA encoding immunoglobulins of  Camelidae  was isolated and transcribed into cDNA according to the procedures described in the above given draft publication and not prior-published European patent application 92402326.0. In each case primers for the PCR reaction directed to the N-terminus of the V H domain and PCR primers that either hybridize with the C-terminal regions of the V H  domain or with the short or large hinge regions as described in the above given draft publication, or with the C-terminal region of the C H 2 or C H 3 domains can be used. In this way structural genes can be obtained encoding the following fragments of heavy chain immunoglobulins of  Camelidae  (Table 2). 
     
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 The various forms of immunoglobulins 
               
               
                 of Camelidae that can be 
               
               
                 expressed in microorganisms. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 a. 
                 the variable domain of a heavy chain; 
               
               
                 b. 
                 the variable domain and the short hinge of a heavy chain; 
               
               
                 c. 
                 the variable domain and the long hinge of a heavy chain; 
               
               
                 d. 
                 the variable domain, the C H 2 domain, and either 
               
               
                   
                 the short or long hinge of a heavy chain; 
               
               
                 e. 
                 a complete heavy chain, including either the short or long hinge. 
               
               
                   
               
             
          
         
       
     
     According to procedures described in detail in the Examples these cDNAs can be integrated into expression vectors. 
     Known expression vectors for  Saccharomyces, Kluyveromyces, Hansenula, Pichia  and  Aspergillus  can be used for incorporating a cDNA or a recombinant DNA according to the invention. The resulting vectors contain the following sequences that are required for expression: (a) a constitutive, or preferably an inducible, promoter; (b) a leader or signal sequence; (c) one of the structural genes as described in Table 2 and (d) a terminator. If the vector is an episomal vector, it preferably comprises an origin of replication as well as a selection marker, preferably a food grade selection marker, (EP-A-487159, UNILEVER/Leenhouts et al.). If the vector is an integration vector, then it preferably comprises sequences that ensure integration and a selection marker in addition to the sequences required for expression of the structural gene encoding a form of the heavy chain immunoglobulin of  Camelidae  or derivatives thereof. The preferred sequences for integration are sequences encoding ribosomal DNA (WO 91/00920, 1991, UNILEVER/Giuseppin et al.) whereas the selection marker will be preferably a food grade marker. 
     For  Saccharomyces  the preferred inducible promoter is the GAL7 promoter (EP-A-0255153, UNILEVER/Fellinger et al.); for  Kluyveromyces  the preferred inducible promoter is the inulinase promoter (not yet published EP application 92203932.6, UNILEVER/Toschka &amp; Verbakel, which is incorporated herein by reference); for  Hansenula  or  Pichia  the preferred inducible promoter is the methanol-oxidase promoter (Sierkstra et al., Current Genetics 19 (1991) 81-87) and for  Aspergillus  the preferred inducible promoter is the endo-xylanase promoter (not prior-published PCT application PCT/EP 92/02896, UNILEVER/Gouka et al., now publicly available as WO-A-93/12237, which is incorporated herein by reference). 
     To achieve efficient secretion of the heavy chain immunoglobulin or parts thereof the leader (secretion) sequences of the following proteins are preferred: invertase and α-factor for  Saccharomyces,  inulinase for  Kluyveromyces,  invertase for  Hansenula  or  Pichia  (Sierkstra et al., 1991 supra) and either glucoamylase or xylanase for  Aspergillus  (not prior-published PCT application WO-A-93/12237, supra). As food-grade selection markers, genes encoding anabolic functions like the leucine2 and tryptophan3 are preferred (Giuseppin et al. 1991, supra). The present invention describes the heterologous production of (functionalized) derivatives or fragments of immunoglobulins in a microorganism, which immunoglobulins in nature occur not as a composite of heavy chains and light chains, but only as a composite of heavy chains. Although the secretion mechanism of mammals and microorganisms is quite similar, in details there are differences that are important for developing industrial processes. 
     To obtain frameworks of the heavy chain immunoglobulins, that are optimally secreted by lower eukaryotes, genes encoding several different heavy chains can be cloned into the coat protein of bacteriophages and subsequently the frameworks of these heavy chain immunoglobulins can be mutated using known PCR technology, e.g. Zhou et al., (1991). Subsequently the mutated genes can be been cloned in  Saccharomyces  and  Aspergillus  and the secretion of the mutated genes can be compared with the wild type genes. In this way frameworks optimized for secretion may be selected. 
     Alternatively these structural genes can be linked to the cell wall anchoring part of cell wall proteins, preferably GPI-linked cell wall proteins of lower eukaryotes, which result in the expression of a chimeric protein on the cell wall of these lower eukaryotes (not prior-published EP application 92202080.5, UNILEVER/Klis et al., now publicly available as International (PCT) patent application WO-A-94/01567, which is incorporated herein by reference). 
     Both methods have the advantage that the binding parts of the immunoglobulins are well exposed to the surrounding of the cell, microorganism, or phage and therefore can bind antigens optimally. By changing the external conditions the binding rates and dissociation rates of this binding reaction can be influenced. Therefore, these systems are very suitable to select for mutated immunoglobulins that have different binding properties. The mutation of the immunoglobulins can either be obtained by random mutagenesis, or directed mutagenesis based on extensive molecular modelling and molecular dynamical studies. 
     mRNAs encoding heavy chains of immunoglobulins raised in  Camelidae  against transition state molecules (Lerner et al., 1991 supra) can be obtained using standard techniques. The structural genes encoding various forms of immunoglobulins according to the invention as summarized in Table 2 can be cloned into the coat protein of bacteriophages or as fusion with the anchoring part of cell wall proteins and can be tested on the catalytic property. In this way immunoglobulins or parts thereof having catalytic properties can be determined and selected. Genes encoding these selected immunoglobulins or parts thereof can be mutated as described before and recloned in bacteriophages, but preferably cloned as chimeric cell wall bound catalysts in lower eukaryotes. By performing appropriate catalytic assays, catalytic immunoglobulins or parts thereof with improved catalytic properties can be determined and selected using standard techniques. 
     An important application of antibodies, especially outside the pharmaceutical industry, will be chimeric proteins consisting of the binding part of antibodies and enzymes. In this way catalytic biomolecules can be designed that have two binding properties, one of the enzyme and the other of the antibody. This can result in enzymes that have superior activity. This can be illustrated with the following examples:
         a. If the substrate of the enzymic reaction is produced by an organism or an enzyme is recognized by the binding domain of the antibody, the local concentration of the substrate will be much higher than for enzymes lacking this binding domain and consequently the enzymic reaction will be improved. In fact this is a mimic of vectorial metabolism in cells (compare e.g. Mitchell, (1979) Science 206 1148-1159);   b. If the substrate of the enzymic reaction is converted into a molecule that kills organisms, then the efficiency and specificity of killing can be increased significantly if the enzyme is equipped with an antibody binding domain that recognizes the target organism (e.g. compare Takahashi et al., (1993) Science 259 1460-1463);       

     The invention will be illustrated by the following Examples without being limited thereto. In previously filed Unilever patent specifications several expression vectors were described, e.g. for the yeasts  S. cerevisiae ,  Kluyveromyces,  and  Hansenula,  and the mould  Aspergillus.  Examples of these publications are EP-A-0173378 (UNILEVER/Ledehoer et al.), EP-A-0255153, supra, and PCT applications WO-A-91/19782 (UNILEVER/van Gorcom et al.) and (not prior-published) WO-A-93/12237, supra. The genes encoding antibodies or (functionalized) fragments thereof according to the invention can be incorporated into the earlier described expression vectors or derivatives thereof using procedures well known to a skilled person in the art. All techniques used for the manipulation and analysis of nucleic acid materials were performed essentially as described in Sambrook et al. (1989) (see also ref. 23 of the above given draft publication), except where indicated otherwise. 
     In the description of the Examples the following endonuclease restriction sites are used: 
     
       
         
               
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 AflH 
                 C↓TTAAG 
                 Mlul 
                 A↓CGCGT 
               
               
                   
                 BspHI 
                 T↓CATGA 
                 Ncol 
                 C↓CATGG 
               
               
                   
                 BspHI 
                 T↓CATGA 
                 Not 
                 GC↓GGCCGC 
               
               
                   
                 BstEH 
                 G↓GTNACC 
                 Nrul 
                 TCG↓CGA 
               
               
                   
                 EagI 
                 C↓GGCCG 
                 SalI 
                 G↓TCGAC 
               
               
                   
                 EcoRI 
                 G↓AATTC 
                 XhoI 
                 C↓TCGAG 
               
               
                   
                 HindIII 
                 A↓AGCTT 
                 BbsI 
                 GAAGAC(N) 2 ↓ 
               
               
                   
                   
                   
                   
                 CTTCTG(N′) 6 ↓ 
               
               
                   
                   
               
             
          
         
       
     
     EXAMPLE 1 
     Construction of Cassettes Encoding V H  Fragments Originating from  Camelidae    
     For the production of V H  fragments originating from  Camelidae,  the antibody gene fragments were isolated and cloned as described above in the draft publication. The thus obtained gene fragments encode the V H  region, a short or a long hinge region and about 14 amino acids of the C H 2 region. By using standard molecular biological techniques (e.g. PCR technology), the V H  gene fragments could be subcloned and equipped at their 5′-ends with a gene fragment encoding the pelB signal sequence and at their 3′-ends with a gene fragment encoding the Flag tail (13 amino acids). Three of these clones were named pB3, pB9 and pB24 and were deposited at the Centraal Bureau voor Schimmelcultures, Baarn on 20 Apr. 1993 with deposition numbers: CBS 270.93, CBS 271.93 and CBS 272.93, respectively. The DNA and amino acid sequences of the  Camelidae -V H  fragments followed by the Flag sequence are presented in FIG. 5(A-C): see SEQ. ID. NO: 32-37. 
     1.1 Construction of pUR4421 
     For the construction of yeast expression plasmids encoding the V H  fragments preceded by the invertase (=SUC2) signal sequence, the α-mating factor prepro-sequence, or the inulinase signal sequence and followed by either nothing; or a Myc tail or Flag tail, the constructs described below can be prepared. 
     The multiple cloning site of plasmid pEMBL9 (Denthe et al., 1983) (ranging from the EcoRI to the HindIII site) wits replaced by a synthetic DNA fragment having the nucleotide sequence as indicated in FIG. 6; see SEQ. ID. NO: 38-41. The 5′-part of this nucleotide sequence comprises an EagI site, the first 4 codons of the  Camelidae  V H  gene fragment and a XhoI site coinciding with codons 5 and 6. The 3′-part comprises the last 5 codons of the  Camelidae  V H  gene (encoding VTVSS; see SEQ. ID. NO: 47) part of which coincides partially with a BstEII site), eleven codons of the Myc tail, and an EcoRI site. The EcoRI site, originally present in pEMBL9, is not functional any more, because the 5′-end of the nucleotide sequence contains AATTT instead of AATTC, indicated in FIG. 6 as “(EcoRI)”. The resulting plasmid is called pUR4421. 
     1.2 Constructs with Flag Tail 
     After digesting the plasmid pB3 with XhoI and EcoRI, a DNA fragment of approximately 425 bp was isolated from agarose gel. This fragment codes for a truncated V H -Flag fragment, missing the first 5 amino acids of the  Camelidae  V H . The obtained fragment can be cloned into pUR4421. To this end plasmid pUR4421 can be digested with XhoI and EcoRI, after which the about 4 kb vector fragment can be isolated from an agarose gel. Ligation with the about 425 bp fragment will result in plasmid pUR4421-03F. 
     1.3 Constructs with Myc Tail 
     After digesting the plasmid pB3 with XhoI and BstEII, a DNA fragment of approximately 365 bp was isolated from agarose gel. This fragment codes for a truncated V H  fragment, missing both the first 4 (QVKL; see SEQ. ID. NO: 46) and the last 5 (VTVSS; see SEQ. ID. NO: 47) amino acids of the  Camelidae  V H  fragment. 
     The obtained fragment was cloned into pUR4421. To this end plasmid pUR4421 was digested with XhoI and BstEII, after which the about 4 kb vector fragment was isolated from an agarose gel. Ligation with the about 365 bp fragment resulted in plasmid pUR4421-03M, in which the gene encoding the V H  fragment is reconstituted. 
     1.4 Constructs Encoding V H  Only 
     Upon digesting pUR4421-03M or pUR4421-03F with BstEII and HindIII, the vector fragments of about 4.4 kb can be isolated from agarose gel and religated in the presence of a synthetic linker peptide having the following sequence: 
                               BstEII                HindIII                GTCACC GTCTCCTCATAATG A                        GCAGAGGAGTATTACTTCGA            
(top strand, SEQ ID NO: 48, is shown 5′ to 3′; bottom strand, SEQ ID NO: 49, is shown 3′ to 5′. In the thus obtained plasmid, pUR4421-03, the Myc tail or Flag tail sequences are removed and the V H  gene fragment is directly followed by a stop codon.
 
     1.5 Other Constructs 
     After isolating the gene fragments encoding V H -hinge-C H 2 fragments as described above in the draft publication, or encoding the intact heavy chain immunoglobulin, it is possible, e.g. by using PCR technology, to introduce an appropriate restriction enzyme recognition site (e.g. EcoRI or HindIII) downstream of the hinge region, downstream of the C H 2 region, or downstream of the total gene. Upon isolating a XhoI-EcoRI or XhoI-HindIII fragment encoding the V H  fragment with a C-terminal extension, the fragment can be cloned into pUR4421 digested with the same restriction enzymes. 
     In analogy with the construction of pUR4421-03, a number of other constructs can be produced encoding functionalized heavy chain fragments in which a second polypeptide is fused to the C-terminal part of the V H  fragment. Optionally, the V H  fragment and the second polypeptide, e.g. an enzyme, might be connected to each other by a peptide linker. 
     To this end either the BstEII-HindIII fragment or the BstEII-EcoRI fragment of either pUR4421-03F or pUR4421-03M has to be replaced by another BstEII-HindIII or BstEII-EcoRI fragment. The latter new fragment should code for the last amino acids (VTVSS, see SEQ. ID. NO: 47) of the V H  fragment, optionally for a linker peptide, and for the polypeptide of interest e.g. an enzyme. Obviously, the introduction of the DNA fragment should result in an in frame fusion between the V H  gene fragment and the other DNA sequence encoding the polypeptide of interest. 
     Alternatively, it is possible to replace the EagI-XhoI fragment of pUR4421-03 with another DNA fragment, coding for a polypeptide of interest, optionally for a peptide linker, and for the first 4 (QVKL, see SEQ. ID. NO: 46) amino acids of the V H  fragment, resulting in an in frame fusion with the remaining part of the V H  fragment. In this way, it is possible to construct genes encoding functionalized V H  fragments in which the second polypeptide is fused at the N-terminal part of the V H  fragment, optionally via a peptide linker. 
     Obviously, it is also possible to construct genes encoding functionalized V H  fragments having a polypeptide fused to the N-terminal as well as fused to the C-terminal end, by combining the above described construction routes. 
     The polypeptides used to functionalize the V H  fragments might be small, like the Myc and the Flag tails, or intact enzymes, like glucose oxidase, or both. 
     From all the above described constructs, derived from pUR4421, an appropriate EagI-HindIII fragment, encoding the functionalized V H  fragment, can be isolated and cloned into a number of different expression plasmids. Several are exemplified in more detail in the following Examples. Although only the V H  fragments are exemplified, similar constructs can be prepared for the production of larger heavy chain fragments (e.g. V H -hinge or V H -hinge-C H 2) or intact heavy chains. The EagI site is introduced before the first codon of the V H  fragment, facilitating an in frame fusion with different yeast signal sequences. 
     In particular cases, were additional EagI and/or HindIII sites are present in the cloned fragments, it is necessary to perform partial digestions with one or both restriction enzymes. 
     Although the above and following constructions only consider the V H  fragment cloned in pB3, a comparable construction route can be used for the construction of expression plasmids for the production of V H  fragments like V H -09 and V H -24, or other V H  fragments. 
     EXAMPLE 2 
     Construction or  S. cerevisiae  Episomal Expression Plasmids for  Camelidae  V H    
     For the secretion of recombinant protein from  S. cerevisiae  it is worthwhile to test in parallel the two most frequently applied homologous signal sequences, the SUC2 invertase signal sequence and the prepro-α mating factor sequence. 
     The episomal plasmid pSY1 and pSY16 (Harmsen et al., 1993) contain expression cassettes for the α-galactosidase gene. Both plasmids contain the GAL7 promoter and PGK terminator sequences. pSY1 contains the invertase (SUC2) signal sequence and pSY16 contains a slightly modified (Harmsen et al., 1993) prepro-α-mating factor signal sequence. 
     Both plasmids, pSY1 and pSY16 can be digested with EagI and HindIII, the about 6500 bp long vector backbone of both plasmids can be isolated and subsequently ligated with the EagI/HindIII fragments from pUR4421-03F ( − 465 bp), pUR4421-03M ( − 455 bp) or pUR4421-03 ( − 405 bp) (See above). 
     This results in a series of 6 different episomal plasmids for expression in  S. cerevisiae , containing behind the SUC2- and the α mating factor prepro-sequence the V H -Flag coding sequence (designated pUR4423F and pUR4426F), the V H -Myc coding sequence (designated pUR4423M and pUR4426M) or the coding sequence of V H  followed by a stop codon (designated pUR4423, FIG. 7 and pUR4426, FIG. 8). 
     Obviously, it is possible to use promoter systems different from the inducible GAL7 promoter, e.g. the constitutive GAPDH promoter. 
     2.1 Production or V H -03-myc and V H -24-myc 
     After introducing the expression plasmids pUR4423M (coding for V H -03-myc, preceded by the SUC2-signal sequence) and pUR4425M (coding for V H -24-myc, preceded by the SUC2-signal sequence) into  S. cerevisiae  via electroporation, transformants were selected from minimal medium agar plates (comprising 0.7% yeast nitrogen base, 2% glucose and 2% agar, supplemented with the essential amino acids and bases). 
     For the production of antibody fragments the transformants were grown overnight in selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with the essential amino acids and bases) and subsequently diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacto pepton and 5% galactose). After 24 and 48 hours of growth, samples were taken for Western blot analysis (FIG. 21). For the immuno detection of the produced V H -myc fragments monoclonal anti-myc antibodies were used. 
     In essentially the same way comparable results were obtained with a yeast transformed with pUR4424M containing a DNA sequence encoding the V H -09-myc protein. 
     EXAMPLE 3 
     Construction of  S. cerevisiae  Multicopy Integration Vectors for the Expression or  Camelidae  V H    
     To combine the benefits of high copy number and mitotically stable expression, the concept of a multicopy integration system into the rDNA locus of lower eukaryotes has already been successfully proven (Giuseppin et al. supra). 
     One of these vectors is pUR2778, a derivative of pUR2774 (Giuseppin et al. supra) from which the pol1-S.O. reporter gene sequence was removed (FIG. 9). 
     This integrating plasmid, pUR2778, can be used for integration of  Camelidae  V H  coding sequences, hence the vector can be digested with SacI and HindIII after which the  − 7.3 kb vector fragment can be isolated. 
     From the in example 2 described pUR4423 or pUR4426 types of plasmids, SacI-HindIII fragments can be isolated encoding a V H  fragment preceded by a signal sequence (SUC2 or α mating factor prepro) and followed by nothing or a Myc or Flag tail. 
     Ligation of these SacI-HindIII fragments with the  − 7.3 kb vector fragment will result in integration plasmids, encoding the (functionalized) V H  fragments under the regulation of the strong and inducible GAL7 promoter. 
     In this way the following expression plasmids were obtained: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4429 
                 P gal7  - SUC2 sig.seq. - V H -03 
               
               
                   
                 pUR4429F 
                 P gal7  - SUC2 sig.seq. - V H -03 - Flag tail 
               
               
                   
                 pUR4429M 
                 P gal7  - SUC2 sig.seq. - V H -03 - Myc tail 
               
               
                   
                 pUR4430 
                 P gal7  - α mat.fac.prepro. - V H -03 
               
               
                   
                 pUR4430F 
                 P gal7  - α mat.fac.prepro. - V H -03 - Flag tail 
               
               
                   
                 pUR4430M 
                 P gal7  - α mat.fac.prepro. - V H -03 - Myc tail 
               
               
                   
                   
               
             
          
         
       
     
     For schematic drawings see FIG. 10 for pUR4429 and FIG. 11 for pUR4430. Obviously, comparable constructs can be prepared for other heavy chain antibodies or fragments thereof. 
     As mentioned before, different promoters might be used, for example, the constitutive GAPDH promoter. 
     EXAMPLE 4 
     Construction of Expression Plasmids for the Production of (Functionalized) V H  Fragments from  Camelidae  by  Kluyveromyces    
     4.1. Construction of  Kluyveromyces lactis  Episomal Expression Plasmids  Camelidae    
     Yeast strains of the genus  Kluyveromyces  have been used for the production of enzymes, such as β-galactosidase for many years, and the growth of the strains has been extensively studied.  Kluyveromyces lactis  is well known for the ability to utilize a large variety of compounds as carbon and energy sources for growth. Since these strains are able to grow at high temperatures and exhibit high growth rates, they are promising hosts for industrial production of heterologous proteins (Hollenberg, C. et al., EP-A-0096430, GIST-BROCADES N.V., 1983). 
     The plasmids pUR2427 and pUR2428 are pTZ19R derivatives with the promoter and the DNA sequence encoding either the signal peptide (=pre-sequence) (in pUR2428), or the natural prepro-sequence (in pUR2427), of inulinase (inu) from  Kluyveromyces marxianus . Both plasmids contain a unique BspMI site suitable to create a perfect joint with EagI or NotI digested DNA-fragments (not yet published European patent application 92203932.6, supra). In both plasmids a unique HindIII site is located a bit further downstream of the BspMI-site, so that EagI-HindIII cut DNA-fragments encoding V H  from  Camelidae  either solely or with Myc- or Flag-tail can be easily ligated into BspMI-HindIII digested pUR2427 or pUR2428. Thereby a set of six plasmids can be created containing the promoter and secretion signals of the  Kluyveromyces marxianus  inulinase gene, joint in frame to  Camelidae  Vh encoding sequences, all on a EcoRI-HindIII restriction fragment: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4445 
                 P inu  Inu prepro seq. - V H  - 03 
               
               
                   
                 pUR4445M 
                 P inu  Inu prepro seq. - V H  -03 - Myc 
               
               
                   
                 pUR4445F 
                 P inu  Inu prepro seq. - V H  - 03 - Flag 
               
               
                   
                 pUR4446 
                 P inu  Inu pre seq. - V H  - 03 
               
               
                   
                 pUR4446M 
                 P inu  Inu pre seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4446F 
                 P inu  Inu pre seq. - V H  - 03 - Flag. 
               
               
                   
                   
               
             
          
         
       
     
     Maps of pUR4445 and pUR4446 are shown in FIG. 12 and FIG. 13. 
     The EcoRI-HindIII fragments of these plasmids can be ligated into the expression vector pSK1 (not yet published European patent application 92203932.6, supra), from which the α-galactosidase expression cassette including the GAL7-promoter is removed with a EcoRI(partial) and HindIII digestion. The resulting plasmids can then be transformed for example in  K. lactis  strain MSK110 (a, uraA, trp1::URA3), as they contain the trp1 marker and the pKD1 episomal plasmid sequences: 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4447 
                 P inu  Inu prepro seq. - V H  - 03 
               
               
                   
                 pUR4447M 
                 P inu  Inu prepro seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4447F 
                 P inu  Inu prepro seq. - V H  - 03 - Flag 
               
               
                   
                 pUR4448 
                 P inu  Inu pre seq. - V H  - 03 
               
               
                   
                 pUR4448M 
                 P inu  Inu pre seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4448F 
                 P inu  Inu pre seq. - V H  - 03 - Flag. 
               
               
                   
                   
               
             
          
         
       
     
     A map of pUR4447 is shown in FIG. 14. 
     Transformation can be performed by standard techniques such as the methods of Beggs (1978) or electroporation, using 0.67% Yeast Nitrogen Base (without amino acids) and 2% glucose is the selection medium for transformants. 
     4.2. Construction of  Kluyveromyces lactis  Multicopy Integration Vectors 
     Alternatively, since all tailed and non-tailed versions of the Vh fragments, joined to the inulinase promoter and secretion signals, are located on EcoRI-HindIII fragments, the rDNA multicopy integration plasmid pMIRKGAL-TΔ1 (Bergkamp et al., 1992) can be used in a similar way as the pSK1 plasmid. In order to replace the α-gal expression cassette present in this plasmid, by a antibody fragment cassette, these plasmids have to be digested with EcoRI(partial) and HindIII. After isolating the vector fragments, they can be ligated with the about 1.2 kb EcoRI-HindIII fragments which can be obtained from the plasmids described in example 4.1. The resulting plasmids can be linearized with SacII and transformed to MSK110, resulting in  K. lactis  strains with potentially high and stable expression of single chain V H  fragments. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4449 
                 P inu  Inu prepro seq. - V H  - 03 
               
               
                   
                 pUR4449M 
                 P inu  Inu prepro seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4449F 
                 P inu  Inu prepro seq. - V H  - 03 - Flag 
               
               
                   
                 pUR4450 
                 P inu  Inu pre seq. - V H  - 03 
               
               
                   
                 pUR4450M 
                 P inu  Inu pre seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4450F 
                 P inu  Inu pre seq. - V H  - 03 - Flag. 
               
               
                   
                   
               
             
          
         
       
     
     4.3. Construction of  Kluyveromyces marxianus Episomal Plasmids    
       Kluyveromyces marxianus  is a yeast which is perhaps even more attractive than  K. lactis  for industrial biotechnology, due to its short generation time on glucose (about 45 minutes) and its ability to grow on a wide range of substrates, and its growth at elevated temperatures (Rouwenhorst et al., 1988). 
     The shuttle vector pUR2434, containing the leu2 marker and the pKD1 plasmid sequences (not yet published European patent application 92203932.6, supra), located on a pUC19 based vector, can be cut with EcoRI(partial) and HindIII to remove the α-galactosidase expression cassette. In this vector the EcoRI-HindIII fragments containing the Vh expression cassettes as described in example 4.1, can be ligated. The resulting plasmids can then be transformed into KMS3, the neat leu2-auxotroph CBS6556  K. marxianus  strain (Bergkamp, 1993) using the method of Meilhoc et al. (1990). 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4451 
                 P inu  Inu prepro seq. - V H  - 03 
               
               
                   
                 pUR4451M 
                 P inu  Inu prepro seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4451F 
                 P inu  Inu prepro seq. - V H  - 03 - Flag 
               
               
                   
                 pUR4452 
                 P inu  Inu pre seq. - V H  - 03 
               
               
                   
                 pUR4452M 
                 P inu  Inu pre seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4452F 
                 P inu  Inu pre seq. - V H  - 03 - Flag. 
               
               
                   
                   
               
             
          
         
       
     
     A map of pUR4451 is shown in FIG. 15. 
     4.4 Construction of  Kluyveromyces marxianus  Multicopy Integration Vectors 
     For high and stable expression in  Kluyveromyces marxianus , the multicopy integration system as described by Bergkamp (1993), can be used. The following cloning route, based on the route for constructing pMIRKM-GAL5 (Bergkamp, 1993), results in suitable expression vectors for production of Vh fragments from  Camelidae.  The EcoRI-NheI(Klenow filled) fragments of pUR4447,-M,-F and pUR4448,-M,-F containing the Vh fragment expression cassettes as described in example 4.1, can be isolated and ligated in EcoRI-EcoRV digested pIC-20H. From the plasmids obtained in this way, and which are equivalents of the pIC-αgal plasmid, the BamHI-NruI fragment can be isolated and ligated with BamHI-SmaI digested pMIRKM4. The result of this will be expression vectors which are equivalent to pMIRKM-GAL5, and contain a tailed or non-tailed Vh fragment from camel under control of inulinase promoter and secretion signals, in a vector which also contains the  K. marxianus  LEU2-gene with defective promoter, and  K. marxianus  rDNA sequences for targeted integration into the genome. These vectors can be used to transform for example KMS3. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 pUR4453 
                 P inu  Inu prepro seq. - V H  - 03 
               
               
                   
                 pUR4453M 
                 P inu  Inu prepro seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4453F 
                 P inu  Inu prepro seq. - V H  - 03 - Flag 
               
               
                   
                 pUR4454 
                 P inu  Inu pre seq. - V H  - 03 
               
               
                   
                 pUR4454M 
                 P inu  Inu pre seq. - V H  - 03 - Myc 
               
               
                   
                 pUR4454F 
                 P inu  Inu pre seq. - V H  - 03 - Flag. 
               
               
                   
                   
               
             
          
         
       
     
     A map of pUR4453 is shown in FIG. 16. 
     EXAMPLE 5 
     Construction of  Hansenula polymorpha  Integrating Vectors for the Expression of (Functionalized) V H  Fragments from  Camelidae    
     In search for productive systems able to carry out authentic posttranscriptional processing and overcoming the limitation of higher eukaryotic expression systems, such as high costs, low productivity and the need for stringent control procedures for the detection of contaminating agents could be overcome by the methylotrophic yeast  H. polymorpha . This strain is able to grow on methanol as its sole carbon and energy source, so the presence of methanol in the growth medium rapidly induces the enzymes of the methanol pathway, such as the key enzymes methanol oxidase (MOX) and dihydroxyacetone synthase (DHAS). 
     While experiments to express foreign genetic information from an episomal plasmid resulted a low plasmid stability, chromosomal integration is the method of choice (Sierkstra et al., 1991). By utilizing the DNA of the mox gene as integration locus the latter were able to express and secrete α-galactosidase regulated by mox promoter and -terminator. Here, the  S. cerevisiae  SUC2 signal sequence was proven to be efficiently functional for secretion. 
     The same approach can be used for expression and secretion of  Camelidae  V H  antibody fragments. Plasmids analogous to pUR3515 (without an origin of replication functional in yeast) and pUR3517 (containing the HARS2 sequence as origin of replication) can be used as expression vectors (Sierkstra et al., 1991). As a starting vector pUR3501 can be used (Sierkstra et al., 1991) in which by means of site directed mutagenesis (e.g. via PCR technology), an EagI restriction site is introduced at the junction between the invertase (=SUC2) signal sequence and the α-galactosidase. From the resulting plasmid, pUR3501Eag, it is possible to replace the EagI-HindIII fragment comprising the α-galactosidase gene by an EagI-HindIII fragment encoding a (functionalized) antibody fragment, obtained as described in example 1. In case of using the EagI-HindIII fragments of the pUR4421-03 series (example 1), this would result in plasmids pUR4437 (FIG. 17), pUR4437M and pUR4437F. In these plasmids the nucleotide sequence encoding the (functionalized) V H  is preceded by a nucleotide sequence encoding the invertase signal sequence and the mox promoter sequence. The obtained plasmids can be digested with BamHI and HindIII and after filling in the sticky ends with Klenow polymerase, the about 2.6 kb fragments can be ligated into plasmid pUR3511 which was digested with SmaI (Sierkstra et al., 1991). In this way the terminator sequence of the mox gene can by fused downstream of the V H  encoding sequences. From the thus obtained plasmids, pUR4438 (FIG. 17) EcoRI-HindIII fragments of about 3 kb can be isolated, containing the mox promoter, the invertase signal sequence, the (functionalized) V H  fragment and the mox transcription terminator. Subsequently these fragments can be cloned into plasmid pUR3513 (no yeast origin of replication) or in pUR3514 (HARS origin of replication) as described by Sierkstra et al. (1991), resulting in two sets of plasmids: 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                 pUR4439 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- no origin 
               
               
                 pUR4439M 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- no origin 
               
               
                 pUR4439F 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- no origin 
               
               
                 pUR4440 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- HARS origin 
               
               
                 pUR4440M 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- HARS origin 
               
               
                 pUR4440F 
                 P mox  - SUC2 sig. seq. - V H  - mox term. -- HARS origin. 
               
               
                   
               
             
          
         
       
     
     Maps of pUR4439 and pUR4440 are shown in FIG. 18. 
     Essentially the same can be done with other EagI-HindIII fragment, obtained as described in example 1. 
     The newly obtained plasmids can be transformed by electroporation of  H. polymorpha  A16 (CBS4732, leu-) and can be selected by growing on selective medium containing 0.68% YNB and 2% glucose. Induction medium should contain 0.5% methanol instead of the glucose. 
     EXAMPLE 6 
     Construction  Aspergillus niger  var.  awamori  Integration Vectors for the Production of V H  Fragments from  Camelidae    
     The multiple cloning site of plasmid pEMBL9 (ranging from the EcoRI to the HindIII site) was replaced by a synthetic DNA fragment having the nucleotide sequence as indicated in FIG. 19; see SEQ. ID. NO: 42-45. The 5′-part of the nucleotide sequence contains a NruI restriction site followed by the first codons of the  Camelidae  V H  gene fragment and a XhoI restriction site. The 3′-part encodes for a BstEII restriction site, the last codons of the  Camelidae  V H  gene, eleven codons of the Myc tail and finally a EcoRI and a AflII site. The resulting plasmid is pUR4432. 
     After digesting plasmid pB3 with XhoI and EcoRI, a DNA fragment of approximately 425 bp can be isolated from agarose gel. This fragment codes for a truncated V H -Flag fragment, missing the first 5 amino acids of the  Camelidae  V H . 
     The obtained fragment can be cloned into pUR4432. To this end plasmid pUR4432 can be digested with XhoI and EcoRI, after which the about 4 kb vector fragment was isolated from an agarose gel. Ligation with the about 425 bp fragment resulted in plasmid pUR4433F. 
     After digesting the plasmids pB3 with XhoI and BstEII, a DNA fragment of approximately 365 bp was isolated from agarose gel. This fragment codes for a truncated V H  fragments, missing the first and last 5 amino acids of the  Camelidae  V H . 
     The obtained fragment was cloned into pUR4432. To this end plasmids pUR4432 can be digested with XhoI and BstEII, after which the about 4 kb vector fragment was isolated from an agarose gel. Ligation with the about 365 bp fragments resulted in plasmids pUR4433M. In a similar way the XhoI-BstEII fragments of pB9 and pB24 were cloned into the pUR4432 vector fragment, resulting in pUR4434M and pUR4435M, respectively. 
     Upon digesting pUR4433M or pUR4433F with BstEII and HindIII, the vector fragments of about 4.4 kb can be isolated from agarose gel and religated in the presence of a synthetic linker peptide having the following sequence: 
                               BstEII                   AflII        HindIII                GTCACC GTCTCCTCATAATGAT CTTAAG GTGAT A                        GCAGAGGAGTATTACTAGAATTCCACTATTCGA            
(top strand, SEQ ID NO: 50, is shown 5′ to 3′; bottom strand, SEQ ID NO: 51, is shown 3′ to 5′. In the thus obtained plasmid, pUR4433, the Myc tail or Flag tail sequences are removed and the V H  gene fragment is directly followed by a stop codon.
 
     Analogous as described in example 1.5, it is possible to clone nucleotide sequences encoding longer fragments of the heavy chain immunoglobulins into pUR4432 or to replace the BstEII-AflII fragments of the above mentioned plasmids pUR4433, pUR4433F or pUR4433M with other BstEII-AflII fragments, resulting in frame fusions encoding functionalized V H  fragments, having a C-terminal extension. 
     Upon replacing the NruI-XhoI fragments of pUR4433, pUR4433F or pUR4433M, in frame fusions can be constructed encoding functionalized V H  fragments, having an N-terminal extension. 
     In the above described constructs an NruI site was introduced before the first codon of the (functionalized) V H  fragment, facilitating an in frame fusion with the precursor-sequence of xylanase, see (not prior-published) WO-A-93/12237, supra. For the construction of  Aspergillus  expression plasmids, from the plasmids pUR4433F, pUR4433M and pUR4433, respectively, an about 455, 445 and 405 bp NruI-AflII fragment has to be isolated encoding the V H  fragment with a Flag, a Myc or no tail. 
     Plasmid pAW14B was the starting vector for construction of a series of expression plasmids containing the exlA expression signals and the genes coding for (functionalized) V H  fragments of  Camelidae  heavy chain antibodies. The plasmid comprises an  Aspergillus niger  var.  awamori  chromosomal 5 kb SalI fragment on which the 0.7 kb exlA gene is located, together with 2.5 kb of 5′-flanking sequences and 2.0 kb of 3′-flanking sequences (see FIG. 20 and (not prior-published) WO-A-93/12237, supra). 
     Starting from pAW14B, pAW14B-10 was constructed by removing the EcoRI site originating from the pUC19 polylinker, and introducing a NotI site. This was achieved by digesting plasmid pAW14B with EcoRI and after dephosphorylation the linear 7.9 kb EcoRI fragment was isolated. The fragment was religated in the presence of the “EcoRI”-NotI linker:
         5′-AATTGCGGCCCC -3′ (see SEQ. ID. NO: 52).       

     Subsequently the AflII site, located downstream of the exlA terminator was removed by partially cleaving plasmid pAW14B-10 and religating the isolated, linearized plasmid after filling in the sticky ends, resulting in plasmid pAW14B-11. 
     Finally, pAW14B-12 was constructed using pAW14B-11 as starting material. After digestion of pAW14B-11 with AflII (overlapping with the exlA stop codon) and BglII (located in the exl promoter) the ˜2.4 kb AflII-BglII fragment, containing part of the exlA promoter and the exlA gene was isolated as well as the ˜5.5 kb AflII-BglII vector fragment. After partial digestion of this ˜2.4 kb fragment with BspHI (located in the exlA promoter and at the exlA start codon) an about 1.8 kb BglII-BspHI exlA promoter fragment (up to the ATG initiation codon) was isolated and ligated with the about 5.5 kb AflII-BglII vector fragment of pAW14B-11 in the presence of the following adaptor: 
                             ( Bsp HI)  BbsI       AflII                CATG CA GTCTTC GGGC                         GTCAGAAGCCCGAATT            
(top strand, SEQ ID NO: 53, is shown 5′ to 3′; bottom strand, SEQ ID NO: 54, is shown 3′ to 5′.
 
     For the construction of the V H  expression plasmids, pAW14B-11 can be partially digested with NruI and digested with AflII, after which the  − 7 kb vector fragment can be isolated from agarose gel and contains the xylanase promoter, the DNA sequence encoding the xylanase signal sequence and the xylanase terminator. Upon ligation of the NruI-AflII fragments of pUR4433M, pUR4434M and pUR4435M with the pAW14B-11 vector, plasmids pUR4436M, pUR4437M and pUR4438M were obtained, respectively. In these plasmids the  Camelidae  V H  polypeptides are preceded by the 27 amino acid long precursor sequence of xylanase and followed by the myc-tail (of 11 amino acids; see Examples 1.3 en 2, FIGS. 6 and 19, and SEQ. ID. NO: 41=45). 
     In a similar way plasmids can be constructed encoding the V H  fragments followed by the FLAG-tail or without a tail. 
     After introducing the amdS and pyrG selection markers into the unique NotI site of pUR4436M, pUR4437M and pUR4438M using conventional techniques, e.g. as described in Examples 2 and 3 of (not prior-published) WO-A-93/12237, supra, the plasmids were transferred to  Aspergillus.    
     Production of the Camel V H  fragments by the selected transformants was achieved by growing the strains in inducing medium essentially as described in example 2.2 of (not prior-published) WO-A-93/12237, supra. Western blot analysis of the culture medium was performed as described in Example 2.1 above and revealed the presence of the antibody fragments. 
     Obviously, expression vectors can be constructed in which different promoter systems, e.g. glucoamylase promoter, and/or different signal sequences, e.g. glucoamylase or glucose oxidase signal sequences, are used. 
     EXAMPLE 7 
     Production of Glucose Oxidase—V H  Fusion Proteins 
     Glucose oxidase catalyses the oxidation of D-glucose to D-gluconate under the release of hydrogen peroxide. Glucose oxidase genes (gox) from  Aspergillus niger  have been cloned (Frederick et al. (1990) J. Biol. Chem. 265 3793, Kriechbaum et al., 1989) and the nucleotide sequences are available from the EMBL data bank under accession numbers J05242 and X16061. The nucleotide sequence of the latter is used as a basis for the following construction route. 
     Upon cloning the gox gene from  A. niger  it is possible, by applying PCR technology, to introduce convenient restriction sites. 
     To introduce a BspHI restriction site, overlapping with the ATG initiation codon, the sequence ATC ATG CAG can be changed to ATC ATG AGG. In the same experiment an EcoRI restriction site can be introduced which is located upstream of the BspHI site. This can be achieved by using the following PCR primer: 
                                  Eco RI      Bsp HI           5′-TCACT GAATTC GGGA TC  ATG  A GG ACT CTC CTT GTG AGC TCG CTT-3′       (see SEQ. ID. NO: 55).       A second PCR primer, having the following sequence can be used:                        Afl II     BbsI    SalI       5′-ATGTCACA AAGCTT AAGCAC GAAGAC A  GTC GAC  CGT GCG GCC GGA GAC-3′                   Hin dIII       (see SEQ. ID. NO: 56)            
in the same PCR experiment, in order to introduce a BbsI site, a AflII site and a HindIII site, downstream of the unique SalI site present in the glucose oxidase gene. After digesting the DNA obtained from this PCR experiment with EcoRI and HindIII, an EcoRI-HindIII fragment of about 160 bp can be isolated and cloned into pEMBL9, which was digested with the same enzymes, resulting in plasmid pGOX1.
 
     From pGOX1 an about 140 bp BspHI-AflII fragment can be isolated and introduced into the 7.2 kb BbsI-AflII vector fragment of pAW14B-12, resulting in pAW14B-GOX. In this plasmid, the 5′-part of the gox gene, encoding the first 43 amino acids, is fused in frame with the ATG initiation codon of the exlA gene. 
     In a second PCR experiment, a MluI restriction site can be introduced near the 3′-end of the gox by changing the sequence TAT GCT TCC to TAC GCG TCC. In the same experiment a HindIII site can be introduced downstream of the MluI site. As a second primer an oligo nucleotide should be used hybridizing upstream of the SalI site. After digesting the DNA obtained from this PCR experiment with SalI and HindIII, an SalI-HindIII fragment of about 1.7 kb can be isolated and cloned into pEMBL9, which was digested with the same enzymes, resulting in plasmid pGOX2. Upon digesting pGOX2 with MluI and HindIII, an about 5.7 kb vector fragment can be isolated. 
     From the plasmids pUR4433, pUR4433F, pUR4433M and the like, XhoI-HindIII fragments can be isolated, encoding the truncated  Camelidae  V H  fragment with or without a tail sequence, and missing the first 4-6 N-terminal amino acids (see Example 1). These fragments can be ligated into the 5.7 kb pGOX2 vector fragment by using MluI-XhoI adaptors. These adaptors are designed in such a way that they result in an in frame fusion between the 3′-end of the gox gene and the restored V H  gene fragment, optionally intersected with a DNA sequence encoding a peptide linker sequence. 
     An example of these designed adaptors is: 
                       MluI                                          XhoI            CGCGT CCATGCAGTCCTCAGGTGGATCATCCCAGGTGAAACTG C                   AGGTACGTCAGGAGTCCACCTAGTAGGGTCCACTTTGACGAGCT            S  M  Q |S  S  G  G  S  S |Q  V  K  L  L  E            
(top strand, SEQ ID NO: 57, is shown 5′ to 3′; bottom strand, SEQ ID NO: 58, is shown 3′ to 5′; the encoded amino acid sequence is SEQ ID NO: 59 which encodes for the last amino acids of GOX, an SSGGSS linker sequence (see SEQ. ID. NO: 62) and the N-terminal amino acids of the Camel V H  fragment of pB3. Instead of the SSGGSS linker (see SEQ. ID. NO: 62) it is possible to use other linkers such as the repeated sequence linkers described in the above indicated European patent application 92402326.0, e.g. a repeated sequence Pro-X, with X being any amino acid, but preferably Gln, Lys or Glu, the sequence containing advantageously at least 3 repeats of Pro-X and especially a fragment composed of a 12-fold repeat of the sequence of Pro-X.
 
     In case the about 435 bp XhoI-HindIII fragment of pUR4433M is used in combination with the above described adaptor, this would result in pGOX2-03M. From this plasmid a SalI-AflII fragment of about 2.1 kb encoding the C-terminal part of glucose oxidase followed by the linker peptide, the Camel V H  fragment of pB3 and finally the Myc tail. 
     Upon digesting pAW14B-GOX partially with BbsI, and with AflII, the about 7.4 kb vector fragment can be isolated. This fragment contains the xylanase promoter, the DNA sequence encoding the N-terminal pan of glucose oxidase and the xylanase promoter. Due to the digestion with BbsI, a SalI sticky end is created, corresponding with the SalI restriction site originally present in the gox gene. Ligation of the SalI-AflII vector fragment with the about 2.1 kb SalI-AflII fragment of pGOX2-03M, resulting in pUR4441M. This expression plasmid encodes for a single chain polypeptide comprising the glucose oxidase enzyme, the (functionalized) Camel V H  fragment and the Myc tail. 
     Introduction of this type of expression plasmids in Aspergillus can be achieved essentially as described in example 6. 
     As the naturally occurring glucose oxidase is a homodimeric enzyme, it might be expected that a fusion protein, comprising glucose oxidase and an antibody fragment as a C-terminal extension, has an increased avidity for the antigen/antibody binding, if this fusion protein is produced as a homodimer. Alternatively, it is possible to produce heterodimers, consisting of one glucose oxidase molecule connected to a V H  fragment and one wild type glucose oxidase molecule. This can be achieved by producing with the same strain both wild type glucose oxidase and the fused glucose oxidase-V H  fragment, or by mixing the two different homodimers produced by different strains under conditions whereby the mixture of dimers are dissociated and subsequently associated. 
     EXAMPLE 8 
     Engineering of  Camelidae  V H  Fragments 
     8.1 Random and Targeted Random Mutagenesis 
     After expressing a number of different  Camelidae  V H  fragments in lower eukaryotic host organisms as described above, or in prokaryotes, fragments produced in relative higher amounts can be selected. Upon subjecting the XhoI-BstEII gene fragments to a (targeted) random mutagenesis procedure, it might be possible to further improve special characteristics of the V H  fragment, e.g. further improvement of the production level, increased stability or increased affinity. 
     To this end the following procedure might be followed. 
     Upon replacing the polylinker of the phagemid vector pHEN1 (Hoogenboom et al., 1991) located on a NcoI-NotI fragment by a new polylinker having the following sequence: 
                           NcoI                 XhoI               BstEII            NotI                CATGG CCAGGTGAAACTG CTCGAG TAAGTGACTAA GGTCACC GTCTCCTCA GC                    CGGTCCACTTTGACGAGCTCATTCACTGATTCCAGTGGCAGAGGAGTCGCCGG            
(top strand, SEQ ID NO: 60, is shown 5′ to 3′; bottom strand, SEQ ID NO: 61, is shown 3′ to 5′ it becomes possible to introduce XhoI-BstEII fragments encoding truncated  Camelidae  V H  fragments in the phagemid.
 
     Following mutagenesis of the V H  encoding sequence (random mutagenesis) or a specific part thereof (targeted random mutagenesis), the mutated V H  fragments can be expressed and displayed on the phage surface in essentially the same way as described by Hoogenboom et al. (1991). Selecting phages displaying (mutant) V H  fragments, can be done in different ways, a number of which are described by Marks et al. (1992). Subsequently, the mutated XhoI-BstEII fragments can be isolated from the phagemid and introduced into expression plasmids for yeast or fungi as described in previous examples. 
     Upon producing the mutant V H  fragments by these organisms, the effects of the mutations on production levels. V H  fragment stability or binding affinity can be evaluated easily and improved V H  fragments can be selected. 
     Obviously, a similar route can be followed for larger antibody fragments. With similar procedures the activity of catalytic antibodies can be improved. 
     8.2 Site-directed or Designed Mutagenesis 
     As an alternative to the methods described above in Example 8.1 it is possible to use the well-known technique of site-directed mutagenesis. Thus, designed mutations, preferably based on molecular modelling and molecular dynamics, can be introduced in the V H  fragments, e.g. in the framework or in the CDRs. 
     8.3 Construction V H  Fragments with Regulatable Binding Efficiencies 
     For particular applications, the possibility to regulate the binding capacity of antibody fragments might be necessary. The introduction of metal ion binding sites in proteins is known from the literature e.g. Pessi et al. (1993). The present inventors envisage that the introduction of a metal binding site in an antibody fragment by rational design can result in a regulatable antibody fragment, when the metal binding site is introduced at a position such that the actual binding of the metal ion results in a conformational change in the antibody fragments due to which the binding of the antigen to the antibody fragment is influenced. Another possibility is that the presence of the metal prevents antigen binding due to steric hindrance. 
     8.4 Grafting of CDR Regions on the Framework Fragments of a  Camelidae  V H  Fragment 
     Grafting of CDR fragments onto framework fragments of different antibodies or fragments thereof is known from the literature (see Jones et al. (1986), WO-A-92/15683, and WO-A-92/01059). In these cases the CDR fragments of murine antibody fragments were grafted onto framework fragments of human antibodies. The sole rationale behind the “humanization” was to increase the acceptability for therapeutic and/or diagnostic applications in human. 
     Essentially the same approach can however also be used for a totally different purpose. Although antibody fragments share some homology in the framework areas, the production levels vain considerably. 
     Once an antibody or an antibody fragment, e.g. a  Camelidae  V H  fragment, has been identified, which can be produced to high levels by an production organism of interest, this antibody (fragment) can be used as a starting point to construct “grafted” antibody (fragments), which can be produced in high levels and have an other specificity as compared to the original antibody (fragment). In particular cases it might be necessary to introduce some modifications in the framework fragments as well in order to obtain optimal transitions between the framework fragments and the CDR fragments. For the determination of the optimal transitions molecular dynamics and molecular modelling can be used. 
     To this end a synthetic gene, encoding the “grafted V H ” fragment, can be constructed and introduced into an expression plasmid. Obviously it is possible to adapt the codon usage to the codons preferred by the host organism. 
     For optimization of the “grafted V H ” fragment, the procedure as described in example 8.1 can be followed. 
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     Additional references to prior-filed but not prior-published patent applications, which are incorporated herein by reference:
         not prior-published PCT application EP 92/02896, filed 9 Dec. 1992 with priority date of 9 Dec. 1991 (UNILEVER/R. J. Gouka et al.), now publicly available as WO-A-93/12237   not prior-published EP application 92202080.5, filed 8 Jul. 1992 (UNILEVER/F. M. Klis et al.), now publicly available as International (PCT) patent application WO-A-94/01567)   not prior-published EP application 92402326.0, filed 21 Aug. 1992 (C. Casterman &amp; R. Hamers), now publicly available as EP-A1-0 584 421   not yet published EP application 92203932.6, filed 11 Dec. 1992 (UNILEVER/H. Y. Toschka &amp; J. M. A. Verbakel).       

     Information on deposits of micro-organisms under the Budapest Treaty is given in Example 1 on page 23, lines 23-25 above. 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.