RECOMBINANT HUMAN MANNAN-BINDING LECTIN

Abstract: The present invention relates to treatment of diseases and disorders of the immune system comprising treatment of deficiencies within the immune defence system. The invention concerns preparation of a novel expression construct encoding human MBL, a novel recombinant preparation of human MBL with high similarity to natural human MBL and futhermore it comprises use of this compound in compositions, medicaments and in methods for treatment of conditions such as those related to immunosuppressive conditions and/or to conditions of MBL deficiencies including latent conditions. Conditions of deficiencies of Mannan-Binding Lectin (MBL) are associated with increased susceptibility to infections. In more particular, the invention concerns treatment of said conditions, including latent conditions, i.e. although there is no present need. The treatment is aimed at humans and at animals, in which the immune system appears to act similarly to the human immune system.

Description: Recombinant Human Mannan-Binding Lectin The invention relates to a process for preparing a composition comprising MBL, and treatment of diseases and disorders of the immune system comprising treatment of deficiencies within the immune defence system In more particular, the invention concerns treatment of conditions or latent conditions of deficiencies of Mannan- Binding Lectin (MBL) which is associated with increased susceptibility to infections The invention further concerns preparation of a novel expression construct encoding human MBL, a novel recombinant preparation of human MBL and furthermore it comprises use of this compound in methods for treatment of conditions such as those related to immunosuppressive chemotherapy and/or conditions of MBL deficiencies including latent conditions, i e not in a present need The treatment are aimed at humans and at animals, in which the immune system appears to act similarly to the human immune system BACKGROUND OF THE INVENTION The immune system comprises a complex array of cellular and molecular mechanisms which recognises and targets pathogenic microorganism or the cells infected with them Recently, MBL has gained great interest as an important part of the innate immune system, that is, the immune system which at time of birth is operational, in contrast to the adaptive immune defence which only during infancy obtains its full power of protecting the body (Janeway et al , 1999) Upon binding of carbohydrates of microbial surfaces, MBL mediates activation of the complement cas- cade, a series of enzymatic activation steps, which eventually label the target for destruction by phagocytosis or by lysis of the microorganism (Law & Reid, 1995) The complement system is being activated through at least three distinct pathways, designated the classical pathway, the alternative pathway, and the MBLectin pathway (Janeway et al , 1999) The classical pathway is initiated when complement factor 1 (C1) recognises surface-bound immunogiobulin The C1 complex is composed of two proteolytic enzymes, C1 r and C1 s, and a non-enzymatic part, C1q, which contains immunoglobuhn-recognising domains C1q and MBL shares structural features, both molecules having a bouquet-like appearance when visualised by electron microscopy Also, like C1q, MBL is found in complex with two proteolytic enzymes, the mannan-bmding lectin associated proteases (MASP) The three pathways all generate a convertase of complement factor 3 (C3 convertase) bound to the surface of the activating surface, / e , the targeted microbial pathogen Conversion of C3 into surface bound C3b is pivotal in the process of eliminating the micro- bial pathogen by phagocytosis or lysis (Janeway et al , 1999) Mannan-bmding lectin (MBL), also named mannan-bmding protein or mannose- binding protein (MBP), was first characterised in rabbits (Kawasaki et al , 1978) Mannan-bmding lectin (MBL) belongs to a group of soluble Ca2 + -dependent (C- type) lectms containing a C-terminal carbohydrate recognition domain and a collagen-like region characterised by repeated triplet-motifs of glycine (Gly) followed by two non-glycine ammo acids Thus, MBL belongs to the group of collectins, i e , C- type lectms with collagen-like regions, which in addition comprises the lung surfactant proteins A and D as well conglutinin and CL-43, these, however, only having been characterised in cattle (Holmskov et al , 1994) The human MBL protein is composed of up to 18 identical 32 kDa polypeptide chains (Lu et al , 1990), each comprising a short N-terminal segment of 21 ammo acids including three cysteine residues, followed by 7 repeats of the collagenous motif Gly-X-Y interrupted by a Gin residues followed by another 12 Gly-X-Y repeats A small 34 residue 'neck- region' joins the C-terminal Ca 2+ -dependent lectin domain of 93 ammo acids with the collagenous part of the molecule (Sastry et a/ , 1989) Three MBL polypeptide chains are joined in a MBL subunit MBL consists of up to six 15 nm stalks of MBL subunits joined at the base of the bouquet Later work characterised MBL in rodents (Mizuno et al , 1981 , Oka et al , 1988), cattle (Holmskov et al , 1993a, Kawai et al , 1997), and chicken (Oka et al , 1985, Laursen et al , 1995, Laursen et al , 1998) In rodents (Dnckamer et al , 1986) and rhesus monkeys (Mogues et al , 1996) two types of MBL genes have been identified, usually designated as A and C forms In rats, an MBL 'B' pseudogene was found (Dnckamer et al , 1986) and recently an MBL pseudogene was also cloned from the human genome, sequence analysis suggesting this to be remnants of a primate MBL-A gene (Guo et al , 1998) Human MBL was characterised by Kawasaki et al in 1983 Only one active human MBL gene has been identified, comprising four exons with three intervening introns of the MBL gene spanning approximately 6 kb and is located at 10q11 2-q21 (Sastry et al , 1989, Taylor et al , 1989) The collagenous regions of the three polypeptide chains combine to form a subunit which is stabilised covalently by disulphide bridges. Individual subunits are joined by disulphide bridges as well as by non-covalently interactions (Lu et al., 1990). The position of these disulphide bridges has, however, not been fully resolved. SDS- PAGE analysis under non-reducing conditions of MBL shows bands with an apparent molecular weight (m.w.) larger than 200 kDa presumably representing blocks of 3, 4, 5 and even 6 assembled subunits (Lu et al, 1990). The actual number of subunits in the natural human MBL protein has been controversial. Lipscombe et al. (1995) obtained data by use of ultracentrifugation suggesting 25% of human serum MBL to be made of 2-3 subunits and only a minor fraction reaching the size of 6 subunits. The relative quantification was carried out by densi- tometry of Western blots developed by chemiluminescence. Lower efficiency in transferring high molecular weight protein onto membranes compared to proteins of lower molecular weight make analysis through this methodology complicated. Lu et al. (1990) found by SDS-PAGE analysis of fractions from ion exchange chromatography that the predominant species of covalently linked MBL subunit chains consisted of tetramers while only pentameric or hexameric complexes activated com- plement. Gel permeation chromatography (GPC) analysis, in contrast, suggests that MBL is comparable in size with the C1 complex. GPC can be carried out under conditions which allow for a study of the importance of weak protein-protein interactions in the formation of MBL molecules and, in combination with standard MBL assay techniques, also allows for unbiased determination of the MBL content in the GPC fractions. The in vivo role of MBL seems mainly to relate to the innate immune system as a humoral factor mediating some anti-microbial activity, which does not require maturation into self/non-seif discrimination like the adaptive immune defence system based on T- and B-cell recognition (Janeway et ah, 1999; Vorup-Jensen et a/., 1998). The recognition of targets for MBL binding is mediated by the C-type lectin domain. C-type CRDs are found in proteins with a widespread occurrence, both in phylogenetic and functional perspective. In the case of MBL, the CRD recognises preferentially hexoses with equatorial 3- and 4-OH groups, such as mannose and N- acetyl glucoseamin while carbohydrates which do not fulfil this sterical requirement, such as galactose and D-fucose, are not bound (Weis et al., 1992). The terminal CRDs are distributed in such a way that to allow for binding of all three domains target surfaces should present binding sites with a spacing of approximately 53 A (Sheriff et a/., 1994; Weis & Drickamer, 1994). This property of 'pattern recognition' may contribute further to the selectively binding of microbial surfaces. The carbohydrate selectivity is obviously an important aspect of the self/non-self discrimination by MBL and is probably mediated by the difference in prevalence of mannose and Λ/-acetyl glucoseamin residues on microbial surfaces, one example being the high content of mannose in the cell wall of yeasts such as Saccharomyces cerevisiae and Candida albicans. Carbohydrate structures in glycosylation of mammalian proteins are usually completed with sialic acid, which prevents binding of MBL to these oligomeric carbohydrates and thus prevents MBL recognition of 'self surfaces. Also, the trimeric structure of each MBL subunit may be of importance for target recognition. Several studies have been carried out on aspects of the structure and biosynthesis of MBL by use of in vitro synthesis systems. The structure of the CRD of rat MBL-A was resolved by crystallising recombinant protein produced in £. coli (Weis et al, 1992), and the structure of rat MBL-C has likewise been resolved by use of recombinant material (Ng et al. , 1996). More recently, the crystal structure of the trimer of CRDs assembled through expressing both the 'neck-region' and the CRD have confirmed the earlier studies on the 'neck-region' as bringing together the three chains by hydrogen bonds between the coiled-coil α-helices (Sheriff et al, 1994; Weis & Drickamer, 1994). The CRDs and 'neck-CRD' fragments of other collectins have likewise been expressed in E. coli based systems without the requirement of refolding protocols to gain functional activity. Thus, the in vitro synthesis of MBL domains can both with regard to the functional properties of carbohydrate recognition and the structural property of trimerisation efficiently be carried out in prokaryotic or simple eukaryotic expression systems. In vitro synthesis of complete collectins by recombinant technique have mainly been attempted by use of mammalian cell lines, e.g., CHO and COS cells, as host cells. Attempts to express MBL in insect cells only resulted in low m.w. MBL (Ma et al , 1996), the recombinant proteins almost entirely consisting of subunits, dimers, and tπmers of the MBL subunit chains Recombinant synthesis of human MBL in mammalian cell lines have been reported in several studies In a study on opsonic function / e , ability to enhance uptake by macrophages, of MBL, Kuhlman et al (1989) used rMBL produced in CHO cells, which showed the same opsonic activity, as natural MBL Characterisation of the rMBL with regard to structure and post-translational modifications was not carried out Likewise, Schweinle et al (1993) produced rMBL and a truncated form of rMBL lacking the collagenous tail by stable transfection of CHO cells Activity of the recombinant proteins was measured by C3 deposition on Salmonella montevideo preincubated with MBL and diluted human serum depleted of antibodies against S montevideo as complement source Surprisingly, the activity of recombinant full- length MBL was as high as for natural MBL though analysis of lodmated rMBL by ultracentπfugation showed the majority of the protein to be low molecular weight material Comparison of the CHO cell produced rMBL with natural MBL concerning size, either through SDS-PAGE, ultracentπfugation or GPC, was not included In a study by Super et al (1992), GPC profiles of rMBL produced in mouse Sp2/0Ag14 hybπdoma cells were presented indicating that the size distribution of rMBL purified from the culture supernatant by carbohydrate affinity chromatography was markedly different from rMBL isolated through anti-MBL antibody affinity chromatography with respect to size distribution Recently, Ohtani et al (1999) published data on human rMBL produced in CHO cells, which gave a yield of 120 μg per mL of culture medium This was obtained using a expression vector allowing for selection of transfectants by G418 resistance and subsequent gene amplification through selection with methionine sulfoximine (MSX) Functional activity concerning carbohydrate selectivity was identical to the natural protein whereas some differences in the ability to activate complement was found as measured through lysis of erythrocytes Both the GPC and SDS-PAGE analysis showed the presence of higher molecular weight forms of MBL though the total size distribution was noted by the authors to differ significantly from MBL purified from plasma Interestingly, the hydroxylation pattern was essentially identical to natural MBL even in the absence of added ascorbic acid Concerning the post- translation modifications, it should be noted from this study that proper hydroxylatioπ of rMBL does not warrant a structure similar to natural MBL. The human MBL locus is polymorphic with three known mutations located in the protein encoding region (Sumiya et al, 1991 ; Lipscombe et al., 1992a; 1992b; Mad- sen et al, 1994) and others affecting regulatory elements of the gene (Madsen et a/., 1995). All mutations apparently leads to a significantly lower level of MBL in body fluids from affected individuals. The spread in MBL concentration as measured in serum is thus about three orders of magnitude, ranging from 2-5 μg MBIJmL to less than 10 ng MBLJmL in individuals homozygous for the mutations affecting protein coding regions. The finding of MBL deficiency being associated with recurrent infections in children which were diagnosed as suffering from an opsonic defect (Super et al, 1989; Sumiya et al, 1991) emphasised the correlation between insufficient MBL levels and reduction in the defence against micro-organisms. In addition to the interaction with micro-organisms, collectins have also been suggested to mediate anti-viral defence (Hartshorn et al, 1993; Malhotra et al, 1994). In vitro studies on interaction with human immunodeficiency virus (HIV) showed MBL to inhibit infection of CD4 positive T and U937 cells. Clinical studies also suggest a role for MBL as a first-line defence against HIV. The period of time from the onset of symp- toms of AIDS until the terminal stage seem to differ between MBL-deficient patients and patients with normal MBL levels. Also, the susceptibility to contract the infection appears to be significantly higher among MBL-deficient individuals as MBL deficiency occurs more frequently among HIV infected patients than healthy controls (Nielsen et al, 1995; Garred et al, 1997a). Hereditary complement deficiencies contribute to development of systemic lupus erythematosus (SLE) as have been shown for cases of C1 , C2, and C4 deficiency (reviewed by Tan & Arnett, 1998). Several studies have suggested MBL deficiency likewise to be a risk factor in development of SLE (Davies et al, 1995; Lau et al, 1996; Ip et al, 1998) although the role of MBL is not well defined. One suggestion, following explanations for the other complement deficiencies as causative agents in SLE, argues that the defect in complement fixation due to MBL deficiency leads to poor immune complex clearance (Ip et al, 1998), thus pointing to MBL as a participant in maintaining homeostasis. Other clinical evidence suggests MBL to play an important role in reproductive biology. Recent reports show association between recurrent miscarriages and the MBL level (Kilpatrick et a/., 1995; Christiansen et ah, 1999). A strategy for treating symptomatic MBL insufficient individuals aiming at reconstituting the MBL pathway has only been descπbed in two studies An opsonic defect observed among some children, at the time only characterised by the clinical mani- festation of frequent infections, was treated by administering plasma (Soothill & Harvey, 1976) while, in a more recent study, plasma-derived MBL was injected into a two-year-old girl (Valdimarsson et al , 1998) In both studies a health improvement was reported though the small number of patients involved in these studies obviously limits conclusions with regard to MBL as therapeutic agent No clinical studies have been conducted with rMBL A study by Ma et al (1999) showed an antitumor activity of rMBL in mice mediated by a virus expression system As appears from the above, several attempts have been made to produce rMBL in in vitro systems in a form either identical to or with a high degree of resemblance to the natural protein However, significant limitations have been demonstrated in the ability of the presently known in vitro synthesis systems to produce such rMBL SUMMARY OF THE INVENTION In one aspect the present invention provides a novel form of MBL that is prepared by recombinant DNA technology in vitro or in vivo More specifically, the invention concerns recombinant MBL (rMBL) in a novel form, which the inventors surprisingly found could be synthesized by the following process A process of producing a human recombinant mannan binding lectin (MBL) composition having a size distribution profile at least 50 % identical to naturally occurring human MBL, comprising the steps of - preparing a gene expression construct encoding human MBL peptide or a functional equivalent thereof, - transforming a host cell culture with the construct, - cultivating the host cell culture, thereby obtaining expression and secretion of the polypeptide into the culture medium, followed by subjecting said culture medium to affinity chromatography using a carbohy- drate-derivatized matrix, said carbohydrate-derivatized matrix having at least a two-fold greater affinity for tetramers, pentamers, and/or hexamers of MBL subunit than for dimers of MBL subunits. obtaining an eluate comprising the human recombinant MBL composition. By a functional equivalent to human MBL is meant a molecule having the function of human MBL, e.g., binding to microbial carbohydrates, mediating opsonising activity and/or activating complement as may be demonstrated by C4 deposition on a man- nan-coated surface The functional activity of MBL may be estimated by its capacity to form an MBIJMASP complex leading to activation of the complement system. When C4 is cleaved by MBIJMASP an active thiol ester is exposed and C4 becomes covalently attached to nearby nucleophilic groups. A substantial part of the C4b will thus become attached to the coated plastic well and may be detected by anti-C4 antibody. A quantitative TRIFMA for MBL functional activity was constructed by 1) coating microtitre wells with 1 mg mannan in 100 ml buffer; 2) blocking with Tween-20; 3) applying test samples, e.g. diluted MBL preparations 4) applying MBL deficient serum (this leads to the formation of the MBIJMASP complex); alternatively the MBL and the MBL deficient serum may be mixed before application with the microtitre wells; 5) applying purified complement factor C4 at 5 mg/ml; 6) incubate for one hour at 37°C; 7) applying Eu-labelled aπti-C4 antibody; 8) applying enhancement solution; and 9) reading the Eu by time resolved fluorometry. Between each step the plate is incubated at room temperature and washed, except between step 8 and 9. Estimation by ELISA may be carried out similarly, e.g. by applying biotin-labelled anti-C4 in step 7; 8) apply alkaline phosphatase-labelled avidin; 9) apply substrate; and 10) read the colour intensity. A calibration curve can be constructed using dilutions of one selected normal plasma. In relation to the present invention the following serum is an example of useful serums: plasma pool LJ 6.57 28/04/97. The func- tionality may be expressed as the specific activity of MBL, such as in units of MBL activity per ng MBL Another assay for determining a functional equivalent to MBL is to determine the ability to bind to receptor/receptors on cells The interaction of MBL with receptor/receptors on cells may be analysed by cyto- fluoπmetry 1) MBL at a concentration of 50 μg/ml is incubated with 2x105 cells The binding is carried out in phosphate buffered salt solution (PBS) containing 1 % FCS and 0 1% Na-azide 2) For detection of cell-bound MBL, biotinylated anti-MBL antibody is applied, 3) followed by the addition of strepavidin-FITC and 4) analysis of the mixture by fluoπmetry A functional equivalent according to the invention fulfils at least one of the criteria discussed above, i e either activation of C4 or interaction with receptor(s) on cells, respectively In a preferred embodiment both criteria are fulfilled By "recombinant human MBL" is meant human MBL which is expressed from engineered nucleic acids and by "MBL gene expression constructs" is meant an expres- sion vector suitable for expression in the host cells By the term "composition having a size distribution profile at least 50 % identical to naturally occurring human MBL" is meant that the size distribution of the various ohgomers of MBL is 50 % identical on SDS-PAGE to human MBL purified from plasma by use of carbohydrate affinity chromatography By 50 % identical is meant that at least 50 % of the ohgomers has an apparent molecular weight higher than 200 kDa, when analysed by SDS-PAGE and/or Western blot In determination of the amount of MBL having an apparent molecular weight higher than 200 kDa densi- tometic analysis may be used wherein protein bands on a SDS-PAGE gel is stained with protein-staining, e g silver staining or Coomassie Blue staining, or specific staining of Western blot using an MBL specific antibody By "purified recombinant MBL" is meant recombinant MBL purified from culture supernatants or body fluids or tissue from transgenic animals by use of carbohydrate affinity chromatography The present invention provides a form of MBL which is similar to natural human MBL, in that it in its purified form has a molecular size distribution profile closer to the natural human MBL than known up till now. Thus, in another aspect the inven- tion relates to a recombinant human MBL composition comprising oligomers of MBL subunits, the size distribution profile of the oligomers being at least 50 % identical to the size distribution of naturally occurring plasma human MBL. In the present context the term "oligomer" refers to the various "mers" of MBL, such as monomer, dimer, trimer, tetramer, pentamer and hexamer. The monomer consists of three identical peptide chains, in the present context referred to as a subunit. The other oligomers are formed as combinations of 2-6 subunits. As to expression of rMBL, the invention relates to a gene expression construct en- coding a recombinant human mannan-binding lectin (MBL) polypeptide, comprising at least one intron sequence from the human MBL gene or a functional equivalent thereof, - at least one exon sequence from the human MBL gene or a functional equivalent thereof, - a promoter region different from the human MBL promoter, and - an expression vector. The expression is preferably carried out in e.g. mammalian cells, the preparation according to the invention results from the use of an expression vector comprising intron sequence(s) from an MBL gene and at least one exon sequence. Regarding the transgenic animals as expression system this term is in this context animals which have been genetically modified to contain and express the human MBL gene or fragments or mimics hereof. By "recombinant MBL with structural properties under non-denaturing similar to natural human MBL" is meant recombinant MBL eluting similarly from gel permea- tion chromatography analysis as human MBL as present in serum By "recombinant MBL with structural properties under denaturing conditions similar to natural human MBL" is meant purified MBL with a size distribution on SDS-PAGE 50% identical to human MBL purified from plasma by use of carbohydrate affinity chromatography (as illustrated by Fig 1) Thus, by "receombinant MBL" is meant MBL substantially free from any impurities naturally associated with MBL purified from plasma In this regard, mannose associated serine proteases (MASP's) are not regarded as impurities Accordingly, in another aspect the present invention relates to a process of producing a human recombinant mannan binding lectin (MBL), comprising the steps of preparing a gene expression construct as defined above encoding a human MBL polypeptide or a functional equivalent thereof, - transforming a host cell culture with the construct, - cultivating the host cell culture, thereby obtaining expression and secretion of the polypeptide into the culture medium, followed by obtaining a culture medium comprising human recombinant MBL Also an aspect of the present invention is a process for isolating or purifying human MBL in order to obtain a composition of polypeptides having a size distribution which is at least 50 % identical with naturally occurring human MBL Thus, the invention relates to a process of isolating a composition of human MBL oligomers having a size distribution at least 50 % identical with naturally occurring plasma MBL comprising - obtaining a preparation of human MBL oligomers, subjecting said preparation to affinity chromatography using a carbohydrate- denvatized matrix, said carbohydrate-deπvatized matrix having a two-fold greater affinity for tetramers, pentamers, and/or hexamers of MBL subunit than for dimers of MBL subunit, and - obtaining an eluate comprising the isolated human recombinant MBL corπ- position In another aspect the invention concerns MBL, including rMBL, fragments or mimics hereof for use in the treatment of cancer and of conditions of diseases and disorders of eg the immune system and reproductive system, said treatment consisting of creation, reconstitution, enhancing and/or stimulating the opsonizmg and bactericidal activity of the complement system, ie enhancing the ability of the immune defence to recognise and kill microbial pathogens The application of rMBL similar to natural MBL in said treatment represents a great advantage over the application of known MBL, in particular plasma-derived human MBL, since the treatment with rMBL confers a much reduced risk of viral infection Said treatment may comprise cure and/or prophylaxis of diseases, disorders and/or conditions in need of treatment of cancer and of conditions of diseases and disorders of e g the immune system and reproductive system by humans and by animals having said functional units acting in this respect like those in humans By condition in need of treatment is to be understood any condition in connection with current and/or expected need or in connection with an improvement of a normal condition In particular, the treatment is a treatment of a condition of deficiency of MBL The invention further comprise pharmaceutical compositions containing MBL prepared according to the invention In yet an aspect the present invention concerns the use of MBL prepared according to the invention including fragments or mimics hereof, in the manufacture of a me- dicament or a pharmaceutical composition for the treatment comprising cure and/or prophylaxis of conditions of diseases and disorders of eg the immune system and reproductive system by humans and by animals having said functional units acting like those in humans Said diseases, disorders and/or conditions in need of treatment with the compounds of the invention comprise eg treatment of conditions of deficiency of MBL, treatment of cancer and of infections in connection with immunosuppressive chemotherapy including in particular those infections which are seen in connection with conditions during cancer treatment or in connection with implantation and/or transplantation of organs. The invention also comprises treatment of conditions in connection with recurrent miscarriage. DETAILED DESCRIPTION OF THE INVENTION The immune system has a considerable ability to prevent infections, which is of decisive importance for homeostasis and consequently survival and preservation of life. Therefore it is of considerable interest to identify compounds that are active in such processes. An intense research within this decade has uncovered Mannan- Binding Lectin (MBL) as a very versatile macromolecule, which eg is believed to be active in the innate immune system. By the present invention it is possible to produce a human recombinant mannan binding lectin (MBL) composition having a size distribution profile at least 50 % identical to naturally occurring human MBL, as described above. The MBL composition is produced as described above and subjected to an affinity chromatography capable of separating higher oligomers of MBL from the other oligomers. The culture medium comprising a variety of MBL oligomers is subjected to an affinity chromatography on a carbohydrate-derivatized matrix. Affinity chromatography is a well-known method for purifying proteins from a protein mixture. The carbohydrate-derivatized matrix has an least two-fold greater affinity for tetramers, pentamers and/or hexamers of MBL subunits than for dimers of MBL subunits. In a preferred embodiment the carbohydrate-derivatized matrix has an least three-fold greater affinity for tetramers, pentamers and/or hexamers of MBL subunits than for dimers of MBL. Also, the carbohydrate-derivatized matrix may have an least two-fold greater affinity for tetramers, pentamers and/or hexamers of MBL subunits than for MBL subunits. Thereby a MBL-composition having a size distribution being at least 50 % identical, such as 60 % identical, such as 70 % identical, such as 80 % identical, such as 90 % identical, such as 95 % identical, to naturally occurring MBL is provided. The size distribution is preferably substantially identical to naturally occurring MBL, whereby is meant that the identity is at least 99 %. The carbohydrate-derivatized matrix has substantially no affinity to subunits and/or dimers of MBL subunits. Preferably the carbohydrate-derivatized matrix has affinity for substantially only tetrameric, pertameric and/or hexameric recombinant MBL subunits. It is preferred that the higher oligomers of the subunit, such as tetramers, pentamers and/or hexamers are dominating the composition. Thus, in the composition the ratio of tetramers, pentamers, and/or hexamers to subunits and/or dimers is preferably at least 2:1 , preferably at least 5:1. In a preferred embodiment, the ratio of the sum of tetramers, pentamers and hexamers to the sum of subunits and dimers is preferably at least 2:1 , more preferably at least 5:1. The matrix may be derivatized with any carbohydrate or carbohydrate mixture whereto MBL binds and for which binding of the higher oligomers of MBL are favoured. The matrix is preferably a hexose-, such as a mannose- or a N-acetyl- glucosamin derivatized matrix, such as most preferably a mannose matrix. The selectivity of the carbohydrate-derivatized matrix is obtained by securing that the matrix as such, i..e the un-derivatized matrix has substantially no affinity to MBL subunits, in particular no affinity to MBL trimers or smaller oligomers. This may be ensured when the matrix as such is carbohydrate-free. In particular the matrix should not contain any Sepharose or the like. It is preferred that the matrix consists of a non-carbohydrate-containing polymer material, such as Fractogel®TSK beads The matrix may be in any form suitable for the chromatography, mostly in the form of beads, such as plastic beads After application of the culture media the column is washed, preferably by using non-denaturing buffers, having a composition, pH and ionic strength resulting in elimination of proteins, without eluting the highed oligomers of MBL Such as buffer may be TBS Elution of MBL is performed with a selective desorbing agent, capable of efficient elution of highed oligomers of MBL, such as TBS containing a desorbing agent, such as EDTA (5 mM for example) or mannose (50 mM for example), and MBL oligomers are collected According to the invention, the sequences from the MBL gene may be from the human MBL gene or from MBL genes of other animal species, in which the immune system in this respect is acting like the human immune system An example of a preferred embodiment of a preparation of a recombinant MBL according to the invention is described in example 1 below, wherein said recombinant MBL is prepared by the use of an expression vector comprising sequences from the human MBL gene An illustration of the expression vector in said preferred embodiment of the preparation method can be found in example 1 and in Figure 2 The invention also concerns the use of expression vectors comprising sequences, which are functional derivatives of the sequences of the human MBL gene By said functional derivatives are meant sequences, which contain base pair alterations that lead to no functional or essentially no functional differences of the expression vector and the in this way prepared MBL has a functionality comparable to the MBL prepared by the use of an expression vector comprising the unaltered sequences from the human MBL gene In addition to the purification method it is preferred that the gene expression con- struct and the host cell also favours production of higher oligomers Accordingly, the gene expression construct preferably comprises at least one intron sequence from the human MBL gene or a functional equivalent thereof Furthermore, the gene expression construct may comprise at least two exon sequences from the human MBL gene or a functional equivalent thereof. More preferably the gene expression construct comprises at least three exon sequences from the human MBL gene or a functional equivalent thereof. When comprising more than one exon, the exon sequences are preferably aligned as in the human MBL gene. Although preferred that the sequence comprises intron sequences, it may for some applications be convenient that the expression construct comprises a cDNA se- quence encoding a MBL subunit or a functional equivalent thereof. The invention features the use of MBL gene expression constructs rather than MBL cDNA constructs for expression of rMBL in mammalian cell lines or transgenic animals to obtain recombinant MBL with structural properties under non-denaturing and denaturing conditions being substantially similar to natural human MBL. By "recombinant human MBL" is meant human MBL which is expressed from engineered nucleic acids and by "MBL gene expression constructs" is meant an expression vector suitable for expression in mammalian cell lines, which contains exon sequences and at least one intron sequence from the human MBL gene or from MBL genes of other animal species, such as but not limited to chimpanzees and rhesus monkeys. Preferably, the DNA sequences encode a polypeptide sequence as shown in SEQ ID NO: 1 or a functional equivalent, whereby a functional equivalent is as defined above. SEQ ID NO:1 corresponds to the MBL sequence having database accession NO: P11226. The equivalent may be obtained by a modification of the peptide sequence shown as SEQ ID NO: 1 , such as a sequence processing a corresponding property as the sequences mentioned in the present invention, but wherein one or more amino acids have been substituted with others. Preferably a functional equivalent contains conservative substitutions, i.e. where one or more amino acids are substituted by an amino acid having similar properties, such that a person skilled in the art of protein chemistry will expect the secondary and tertiary structure of the protein to be unchanged. Amino acids suitable for conservative substitutions include those having functionally similar side chains. For example, hydrophobic residues: e.g. glycine, alanine, valine, ieucine, isoleucine and methionine may replace another such residue. Similarly, conservative substitutions may involve interchanging hydro- philic residues (e g arginme and lysme, glutamine and aspargine, threonine and serine), basic reduces (e g , lysme, arginme and histidine), and/or acidic residues (e g , aspartic acid and glutamic acid) Functional equivalents may also, or alternatively, be modified by for example the deletion or addition of ammo acids, or the chemical modification of ammo acids, as long as the function of the polypeptide is preserved The isolated MBL peptide including any functional equivalents thereof, may in one embodiment comprise at least 80 am o acid residues, such as at least 100 am o acid residues, such as at least 150 ammo acid residues, such as at least 200 ammo acid residues, for example at least 220 ammo acid residues, such as at least 250 ammo acid residues In a preferred embodiment the expression vector is suitable for expression in mam- malian cell lines or transgenic animals, which contains exon sequences and at least one intron sequence from the human MBL gene or from MBL genes of other animal species, such as, but not limited to, chimpanzes and rhesus monkeys In one embodiment the host cell culture is cultured in a transgene animal By a transgenic animal in this context is meant an animal which has been genetically modified to contain and express the human MBL gene or fragments or mimics hereof In a preferred embodiment the expression construct of the present invention comprises a viral based vector, such as a DNA viral based vector, a RNA viral based vector, or a chimeric viral based vector Examples of DNA viruses are cytomegalo virus, Herpex Simplex, Epstem-Barr virus, Simian virus 40, Bovine papillomavirus, Adeno-associated virus, Adenovirus, Vaccinia virus, and Baculo virus In mammalian host cells, a number of viral-based expression systems may be utilized In cases where an adenovirus is used as an expression vector, the nucleic acid molecule of the invention may be ligated to an adenovirus transcription/translation control complex, for example, the late promoter and tripartite leader sequence This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non-essential region of the viral genome (for example, region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a MASP-3 gene product in infected hosts (for example, see Logan and Shenk, Proc Natl Acad Sci USA 81 3655-3659, 1984) Specific initiation signals may also be required for efficient translation of inserted nucleic acid molecules These signals include the ATG initiation codon and adjacent sequences In cases where an entire gene or cDNA, including its own initiation codon and adja- cent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided Furthermore, the initiation codon must be in phase with the reading frame of the desired coding se- quence to ensure translation of the entire insert These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc (see Bittner et al , Methods in Enzymol 153 516-544, 1987) Examples of RNA virus are Semhki Forest virus, Smdbis virus, Poko virus, Rabies virus, Influenza virus, SV5, Respiratory Syncytiai virus, Venezuela equine encephalitis virus, Kunjin virus, Sendai virus, Vesicular stomatitisvirus, and Retroviruses Examples of chimetic viruses are Adeno-virus, Smdbis virus and Adenovirus - adeno-associated virus Regarding specific vectors reference is made to Makπdes, S C , "Components of vectors for Gene Transfer and Expression in Mammalian Cells", which is hereby incorporated by reference In particular, an Epstem-Barr virus origin of replication or functional derivatives or mimics hereof including the pREP9 vector is used in one aspect the invention provides an expression construct encoding human MBL, featured by comprising one or more intron sequences from the human MBL gene including functional derivatives hereof Additionaly, it contains a promoter region selected from genes of virus or eukaryotes, including mammalia and insects The promoter region is preferably selected to be different from the human MBL promoter, and preferably in order to optimize the yield of MBL and size distribution of MBL oligomers, the promoter region is selected to function most optimally with the vector and host cells in question In a preferred embodiment the promoter region is selected from a group comprising Rous sarcoma virus long terminal repeat promoter, and cytomegalovirus immediate- early promoter, and elongation factor-1 alpha promoter In another embodiment the promoter region is selected from genes of microorganisms, such as other viruses, yeasts and bacteπae In order to obtain a greater yield of recombinant MBL, the promoter region may comprise enhancer elements, such as the QBI SP163 element of the 5' end un- translated region of the mouse vascular endothelian growth factor gene The construct is used for transforming a host cell to obtain a host cell culture capable of expressing MBL. The synthesis of the recombinant MBL may be by use of in vitro or in vivo cultures The host cell culture is preferably an eucaryotic host cell culture. By transformation of an eukaryotic cell culture is in this context meant introduction of recombinant DNA into the cells The expression construct used in the process is characterised by having the MBL encoding region selected from mammalian genes including human genes and genes with big resemblance herewith such as the genes from the chim- panzee The expression construct used is furthermore featured by the promoter region being selected from genes of virus or eukaryotes, including mammalian cells and cells from insects The present invention provides thus recombinant MBL, which is a form of MBL similar to natural human MBL, in that it in its purified form has a molecular size distribution profile closer to the natural human MBL than known up till now The process for producing recombinant MBL according to the invention is characterised in that the host cell culture is preferably eukaryotic, and for example a mam- malian cell culture A preferred host cell culture is a culture of human kidney cells and in an even more preferred form the host cell culture is a culture of human embryonal kidney cells (HEK cells) The invention features the use of HEK 293 cell lines for production of recombinant human MBL By "HEK 293 cell lines" is meant any cell line derived from human embryonal kidney tissue such as, but not limited to, the cell lines deposited at the American Type Culture Collection with the numbers CRL-1573 and CRL-10852 Other cells may be chick embryo fibroblast, hamster ovary cells, baby hamster kidney cells, human cervical carcinoma cells, human melanoma cells, human kidney cells, human umbilical vascular endothehum cells, human brain endothe um cells, human oral cavity tumor cells, monkey kidney cells, mouse fibroblast, mouse kidney cells, mouse connective tissue cells, mouse oligodendπtic cells, mouse macro- phage, mouse fibroblast, mouse neuroblastoma cells, mouse pre-B cell, mouse B lymphoma cells, mouse plasmacytoma cells, mouse teratoca noma cells, rat astro- cytoma cells, rat mammary epithelium cells, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired Such modifications (for example, glycosylation) and processing (for example, cleavage) of protein products may be important for the function of the protein Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcπpt, glycosylation, and phosphorylation of the gene product may be used The mammalian cell types listed above are among those that could serve as suitable host cells In another aspect the present invention provides MBL that is prepared by recombinant DNA technology m e g mammalian cells or in transgenic animals More specifically, the invention concerns recombinant MBL (rMBL), which is synthesized by - preparing a gene expression construct as defined above encoding a human MBL polypeptide or a functional equivalent thereof, - transforming a host cell culture with the construct, - cultivating the host cell culture, thereby obtaining expression and secretion of the polypeptide into the culture medium, followed by - obtaining a culture medium comprising human recombinant MBL Furthermore, the invention relates to the synthesis of MBL by (a) preparation of an expression construct encoding human MBL, (b) transformation of a eukaryotic cell culture with said expression construct and thereby obtain an eukaryotic recombinant host cell culture, (c) growth of said recombinant host cell culture either in vitro or in form of a transgenic animal and thereby express said human MBL, (d) harvest of said expressed human MBL and purification hereof by means of affinity chromatography as described above The host cell culture may be cultured in any suitable culture medium Example of culture medium are RPMI-1640 or DMEM supplemented with, e g , insulin, transferrin, selenium, and foetal bovine serum The invention further consists of a step for purification or isolation of human MBL, such as human MBL from culture supernatants or from body fluids or tissue from transgenic animals by use of carbohydrate affinity chromatography as described above In a preferred embodiment of the invention the affinity chromatography is performed by means of matrices of mannose, hexose or N-acetyl-glucoseamm derivatized matrices, which are suitable for affinity chromatography as described above In particular, an affinity chromatography is used, in which the matrices have been deπvatizised with mannose Previously the use of matrices deπvatizised with mannan has been employed to harvest recombinant MBL from culture supernatant While mannan-deπvatized matrices binds both high and low molecular weight forms of MBL, the use of matrices deπvatizised as described above, such as with mannose offers an surprising advantage in the purification of recombinant MBL as this matrix selectively bind the form of recombinant MBL that resemble natural MBL The degree of similarity to the natural MBL is measured to be more than 50% in analysis of the size distribution by SDS-PAGE followed by protein staining of the gel as known in the prior art, see e g Ma et al (1997) Purified recombinant MBL is in this context to be understood as recombinant MBL purified from cell culture supernatants or body fluids or tissue from transgenic animals purified by use of carbohydrate affinity chomatography The process of isolating or purifying a composition of human MBL oligomers having a size distribution at least 50 % identical with naturally occurring plasma MBL preferably comprises - obtaining a preparation of human MBL ohgomers, - subjecting said preparation to affinity chromatography using a carbohydrate- derivatized matrix, said carbohydrate-derivatized matrix having a two-fold greater affinity for tetramers, pentamers and/or hexamers of MBL than for dimers of MBL, and - obtaining an eluate comprising the isolated human recombinant MBL composition The present invention provides a novel form of recombinant MBL composition comprising oligomers of MBL subunits, the size distribution profile of the ohgomers being at least 50 % identical to the size distribution of naturally occurring plasma human MBL In a preferred embodiment the size distribution is at least 60 % identical, such as 70 % identical, such as 80 % identical, such as 90 % identical, such as 95 % identical, to naturally occurring MBL is provided It is preferred that the higher ohgomers of the subunit, such as tetramers, pentamers and/or hexamers are dominating the composition Thus, in the composition the ratio of tetramers, pentamers, and/or hexamers to subunits and/or dimers is preferably at least 2:1 , preferably at least 5:1. In a preferred embodiment, the ratio of the sum of tetramers, pentamers and hex- amers to the sum of subunits and dimers is preferably at least 2:1 , more preferably at least 5:1. When estimating the size distribution of the oligomers of the MBL composition the size distribution is assessed by Western blotting on a SDS-PAGE. Hereby it may be seen that 50 % means that more than 50 % of the rMBL has an apparent molecular weight higher than 200 kDa when analysed by SDS-PAGE and/or Western blot. The recombinant MBL composition may be purified by any suitable means from the culture medium, such as by any physico-chemical isolating method, including but not limited to filtration methods, chromatography, such as ion-exchange chromatography based on size, gel permeation chromatography or affinity chromatography. The recombinant MBL composition is preferably purified from the culture medium by means of affinity chromatography as discussed above. The functionality of the recombinant MBL composition is preferably resembling the functionality of plasma or serum MBL. In the present context the functionality of MBL is meaning the capability of activating the complement system as discussed above in relation to functional equivalents. The functionality may be expressed as the specific activity of MBL, such as units of MBL activity per ng MBL. The functionality the recombinant MBL composition as expressed as specific activity is preferably at least 25 % of the specific activity of MBL purified from serum, such as at least 50 % of the specific activity of MBL purified from serum, more preferred at least 75 % of the specific activity of MBL purified from serum. The MBL composition according to the invention is substantially free from any impurities naturally associated with the MBL when produced in a native host organism, such as from any impurities naturally associated with the MBL purified from serum By a native host organism is meant that MBL is produced by a cell normally expressing MBL. The eluant obtained from the affinity chromatography may be used as such for preparing a pharmaceutical composition, or the eluant may be subjected to further purification steps before being used The MBL composition obtained by the present invention may be used for the preparation of a pharmaceutical composition for the prevention and/or treatment of various diseases or conditions In addition to the MBL o gomers, the pharmaceutical composition may compπse a pharmaceutically acceptable carrier substance and/or vehicles In particular, a stabilising agent may be added to stabilise the MBL proteins The stabilising agent may be a sugar alcohol, sacchaπde, protein and/or ammo-acids An example of a stabilising agent may be albumin or maltose Other conventional additives may be added to the pharmaceutical composition depending on administration form for example In one embodiment the pharmaceutical composition is in a form suitable for injec- tions Conventional carrier substances, such as isotonic saline, may be used In another embodiment the pharmaceutical composition is in a form suitable for pulmonal administration, such as in the form of a powder for inhalation or creme or fluid for topical application The present invention is based upon the synthesis of a novel form of recombinant MBL, which form is closer to the natural human MBL than achieved up till now and which can be synthesised by use of in vitro or in vivo technology Based upon the evidence for the important role of MBL in the immune defence, this provided form of recombinant MBL offers the surprising possibility of treatment of conditions such as cancer and of infections in connection with immunosuppressive chemotherapy The structural differences between the previously reported preparations of recombinant MBL compared to the natural form of MBL made any such treatment unlikely Also, the application of rMBL similar to natural MBL in said treatment represents a great advantage over the application of known MBL, in particular plasma-derived human MBL, since the treatment with rMBL confers a much reduced risk of viral infection The treatment needs not be a treatment of a diagnosed disease, disorder or condi- tion in a presently or apparently need of treatment but may be used to prevent the disease or condition to occur A treatment in this context may comprise cure and/or prophylaxis of e g the immune system and reproductive system by humans and by animals having said functional units acting in this respect like those in humans By conditions to be treated are not necessarily meant conditions presently known to be in a need of treatment, but comprise generally any condition in connection with current and/or expected need or in connection with an improvement of a normal condition In particular, the treatment is a treatment of a condition of deficiency of MBL In another aspect of the present invention the manufacture is provided of a medicament consisting of said pharmaceutical compositions of MBL including compositions of rMBL fragments or mimics hereof intended for treatment of conditions comprising cure and/or prophylaxis of conditions of diseases and disorders of e g the immune system and reproductive system by humans and by animals having said functional units acting like those in humans Said diseases, disorders and/or conditions in need of treatment with the compounds of the invention comprise eg treatment of conditions of deficiency of MBL, treatment of cancer and of infections in connection with immunosuppressive chemotherapy including in particular those infections which are seen in connection with conditions during cancer treatment or in connection with implantation and/or transplantation of organs The invention also comprises treatment of conditions in connection with recurrent miscarriage Thus, in particular the pharmaceutical composition may be used for the treatment and/or prevention of clinical conditions selected from infections, MBL deficiency, cancer, disorders associated with chemotherapy, such as infections, diseases associated with human immunodeficiency virus (HIV), diseases related with congenital or acquired immunodeficiency More particularly, chronic inflammatory demyelinating polyneuropathy (CIDP, Multifocal motoπc neuropathy, Multiple scelrosis, Myasthenia Gravis, Eaton-Lambert's syndrome, Opticus Neuritis, Epilepsy, Primary antiphosho- hpid syndrome, Rheumatoid arthritis, Systemic Lupus erythematosus, Systemic scleroderma, Vascuhtis, Wegner's granulomatosis, Sjøgren's syndrome, Juvenile rheumatiod arthritis, Autoimmune neutropenia, Autoimmune haemolytic anaemia, Neutropenia, Crohn's disease, Colitis ulcerous, Coeliac disease, Asthma, Septic shock syndrome, Chronic fatigue syndrome, Psoriasis, Toxic shock syndrome, Diabetes, Smuitis, Dilated cardiomyopathy, Endocarditis, Atherosclerosis, Primary hypo/agammaglobuhnaemia including common variable immunodeficiency, Wiskot- Aldπch syndrome and serve combined immunodefiency (SCID), Secondary hypo/agammaglobuhnaemia in patients with chronic lymphatic leukaemia (CLL) and multiple myeloma, Acute and chronic idiopathic thrombocytopenic purpura (ITP), Allogenic bone marrow transplantation (BTM), Kawasaki's disease, and Guillan- Barre's syndrome The route of administration may be any suitable route, such as intravenously, mtra- musculary, subcutanously or intradermally Also, pulmonal or topical administration is envisaged by the present invention In particular the MBL composition may be administered to prevent and/or treat infections in patients having clinical symptoms associated with congenital or acquired MBL deficiency or being at risk of developing such symptoms A wide variety of conditions may lead to increased susceptibility to infections in MBL-deficient individuals, such as chemotherapy or other therapeutic cell toxic treatments, cancer, AIDS, genetic disposition, chronic infections, and neutropenia It appears that cancer patients treated by chemotherapy are often susceptible to infection due to adverse effects of the drug regime on cells of the immune system, which is the background for the use of MBL therapy in the treatment of this condi- tion The observed low plasma concentrations of MBL (below 500 ng/mL) are indicative for an increased susceptibility to clinical significant infections, and the immune defence of these patients can be reinforced by administration of recombinant or natural plasma-derived MBL The pharmaceutical composition may thus be administered for a period before the onset of administration of chemotherapy or the like and during at least a part of the chemotherapy The MBL composition may be administered as a general "booster" before chemotherapy, or it may be administered to those only being at risk of developing MBL deficiency The group of patients being at risk may be determined be measuring the MBL level before treatment and only subjecting those to treatment whose MBL level is below a predetermined level The limit for determining a low MBL level is evalu- ated to be below 500 ng/ml for most groups The MBL level may be determined by time resolved immunofluorescent assay as described in Example 9, ELISA, RIA or nephelometry Another indication for administering MBL is when the MBL level is below 50 % of the normal level, such as below 300 ng/ml, or below 200 ng/ml The MBL composition is administered in suitable dosage regimes, in particularly it is usually administered at suitable intervals, eg once or twice a week during chemotherapy Normally from 1-100 mg is administered per dosage, such as from 2-10 mg, mostly from 5-10 mg per dosage Mostly about 0 1 mg/kg body weight is administered Thus, in one aspect the invention concerns MBL, including rMBL, fragments or mimics hereof for use in the treatment of cancer and of conditions of diseases and disorders of e g the immune system and reproductive system, said treatment consisting of creation, reconstitution, enhancing and/or stimulating the opsonic and/or bactericidal activity of the complement system, i e enhancing the ability of the immune defence to recognise and kill microbial pathogens Furthermore, an aspect of the present invention is the use of a recombinant composition according to the present invention in a kit-of-parts further compπsing another medicament In particular the other medicament may be an anti-microbial medicament, such as antibiotics Concerning miscarriage, it has been reported that the frequency of low plasma levels of MBL is increased in patients with otherwise not explained recurrent miscarriages, which is the background for lowering of the susceptibility to miscarriage by a reconstitution of the MBL level by administration of recombinant MBL in these cases. As to the nature of compounds of the invention, it appears, that in its broad aspect, the present invention relates to compounds which are able to act as opsonins, that is, able to enhance uptake by macrophages either through direct interaction be- tween the compound and the macrophage or through mediating complement deposition on the target surface. A particular example hereof is MBL, a fragment or a mimic hereof. The present invention is based upon the disclosure of a synthesis of a recombinant human MBL which appears to be closer to the structure of the natural human MBL than achieved in the past. The invention has now been explained and accounted for in various aspects and in adequate details, but additionally it will be illustrated below by figure 1 and 2 and the non-limiting examples of preferred embodiments. BRIEF DESCRIPTION OF THE FIGURE Figure 1 shows purified recombinant and native MBL compared analysed in the non- reduced state by SDS-PAGE and silver staining (stacking gel included). Lane 'S' was loaded with 24 ng of natural MBL (MO-04, The State Serum Institute), Lane 'HEK' with 60 ng of rMBL produced in HEK 293EBNA cells and Lane Ηyb' with 12 ng of rMBL produced in Sp2/0Ag14 cells (supplied by Dr. R.A.B. Ezekowitz). Arrow shows the position of the predominant form in the hybridoma cell-produced rMBL. Although the amount rMBL in Lane 'HEK' is 5 times higher than in Lane 'Hyb', no band 55 kDa band can be seen in Lane 'HEK', suggesting a significant difference in the disulfide bridge formation of the two rMBL subunits. ( As discussed in Example 6 Figure 2 shows a sketch of a preferred expression construct according to the invention viz. the MBUpREP9 construct as discussed in Example 1. Figure 3 shows MBL concentrations in fractions (stated as elution volume) from gel permeation chromatography analysis (Example 7). Figure 4 shows a Western blot analysis of rMBL purified by mannose-TSK beads, mannan-TSK beads, mannose-Sepharose beads, mannan-Sepharose beads and underivatizised Sepharose 4B beads. For comparison unfractionated culture supernatant from MBlJpREP9 transfected cells was included (Example 3). Figure 5 shows a Western blot of two preprations of rMBL analysed in the non- reduced state: 50 ng rMBL produced according to the present invention ('293 EBNA cells') and 50 ng rMBL preparations produced according to the method described by Super et a/., 1992 ('Sp2/0Ag14 cells'). Natural MBL purified from human serum as described by Lu et al. 1990 ('Natural MBL') (Example 6). EXAMPLES EXAMPLE 1 Design of the MBL gene expression construct: Human genomic DNA was isolated from 20 mL of blood drawn into EDTA. The EDTA/blood was mixed with 80 mL of cold (4 °C) 10 mM Tris-HCI, 5 m MgCl2, 1% (v/v) Triton X-100, 0.32 mM sucrose, and left to incubate at 4 °C for 30 min with occasional agitation. The sample was centrifuged at 1000*g for 30 min at 4 °C, followed by discharge of the supernatant and a brief rinse of the nuclei-containing pel- let in approximately 10 mL of 0.9% (w/v) of NaCI. The collected nuclei were resuspended in 6 mL of 75 mM NaCI, 24 mM EDTA [pH 8.0], and 460 μL of 10% (w/v) SDS and 1 mg pronase (cat.no. 165921, Boehringer-Mannheim, Mannheim, Germany) was added. After o/n incubation at RT with gentle agitation, 2 mL of a saturated (~ 6 M) NaCI solution was added. Following vigorous agitation, the debris was removed by centrifugation at 1000 χ g. Isopropanol was added 1:1 to the supernatant and the vial was gently shaken until the DNA precipitate appeared. The thread-like precipitate was collected on a sealed Pasteur pipette, washed on the sealed tip in 70% (v/v) ethanol, air-dried and finally resuspended in 1 mL of TE-buffer. PCR amplification of the MBL gene was carried out with a sense-primer modified to contain an Xbol site: 5'-AGATTAACCTTCCtcGAGTTTTCTCACACC-3'. The anti-sense primer was modified to contain a SamHI site: 5'- TAACqqaTCCACTCCAATAATACATACAC-3' (restriction sites engineered into the sequences are underlined and modified basepairs compared to the natural gene sequence as published by Sastry et a/. (1989) are show as lower case letters in both primer sequences. Primers were produced by DNA technology, Aarhus, Denmark). The sense and anti-sense primers were located in the 5' and 3' UTRs respectively. Reaction conditions for the PCR consisted of 200 μ dATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP (cat.no. 1969064, Boehringer-Mannheim), reaction buffer at 1 * strength, 0.75 μL of enzyme (cat.no. 1732641 , Expand High Fidelity™, Boehringer- Mannheim, reaction buffer supplied with enzyme), genomic DNA (100 ng), and water to a final volume of 50 μL. The PCR thermal program performed 1 cycle at 96 °C for 3 min, 10 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 5 min, followed by 20 cycle of 94 °C for 15 s, 60 °C for 30 s, and 68 °C for 5 min with a time increment of 20 s per cycle, followed by a final elongation step at 72 °C for 5 min (Omn- E™ thermal cycler, Hybaid, Ashford, UK). Cloning of the 6.2 kb PCR product into the pCR2 vector by use of the TA cloning kit™ (cat.no. K2000-01 , Invitrogen, Leek, The Netherlands) was carried out according to the manufactures' recommendations. The MBL gene was isolated from the pCR2 by digesting the vector with Not\ and BambU enzymes (cat.nos. 15441-025 & 15201-023, GibcoBRL, Paisley, UK) for 6 h at 37 °C in an appropriate buffer. The insert DNA, i.e., the 5'-Not \-MBL-Bamh\-3' fragment, was isolated from an 1 % (w/v) agarose gel by use of Pharmacia's Bandprep™ kit (cat.no. 27-9285-01 , Pharmacia, Uppsala, Sweden). The pREP9 vector (cat.no. V009-50, Invitrogen) was digested as described for the MBUpCR2 vector and the restriction product, i.e., the linearised vector, was likewise isolated from agarose to minimise contamination with undigested vector. In a final volume of 10 μL, 1 μL (~ 100 ng) of digested pREP9 vector, 4 μL (~ 400 ng) of 5'-Λ/otl-MB -βamHI-3', ligation buffer at 1 strength and 20 units of T4 ligase (cat.no. 202S, New England Biolabs, Beverly, MA, reaction buffer supplied with enzyme) was incubated o/n at 14 °C. E. coli TOPF10 cells were made transformation competent by CaCl2 permeabilising and transformed with the ligation product by heat-shock as described by Hanahan (1983). The bacteria were spread onto LB-agar plates supplemented with 50 μg ampicilin/mL, and colonies appeared after o/n incubation at 37 °C. Figure 2 shows a sketch of a preferred expression construct according to the inven- tion viz. the MBUpREPd construct. The PCR amplified (the location of sense and antisense primers are indicated black with arrows) part of the MBL gene contained the four exons, including the entire known 3' untranslated of exon 4 as determined by Sastry et al. 1989. The fragment was merged with the expression vector, named pREP9, in the Λ/otl and Bam HI endonuclease restriction sites (the EcoRV, Xbal, BglW, Xmn\, C/al, Stul and Sac\ restriction endonuclease sites in the vector sequence are also indicated). Transcription of the MBL fragment cloned into the pREP9 expression vector was controlled by the Rous sarcoma virus long terminal repeat promoter (PRSV) located upstream to the cloning site (direction of the transcription for elements of the vector are indicated with arrow heads) and with a sim- ian virus 40 (SV40) polyadenylation site. Bacterial propagation, that is, production of vector DNA in E. coli, was obtained through an ampicilin resistance marker (Amp) and a ColE1 prokaryot 'origin of replication'. Maintenance of the vector in HEK293EBNA cells was conferred by the Epstein-Barr virus origin of replication (OriP) and expression of the Epstein-Barr virus nuclear antigen 1 (EBNA-1), which attaches the vector to the host cell chromosomes. EXAMPLE 2 Expression of recombinant MBL in mammalian cells: HEK 293EBNA cells, grown until confluence in 150 cm 2 flasks (cat.no. 9075, TPP AG) containing DMEM (Biological Industries) supplemented with 10% FBS, gluta- min, 200 μg G418/mL (Geneticin™, cat.no. G9516, Sigma) and antibiotics (10,000 units penicillin and 0.1% (w/v) streptomycin), were harvested by trypsination and seeded in a four fold lower density (-25% confluence) into 150 cm 2 flasks (cat.no. 90150, TPP) 24 h prior to transfection. Three hours prior to transfection all medium was renewed. In a total volume of 1680 μL, 60 μg of the MBlJpREP plasmid DNA (reference is made to Example 1) was mixed with 150 mM NaCI, 1 mM EDTA, 10 m Tris-HCI (pH 7.12), 268 mM CaCl2. The DNA, calcium solution was added into 1680 μL of 50 mM HEPES, 250 mM NaCI, 1.5 mM Na 2 HPθ4+NaH 2 Pθ4 adjusted to pH 7.12. The cells were incubated for 14-16 h with the DNA/Ca PO4 precipitate, rinsed twice in PBS and further incubated with RPMI-1640 medium (50 mL per bottle) supplemented with glutamin, antibiotics (penicillin and streptomycin) and insulin- transferrin-selenium (ITS) solution (cat.no. 51300-44, GibcoBRL) for 6-8 d. EXAMPLE 3 Purification of rMBL by use of carbohydrate-derivatizised matrices. Fractogel TSK HW-75 beads (cat.no. 14985, Merck KgaA, Darmstadt, Germany) were activated by mixing 50 mL of beads with 50 mL of 0.5 M Na2CO3 (pH 11) and 10 mL of vinyl sulfone (cat.no. V370-0, Aldrich, Milwaukee, WI), followed by incubation with agitation for VΛ h at RT. The beads were collected on a glass filtre and washed once in H2O and once in 0.5 M Na2CO3 (pH 1 1). Beads were resuspended in 0.5 M Na2CO3 (pH 11) and mannose added to a final concentration of 10% (w/v). The mixture was left to incubate with agitation o/n at RT. Following rinse in H 2O, residual reactive groups were blocked by incubating the beads in 0.1 M ethanola- mine (pH 9) for 2 h. After a rinse in H2O and TBS, 5 mL of mannose-coupled TSK- 75 was packed on a chromatography column and washed in 15 mL of 0.1 M glycin (pH 3.0) and equilibrated in TBS/0.05% (v/v) Tween-20, 2 mM CaCl2 until UV- absorption remained stable. Culture medium from MBL/pREP9 transfected cells, cleared by centrifugation at 5000 χ g, was passed over the column at 0.4 mLJmin. The column was washed in 75 mL of TBS, 2 mM CaCl2, and the bound rMBL eluted in TBS, 5 mM EDTA and collected in fractions of 500 μL. To prepare TSK beads with mannan (purified from yeast as described by Nakajima & Ballou, 1974), the beads were activated as described above and incubated with 25 mg mannan/mL 0.5 M Na2CO3 (pH 11), followed by the same treatment as for preparation of mannose- derivatized beads. Sepharose 4B (cat.no. 17-0120-01 , Pharmacia, Uppsala, Sweden) beads were derivatized with mannose and mannan as described for the TSK beads. Eluates from the three matrices were anlysed Western blotting as described in Example A, loading 7 ng of rMBL purified by mannose-TSK beads, mannan-TSK beads, mannose-Sepharose beads, and mannan-Sepharose beads into separate lanes of the SDS-PAGE gel. For comparison unfractionated culture supernatant corresponding to 7 ng of rMBL was loaded in a lane. The Western blot is shown in Figure 4. EXAMPLE 4 Analysis of MBL under non-denatuπng conditions by gel permeation chromatography Permeation chromatography of purified rMBL or culture medium containing rMBL was performed on a Superose-6 column (Superose 6™ HR 10/30, cat.no. 17-0537- 01 , Pharmacia) equilibrated in 5 mM EDTA, TBS,0.01% (v/v) Tween-20 [pH 7.4]. A sample volume of 100 μL was passed over the column with a flow rate of 0.5 mlJmin. Fractions were collected 5 mL post sample injection in volumes of 250 μL. EXAMPLE 5 Analysis of MBL under denatunng conditions by SDS-PAGE/silver-staining SDS-PAGE 4%-20% gradient gels were prepared and used in electrophoresis as described by Jensen et al. (1997). The procedure for silver staining was adopted from Nesterenko et al. (1994). The PAGE gels were fixed in 100 mL 50% (v/v) acetone, 6.25 mL tricholoroacetic acid, 0.85% (v/v) formaldehyde for 15 min, followed by rinsing trice in H2O, and washing for 15 min in H2O. The gel was incubated in 100 mL of 50% (v/v) acetone for 15 min followed by incubation for 1 min in 0.67 mM Na2S2θ3, and three rinses in H2O. Impregnation of the gel with silver nitrate was carried out in 15 mM AgNO3, 0.9% (v/v) formaldehyde, followed by rinsing three times in H2O. The gel was developed by incubating in 0.19 mM Na2CO3, 0.9% (v/v) formaldehyde, 0.67 mM Na2S2θ3 for approx. 3 min. The precipitation was stopped by incubation of the gel for 3 min in 1 % (v/v) acetic acid and preserved in 25% (v/v) ethanol, 3% (v/v) glycerol. EXAMPLE 6 Analysis of rMBL by SDS-PAGE followed by Western blotting Tris buffered saline (TBS) and phosphate buffered saline (PBS), all adjusted to pH 7.4 were prepared as described by Sambrook et al. (1989). For blocking nonspecific protein interactions, the buffers were supplemented with 0.05% (v/v) Tween- 20 (TBS/Tw and PBS/Tw). SDS-PAGE 4%-20% gradient gels were prepared and used in electrophoresis as described by Jensen et al. (1997). 50 ng of rMBL pro- duced in HEK 293EBNA cells (prepared according to the present invention), 50 ng of rMBL prepared as described by Super et al (1992), and 50 ng of MBL prepared from human plasma (MO012, State Serum Institute) was loaded onto the gel in the non-reduced state Proteins were transferred onto Amersham's Hybond™-P mem- brane (cat no RPN303F) by semi-dry blotting in 50 mM Tπs-base, 40 mM glycin, 3 7% (w/v) SDS, 20% (v/v) ethanol for 60 Vxh Non-specific protein interactions were blocked by incubating the membrane with 0 1 % Tween-20 for Y∑ h For detection of MBL, the membrane was incubated with a monoclonal antibody raised against human MBL (Hyb 131-1 , State Serum Institute) diluted to 1 μg/ ml in TBS, 0 05% Tween-20, 1 mM EDTA, 1 mg HSA/mL (cat no 44 05 11 , State Serum Institute), 1 mg human IgG/mL (cat no 00 7740, Centeon Pharma GmbH, Marburg, Germany) o/n at RT After 3 washes in TBS/Tw buffer, the membrane was incubated with HRP conjugated rabbit anti mouse IgG (cat no P0260, Dako A S, Copenhagen, Denmark), diluted 2,500 fold in TBS/Tw, 1 mM EDTA with 100 μg human IgG/mL for 2 h at RT Finally, the membrane was washed 5 times in TBS/Tw and developed with Pierce's chemiluminescence substrate (SuperSignal™, cat no 34080, Pierce, Rockford, IL) on photographic films (Kodak, NY) EXAMPLE 7 Analysis of rMBL by gel permation chromatography Gel permeation chromatography of purified rMBL or culture medium containing rMBL was performed on a Superose-6 column (Superose 6™ HR 10/30, cat no 17- 0537-01 , Pharmacia) equilibrated in 5 mM EDTA, TBS,0 01 % (v/v) Tween-20 [pH 7 4] A sample volume of 100 μL (corresponding to 100 ng of rMBL or natural MBL) was passed over the column with a flow rate of 0 5 mlJmin Fractions were collected 5 mL post sample injection in volumes of 250 μL The fractions were diluted 2 fold in TBS/Tw, 10 mM EDTA and analysed by a time-resolved immunofluorometπc assay (TRIFMA) as described in Example 8 EXAMPLE 8 Analysis of rMBL by a time-resolved immunofluorometπc assay (TRIFMA) Flourosorp™ microtitre plates (cat no 437958, Nunc, Kamstrup, Denmark) were coated o/n with 100 μL (per well) of MAb 131-1 anti-MBL (Statens Serum Institut, Copenhagen, Denmark) at 5 μg/mL PBS and washed trice in TBS/Tw A standard serum with a known MBL concentration (3 6 μg MBL/mL) was diluted 20 fold in 10 mM EDTA, TBS/Tw and used as such as well as in further 5-fold dilutions with duplicate wells, each receiving 100 μL of the dilution Three control sera, one with a high cone of MBL (1 2 μg MBL/mL), one with a medium concentration of MBL (250 ng MBL/mL), and one with a low concentration of MBL (50 ng MBL/mL), were applied diluted 20 fold in TBS/Tw/EDTA An MBL defi- cient serum was likewise diluted 20 fold and added to the plate as a negative control Culture supernatants, either FBS containing or serum-free, were typically diluted 20 and 100 fold After o/n incubation, the plate was washed trice in TBS/Tw and each well received 100 μL of Eu 3+ -labelled MAb 131-1 anti-MBL at 125 ng/mL 25 μM EDTA, TBS/Tw Labelled antibody was prepared essentially according to Wallac's protocol (Wallac, Turku, Finland) Development and measurement of bound europium was carried out by standard procedures for time resolved immunofluorometry as descπbed elsewhere (Hemmila et al , 1993) REFERENCES Davies, E J , Snowden, N , Hillarby, M C , Carthy, D , Grennan, D M , Thomson, W & W E Oilier (1995) Mannose-bmding protein gene polymoφhism in systemic lupus erythematosus Adhntis Rheum 38 110-114 Drickamer, K , J , Dordal, M S & L Reynolds (1986) Mannose-bmding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails J Biol Chem 261 6878-6887 Christiansen, O B , Kilpatπck, D C , Souter, V , Warming, K , Thiel, S & J C Jen- senius (1999) Mannan-bmding lectin deficiency is associated with unexplained recurrent miscarriage Scand J Immunol 49 193-196 van Emmeπk, L C , Kuijper, E J , Fijen, C A P , Dankert, J & S Thiel (1994) Binding of mannan-bmding protein to various bacterial pathogens of menigitis Clin Exp Immunol 97 411-416 Garred, P , Richter, C , Andersen, A B , Madsen, H O , Mtoni, I , Svejgaard, A & J Shao (1997a) Mannan-bmding lectin in the sub-saharan HIV and tuberculosis epi- demies Scand J Immunol 46 204-208 Garred, P , Madsen, H O , Balslev, U , Hofmann, B , Pedersen, C , Gerstoft, J & A Svejgaard (1997b) Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-bmding lectin Lancet 349 236-240 Guo, N , Mogues, T , Weremowicz, S , Morton, C C & K N Sastry (1998) The human ortholog of rhesus mannose-bmding protem-A gene is an expressed pseudogene that localizes to chromosome 10 Mamm Genome 9 246-249 Hanahan, D (1983) Studies on transformation of Escherichia coli with plasmids J Hartshorn, K L , Sastry, K , Brown, D , White, M R , Okarma, T B , Lee, Y -M & A I Tauber (1993) Conglutmin acts as an opsonm for influenza A viruses J Immunol 151 6265-6273 Haurum, J , Thiel, S , Jones, I M , Fischer, P , Laursen, S B & J C Jensenius (1993) Complement activation upon binding of mannan-bmding protein to HIV envelope glycoporteins AIDS 7 1307-1313 Hemmila, I , Mukkala, V M , Latva, M & P Kiilholma (1993) Di- and tetracarboxylate derivatives of pyπdines, bipyπdines and terpyπdines as lummogenic reagents for time-resolved fluorometπc determination of terbium and dysprosium J Biochem Biophys Methods 26 283-290 Holmskov, U , Holt, P , Reid, K B M , Willis, A C , Teisner, B & J C Jensenius (1993a) Purification and characterization of bovine mannan-bmding protein Glyco- biology 3 147-153 Holmskov, U , Malhotra, R , Sim, R B & J C Jensenius (1994) Collectins collagenous C-type lectms of the innate immune defense system Immunol Today 15 67- 74 Huang, Y , Kong, Y , Wang, Y , Qi, G & C Lu (1996) Stable expression of anti-HPV 16 E7-πbozyme in CV-1 cell lines Chin J Biotechnol 12 215-20 lobst, S T , Wormald, M R , Weis, W I , Dwek, R A & K Drickamer (1994) Binding of sugar ligands to Ca(2+)-dependent animal lectms I Analysis of mannose binding by site-directed mutagenesis and NMR J Biol Chem 269 15505-15511 Ip, W K , Chan, S Y , Lau, C S & Y L lau (1998) Association of systemic lupus erythematosus with promoter polymorphisms of mannose-bmding lectin gene Ar- thntis Rheum 41 1663-1668 Janeway, C A , Travers, P , Walport, M & J D Capra (1999) Immunobiology (4 th ed ) Churchill Livingston, Edmburg, 634 pp Ji, Y -H , Fujita, T , Hatsuse, H , Takahashi, A , Matsushita, M & M Kawakami (1993) Activation of the C4 and C2 components of complement by a proteinase in serum bactericidal factor, ra-reactive factor J Immunol 150 571-578 Kawai, T , Suzuki, Y , Eda, S , Ohtani, K , Kase, T , Sakamoto, T , Uemura, H & N Wakamiya (1998) Molecular and biological characterization of rabbit mannan- bindmg protein Glycobiology 8 237-244 Kawasaki, T , Etoh, R & I Yamashma (1978) Isolation and characterisation of a mannan-bmding protein from rabbit liver Biochem Biophys Res Comm 3 1018- 1024 Kilpatπck, D C (1997) Mannan binding protein is sera positive for rheumatoid factor Br J Rheumatol 36 207-209 Kuhlman, M , Joiner, K & R A B Ezekowitz (1989) The human mannose-bmding protein function as an opsonm J Exp Med 169 1733-1745 Kurata, H , Cheng, H M , Kozutsumi, Y , Yokota, Y & T Kawasaki (1993) Role of the collagen-like domain of the human serum mannan-bmding protein in the activation of complement and the secretion of this lectin Biochem Biophys Res Comm 191 1204-1210 Kurata, H , Sannoh, T , Kozutsumi, Y , Yokota, Y & T Kawasaki (1994) Structure and function of mannan-bmding proteins isolated from human liver and serum J Biochem 115 1148-1154 Lau, Y L , Lau, C S , Chan, S Y , Karlberg, J & M W Turner (1996) Mannose- bmding protein in Chinese patients with systemic lupus erythematosus Arthπtis Rheum 39 706-708 Laursen, S B , Hedemand, J E , Thiel, S , Willis, A C , Skπver, E , Madsen, P S & J C Jensenius (1995) Collectm in a non-mammalian species isolation and characterization of mannan-bmding protein (MBP) from chicken serum Glycobiology 5 553-561 Laursen, S B , Dalgaard, T S , Thiel, S , Lim, B L , Jensen, T V , Juul-Madsen, H R , Takahashi, A , Hamana, T , Kawakami, M & J C Jensenius (1998) Cloning and sequencing of a cDNA encoding chicken mannan-bmding lectin (MBL) and compan- son with mammalian analogues Immunology 93 421-430 Law, S K A & K B M Reid (1995) Complement, IRL Press, Oxford Lipscombe, R J , Lau, Y L , Levmsky, R J , Sumiya, M , Summerfield, J A & M W Turner (1992a) Identical point mutation leading to low levels of mannose binding protein and poor C3b mediated opsonisation in Chinese and Caucasian populations Immunol Lett 32 253-257 Lipscombe, R J , Sumiya, M , Hill, A V , Lau, Y L , Levmsky, R J , Summerfield, J A & M W Turner (1992b) High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene Hum Mol Genet 1 709-715 Lipscombe, R J , Sumiya, M , Summerfield, J A & M W Turner (1995) Distinct physicochemical characteristics of human mannose-bmding protein expressed by individuals of differing genotype Immunology 85 660-667 Lu, J , Thiel, S , Wiedemann, H , Timpl, R & K B M Reid (1990) Binding of the pentamer/hexamer forms of mannan-bmding protein to zymosan activates the pro- enzyme C1 r^CI ≤2 complex, of the classical pathway of complement without involvement of C1q J Immunol 144 2287-2294 Ma, Y , Yokota, Y , Kozutsumi, Y & T Kawasaki (1996) Structural and functional roles of the ammo-terminal region and collagen-like domain of human serum man- nan-binding protein Biochem Mol Biol Int 40 965-974 Ma, Y , H Shida & T Kawasaki (1997) Functional expression of human mannan- binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vac- cinia virus: post-translational modification, molecular assembly, and differentiation of serum an liver MBP. J.Biochem. (Tokyo) 122: 810-818. Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N. & T. Kawasaki (1999) Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding protein-dependent cell-mediated cytotoxicity. PNAS 96:371-375. Madsen, H.O., Garred, P., Kurtzhals, J.A., Lamm, L.U., Ryder, L.P., Thiel, S. & A. Svejgaard (1994) A new frequent allele is the missing link in the structural polymorphism of the human mannan-binding protein. Immunogenetics 40:37-44. Madsen, H.O., Garred, P., Thiel, S., Kurtzhals, J.A., Lamm, L.U., Ryder, L.P., & A. Svejgaard (1995) Inteφlay between promoter and structural gene variants control basal serum level of mannan-binding protein. J. Immunol. 155:3013-3020. Malhotra, R, Haurum, J.S., Thiel, S. & R.B. Sim (1994) Binding of human collectins (SP-A and MBP) to influenza virus. Biochem. J. 304:455-461. Matsushita, M. & T. Fujita (1992a) Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like protease. J. Exp. Med. 176:1497-1502. Matsushita, M., Ezekowitz, R.A.B. & T. Fujita (1995a) The Gly-54→Asp allelic form of human mannose-binding protein (MBP) fails to bind MBL-associated serine protease. Biochem. J. 311 :1021-1023. Mizuno, Y., Kozutmi, Y., Kawasaki, T. & I. Yamashina (1981) Isolation and characterization of a mannan-binding protein from rat liver. J. Biol. Chem. 256:4247-4252. Mogues, T., Ota, T., Tauber, A.I. & K.N. Sastry (1996) Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications. Glycobiology 6:543-550. Makrides, S.C., "Components of vectors for Gene Transfer and Expression in Mammalian Cells" Nakajima, T. & CE. Ballou (1974) Characterization of the carbohydrate fragments obtained from Saccharomyces cerevisiae mannan by alkaline degradation. J. Biol. Chem. 249:7679-7684 Ng, K.K., Drickamer, K. & W.I. Weis (1996) Structural analysis of monosaccharide recognition by rat liver mannose-binding protein. J. Biol. Chem. 271 :663-674 . Nielsen, S.L., Andersen, P.L, Koch, C, Jensenius, J.C. & S. Thiel (1995) The level of the serum opsonin, mannan-binding protein in HIV- antibody-positive patients. Clin. Exp. Med. 100:219-222. Ohta, M., Okada, M., Yamashina, I. & T. Kawasaki (1989) The mechanism of carbohydrate-mediated complement activation by the serum mannan-binding protein. J. Biol. Chem. 265:1980-1984. Ohtani, K., Suzuki, Y., Eda, S., Kawai, T., Kase, T., Keshi, H., Sakai, Y., Yamamoto, S., Sakamoto, T. & N. Wakamiya (1999) High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells. J. Immunol. Methods. 222:135-44 . Oka, S., Ikeda, K., Kawasaki, T. & I. Yamashina (1985) Isolation and characterization of mannan-binding proteins from chicken liver. Arch. Biochem. Biophys. 241 :95- 105. Oka, S., Ikeda, K., Kawasaki, T. & I. Yamashina (1988) Isolation and characteriza- tion of two distinct mannan-binding proteins from rat serum. Arch. Biochem. Biophys. 260:257-266. Pastinen, T., Liitsola, K., Niini, P., Salminen, M. & A.-C. Syvanen (1998) Contribution of the CCR5 and MBL genes to susceptibility to HIV type 1 infection in the Fin- nish population. AIDS Res. Hum. Retroviruses 14:695-698. Reddy, P S & R B Corley (1998) Assembly, sorting, and exit of oligomeric proteins from the endoplasmic reticulum Bioessays 20 546-554 Sastry, K , Herman, G A , Day, L , Deignan, E , Bruns, G , Morton, C C & R A B Ezekowitz (1989) The human mannose-bmding protein gene J Exp Med 170 1175-1189 Sato, T , Endo, Y , Matsushita, M & T Fujita (1994) Molecular characterization of a novel serine protease involved in activation of the complement system by mannose- bmding protein Int Immunol 6 665-669 Schweinle, J E , Nishiyasu, M , Ding, T Q , Sastry, K , Gillies, S D & R A B Ezekowitz (1993) Truncated forms of mannose-binding protein multimeπze and bind to mannose-πch salmonella montevideo but fail to activate complement in vitro J Biol Chem 268 364-370 Sheriff, S , Chang, C Y & R A B Ezekowitz (1994) Human mannose-bmding protein carbohydrate recognition domain tπmeπzes through a triple α-hehcal coiled-coil Structural biol 1 789-794 Soothill, J F & B A Harvey (1976) Defective opsonization A common immunity deficiency Arch Dis Child 51 91-99 Stover, C M , Thiel, S , Thelen, M , Lynch, N J , Vorup-Jensen, T , Jensenius, J C & W J Schwaeble (1999) Two constituents of the initiation complex of the mannan- bmding lectin activation pathway of complement are encoded by a single structural gene J Immunol 162 3481-3490 Sumiya, S , Super, M , Tabona, P , Levmsky, R J , Arai, T , Turner, M W & J A Summerfield (1991) Molecular basis of opsonic defect in immunodeficient children Lancet 337 1569-1570 Super, M., Thiel, S., Lu, J., Levinsky, R.J. & M.W. Turner (1989) Association of low levels of mannan-binding protein with a common defect opsonisation. Lancet ii:1236-1239. Super, M., Gillies, S.D., Foley, S., Sastry, K., Schweinle, J.E., Silverman, V.J. and R.A.B. Ezekowitz (1992) Distinct and overlapping functions of allelic forms for human mannose-binding protein. Nature Genet. 2:50-55. Tabona, P., Mellor, A. & J.A. Summerfield (1995) Mannose binding protein is in- volved in first-line host defence: evidence from transgenic mice. Immunology 85:153-159. Takada, F., Takayama, Y., Hatsuse, H. & M. Kawakami (1993) A new member of the C1s family of complement proteins found in a bactericidal factor, ra-reactive factor, in human serum. Biochem. Biophys. Res. Commun. 196:1003-1009. Takayama, Y., F. Takada, Takahashi, A. and M. Kawakami (1994) A 100-kDa protein in the C4-activating component of ra-reactive factor is a new serine protease having module organisation similar to Clr and C1s. J. Immunol. 152: 2308-2316. Tan, F.K. & F. C. Arnett (1998) The genetics of lupus. Curr. Opin. Rheumatol. 10:399-408. Taylor, M.E., Brickell, P.M., Craig, R.K. & J.A. Summerfield (1989) Structure and evolutionary origin of the gene encoding a human serum mannose-binding protein. Biochem. J. 262:763-771. Thiel, S., Vorup-Jensen, T., Stover, CM., Schwaeble, W., Laursen, S.B., Poulsen, K., Willis, A.C., Eggleton, P., Hansen, S., Holmskov, U., Reid, K.B. & J.C. Jensenius (1997). A second serine protease associated with mannan-binding lectin that activates complement. Nature 386:506-510. Turner, M.W. (1996) Mannose-binding lectin: the pluripotent molecule of the innate immune system. Immunol. Today 17:532-540. Valdimarsson, H., Stefansson, M., Vikingsdottir, T., Arason, G.J., Koch, O, Thiel, S. & J.C. Jensenius (1998) Reconstitution of opsonizing activity by infusion of mannan- binding lectin (MBL) to MBL-deficient humans. Scand. J. Immunol. 48:116-23. Vorup-Jensen, T., Jensenius, J.C. & S. Thiel (1998) MASP-2, the C3 convertase generating protease of the MBLectin complement activating pathway. Immunobiol- ogy 199: 348-357. Voss, T., Melchers, K., Scheirie, G. & K.P. Schafer (1991) Structural comparison of recombinant pulmonary surfactant protein SP-A derived from two human coding sequences: implications for the chain composition of natural human SP-A. Am. J. Respir. Cell Mol. Biol. 4:88-94. Weis, W.I., Drickamer, K. & W.A. Hendrickson (1992) Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature 360:127-134. Weis, W.I. & K. Drickamer (1994) Trimeric structure of a C-type mannose-binding protein. Structure 2:1227-1240.

Claims: CLAI M S : 1. A process of producing a human recombinant mannan binding lectin (MBL) composition having a size distribution profile at least 50 % identical to natural occurring human, comprising the steps of: preparing a gene expression construct encoding human MBL peptide or a functional equivalent thereof, transforming a host cell culture with the construct, - cultivating the host cell culture, thereby obtaining expression and secretion of the polypeptide into the culture medium, followed by subjecting said culture medium to affinity chromatography using a carbohydrate-derivatized matrix, said carbohydrate-derivatized matrix having at least a two-fold greater affinity for tetramers, pentamers and/or hexamers of MBL subunit than for dimers of MBL subunit, - obtaining an eluate comprising the human recombinant MBL composition. 2. The process according to claim 1 , wherein the gene expression construct comprises at least one intron sequence from the human MBL gene or a functional equivalent thereof. 3. The process according to claim 1 , wherein the gene expression construct comprises at least two exon sequences from the human MBL gene or a functional equivalent thereof. 4. The process according to claim 1 , wherein the gene expression construct comprises a cDNA sequence encoding a MBL subunit or a functional equivalent thereof. 5 The process according to claim 1 , wherein the host cell culture is cultured in vitro 6 The process according to claim 1 , wherein the host cell culture is cultured in I The process according to claim 6, wherein the host cell culture is cultured in a transgene animal 8 The process according to claim 1 , wherein the host cell culture is an eucaryotic host cell culture 9 The process according to claim 8, wherein the host cell culture is a mammalian host cell culture 10 The process according to claim 1 , wherein the matrix as such has substantially no affinity to MBL I I The process according to claim 10, wherein the matrix as such is carbohydrate free 12 The process according to claim 11 , wherein the matrix as such is consisting of a non-carbohydrate containing polymer material 13 The process according to claim 12, wherein the matrix is consisting of plastic beads 14 The process according to claim 1 , wherein the isolation of the polypeptide composition is performed on a hexose-deπvatized matrix 15 The process according to claim 14, wherein the isolation of the polypeptide composition is performed on a mannose-deπvatized matrix 6 The process according to the claim 1 , wherein the carbohydrate-derivatized ma- tπx has substantially no affinity for subunits and/or dimers of MBL subunits 17 The process according to any of the preceding claims, wherein the carbohydrate-derivatized matrix has affinity for substantially only tetrameric recombinant MBL subunits 18 The process according to any of the preceding claims, wherein the carbohydrate-derivatized matrix has affinity for substantially only pentameπc recombinant MBL subunits 19 The process according to any of the preceding claims, wherein the carbohydrate-derivatized matrix has affinity for substantially only hexameric recombinant MBL subunits 20 The process according to any of the preceding claims, wherein the carbohydrate-derivatized matrix has affinity for substantially only tetramers, pentamers, and/or hexamers of recombinant MBL subunits 21 The process according to any of the preceding claims, wherein the size distribution profile is substantially identical to naturally occurring human plasma MBL 22 A recombinant human MBL composition comprising ohgomers of MBL subunits, the size distribution profile of the ohgomers being at least 50 % identical to the size distribution of naturally occurring plasma human MBL 23 The composition according to claim 22, werein the size distribution is at least 95 % identical to the size distribution of naturally occurring plasma human MBL 24 The composition according to claim 22, wherein the ratio of tetramers, pentamers and/or hexamers to dimers of MBL subunits is at least 2 1 , preferably at least 5 1 25 The composition according to claim 22, wherein MBL composition is substantially free from any impurities naturally associated with the MBL when produced in a native host organism 26 The composition according to any of the claims 22-25, wherein the size distribution is assessed by SDS-PAGE and/or Western Blotting 27 The composition according to any of the claims 22-26, being in a non-denatured state 28 The composition according to any of the claims 22-26, being in a denatured state 29 The composition according to claim 22, wherein the MBL subunit is assembled of three peptide sequences comprising the sequence as shown i SEQ ID NO 1 or a functional equivalent thereof 30 A gene expression construct encoding a recombinant human mannan-bmding lectin (MBL) polypeptide, comprising at least one intron sequence from the human MBL gene or a functional equivalent thereof, - at least one exon sequence from the human MBL gene or a functional equivalent thereof, - a promoter region different from the human MBL promoter, and - an expression vector 31 The gene expression construct according to claim 30, wherein the promoter region is selected from genes of eucaryotes, such as mammals and insects 32 The gene expression construct according to claim 30, wherein the promoter region is selected from viral genes 33 The gene expression construct according to claim 32, wherein the promoter region is selected from a group comprising Rous sarcoma virus long terminal repeat promoter, and cytomegalovirus immediate-early promoter, and elongation factor- 1 alpha promoter 34 The gene expression construct according to claim 30, wherein the promoter re- gion is selected from genes of microorganisms, such as viruses, yeast or bacteria 35 The gene expression construct according to claim 30, wherein the promoter region comprises enhancer elements 36 The gene expression construct according to claim 30, wherein the vector is a pREP9 vector 37 A process of producing a human recombinant mannan binding lectin (MBL), comprising the steps of preparing a gene expression construct as defined in any of claims 30-36 encoding a human MBL polypeptide or a functional equivalent thereof, - transforming a host cell culture with the construct, cultivating the host cell culture, thereby obtaining expression and secretion of the polypeptide into the culture medium, followed by - obtaining a culture medium comprising human recombinant MBL 38 A process of isolating a composition of human MBL o gomers having a size distribution at least 50 % identical with naturally occurring plasma MBL comprising - obtaining a preparation of human MBL ohgomers, - subjecting said preparation to affinity chromatography using a carbohydrate- derivatized matrix, said carbohydrate-derivatized matrix having a two-fold greater affinity for tetramers, pentamers, and/or hexamers of MBL subunit than for dimers of MBL subunit, and - obtaining an eluate comprising the isolated human recombinant MBL composition. 39. The process according to claim 38, wherein the matrix as such has substantially no affinity to MBL. 40. The process according to claim 39, wherein the matrix as such is carbohydrate free. 41. The process according to claim 39, wherein the matrix as such is consisting of a non-carbohydrate containing polymer material. 42. The process according to claim 41 , wherein the matrix is consisting of plastic beads. 43. The process according to claim 38, wherein the isolation of the polypeptide composition is performed on a mannose-matrix. 44. The process according to claim 38, wherein the isolation of the polypeptide com- position is performed on a hexose-matrix. 45. The process according to the claim 38, wherein the carbohydrate-derivatized matrix has substantially no affinity for subunits and/or dimers of MBL subunits. 46. The process according to claim 38, wherein the the carbohydrate-derivatized matrix has affinity for substantially only tetramers, pentamers and/or hexamers of MBL subunits. 47. A pharmaceutical composition comprising a human recombinant MBL composi- tion as defined in any of the claims 22-29, optionally further comprising a pharmaceutically acceptable carrier substance. 48.The composition according to claim 47, in a form suitable for injections. 49 The composition according to claim 48, wherein the carrier substance is saline, human serum albumin, or mannose 50 The composition according to the claims 47, in a form suitable for pulmonal ad- ministration 51 The composition according to claim 50, in the form of a powder for inhalation 52 The composition according to claim 47, in the form of a creme or lotion for topi- cal application 53 Use of a gene expression construct as defined in the claims 30-36 for the production of a human recombinant MBL composition 54 Use of a human recombinant MBL composition as defined in the claims 22-29 for the production of a pharmaceutical composition 55 The use of a composition according to claim 54, for the production of a pharmaceutical composition for the treatment, in an individual, of clinical conditions selected from infections, MBL deficiency, cancer, disorders associated with chemotherapy, miscarriages, disorders associated with neutropenia, and human immunodeficiency virus (HIV) 56 The use according to claim 55, wherein the pharmaceutical composition is ad- ministered intravenously, mtramusculary, subcutanously, or intradermally 57 The use according to claim 55, wherein the pharmaceutical composition is administered pulmonally 58 The use according to claim 55, wherein the pharmaceutical composition is administered topically 59 The use according to claim 55, wherein the pharmaceutical composition is administered prophylactically before initiation of chemotherapy or other therapeutic cell toxic treatments 60 The use according to claim 59, wherein the treatment is continued for a suitable period after initiation of the of chemotherapy or other therapeutic cell toxic treatment 61 The use according to claim 60, wherein the serum MBL level in the individual is measured before initiating treatment 62 The use according to claim 61 , wherein the serum MBL level is below 50 % of the normal level before initiating treatment 63 The use according to claim 55, wherein the amount of MBL composition administered is from 1-100 mg/dosage

Inventors: JENSEN THOMAS VORUP, JENSENIUS JENS CHRISTIAN, THIEL STEFFEN, JENSEN, THOMAS, VORUP, JENSENIUS, JENS, CHRISTIAN, THIEL, STEFFEN
Applicants: JENSEN THOMAS VORUP, JENSENIUS JENS CHRISTIAN, THIEL STEFFEN, JENSEN, THOMAS, VORUP, JENSENIUS, JENS, CHRISTIAN, THIEL, STEFFEN
Application year: 2000
Main classification: C12N15
IPC codes: A61K9, A61K38, A61K47, A61P31, A61P35, A61P37, C07K1, C07K14, C12N15, C12P21, C12R1