|Title of Invention||
"A METHOD FOR THE MASS PRODUCTION OF MULTIMERIC RECOMBINANT HUMAN MBL"
|Abstract||The present invention relates to a method for the mass-production of multimeric recombinant human mannose binding lectin (rhMBL) in a CHO cell line transfected with a vector containing the sequence of human MBL coding region. Further this invention relates to the construction of a recombinant cell line from which MBL can be produced more than 80% as highly functional polymeric form. Further this invention relates to the construction of a recombinant cell line that can produce 50 ug/million cells/day in a flask culture systEm for the production of MBL. Further this invention describes methods for high density culture of the cell in a protein free media using bioreactor and for selective purification of high molecular form of MBL from the culture media. This invention also describes a method for production of functional rhMBL that could be used for the development of a therapeutic agent for the treatment of viral, bacterial, or fungal infections.|
|Full Text||METHOD FOR THE MASS PRODUCTION OF MULTIMERIC MANNOSE BINDING LECTIN
The present invention relates to the development of processes by which the mass-production of multimeric recombinant human mannose binding lectin (rhMBL) possible.
Background of the Invention
Mannose binding lectin (referred to as "MBL" hereinafter) is a serum protein involved in natural immunity (innate immunity). The molecular weight of MBL is 32 kDa. MBL is composed of C-terminal carbohydrate recognition domain (CRD), collagen domain, and cystein rich region in the amino terminal end. Three MBL molecules form a complex via triple helix formation using collagen domains, and then up to 6 complexes can combine together by inter-molecular disulfide bond (-s-s-) among cystein residues on the N-terminal end, resulting in the formation of multimeric MBL molecule consisting of up to 18 MBL molecules.
Oligomeric form of MBL can form a complex with other proteins such as MBL associated serine proteases (MBSP-1, MASP-2 or MASP-3) or MBL associated protein (Map 19). Although the general molecular structure and the function of MBL involved in complement activation are similar to Clq, which is the first component of complement system, unlike Clq MBL activates complement system by cleaving C4 and C2. This complement activation process called lectin pathway is accomplished through MBL associated serine protease activated upon MBL binding to microbe. Activation of
complement system by MBL is one of MBL functions in the primary defense against microbial infection before taking place adaptive immune responses. MBL binding to microorganism through carbohydrate-recognizing domains (CRD) that recognize unique glycosylation patterns of surface protein of a microorganism is the prerequisite of complement activation. MBL binding to microorganism activates the MBL associated precursors of serine proteases, MASP-1 and MASP-2, leading to the cleavage of C4 and C2 of the complement system, generating C4b2a and C3b. Some of the proteins generated in the complement activation directly attach to the surface of a microorganism as opsonin, and MBL can also function as opsonin to make the phagocytosis of MBL bound microbes more efficient by phagocytic cells. Some activated complement proteins directly lyses invading microorganisms by forming membrane attacking complex (MAC), and other complement protein fragments produced during the activation process promote the secretion of cytokine causing inflammation.
Previous reports described expression of MBL gene hi a variety of cells such as CHO cells, HLF hepatoma cells or HEK293 EBNA cells. In particular, the expression level in CHO cells was very high but the produced MBL was mainly monomers and dimmers (Katsuki Ohtani et al, J. Immunol. Methods, 222: 135-144, 1999). The production of oligomeric form of MBL in HEK293 EBNA cells has been accomplished, but the gross productivity was less than 1 |ig/M£ (T. Vorup-Jensen et al., International Immunopharinacology, 1: 677-687, 2001). Production of oligomeric form of MBL is critically important, for the monomers and dimmers consisting of 3 subunits and 6 subunits, respectively, have little, if any, activities. It is also an important matter in developing a pharmaceutical product from MBL to develop a high producer cell line, since it is required in many milligram quantities.
MBL may also play an important role in adaptive immunity. Cells involved in
phagocytosis such as neutrophils and macrophages play an important role in eliminating invading foreign microorganisms by phagocytosis. In addition to this, macrophage is an antigen presenting cell (APC), which activates T-cells, resulting in induction of acquired immune response. Therefore, multimeric MBL involved in activating complement system and as opsonin plays an important role not only in the primary defense as a component of innate immune system but also in the induction of acquired immune responses.
The amount of MBL in human serum varies according to individuals, ranging from 50 ng/M£ to several \iQ/M due to genetic variation of MBL gene. For example, when a point mutation occurred at codon 52, 54, or 57 of exon 1 of MBL gene, MBL molecules do not form oligomeric forms, producing non-functional smaller complexes consisting of not more than 3 MBL molecules. When mutation occurs in the promoter regions of MBL gene, the expression level of MBL was decreased, resulting in tremendous variation of blood level MBL among affected individuals. In general, those infants or patients with neutropenia who have below certain level of MBL have weak immunity, becoming highly susseptible to microbial infections (Sumiya, M. et al., Lancet, 337: 1569-1570, 1991; Summerfield, J. A. etal., Lancet, 345: 886, 1995; Garred, P. et al., Lancet, 346: 941, 1995; Summerfield, J. et al., Bi: Med. J., 314: 1229, 1997; Mullighan, C. G. et al., Scand. J. Immunol., 51: 111-122, 2000; Neth, O. et al., Lancet, 358: 614-618, 2001; Peterslund, N. A. etal., Lancet, 358: 637-638, 2001; Mullighan C. G. et al., Blood, 99: 3524-3529, 2002).
According to a study on hepatitis B virus chronic patients, the level of MBL in blood is a critical factor in the prognosis of the disease (Hakozaki Y. et al., Liver 22, 29-34, 2002). Particularly, among patients developed fulminant hepatic failure from hepatitis B virus chronic infection, mortality of those who had over 3 HQ/M£ of blood
level MBL was 0%, whereas those who had 1.5 \IQ/M and under 0.5 \IQ/M of blood level MBL it was 58% and over 80%, respectively. This study shows dramatic relationship between the blood level of MBL and mortality of patients with an infectious disease. This study also suggests that MBL supplement to the patients with low or no MBL may be beneficial.
In order for recombinant MBL to be used as a therapeutic agent, the recombinant MBL protein has to be produced in massive quantity for a reasonable price in the form of active oUgomeric molecules. Unlike cytokines it is required in many milligrams per patients in each application. For example, human blood volume is average about 5 liters, requiring 25 mg to make 5 HQ/ml MBL. Taking account of other body fluid volume, it could require much more in each application. Therefore, it had been a critically important issue in the commercial product development out of MBL that one establishes not only a high producer recombinant cell line but also the cell line that produces active forms of polymeric MBL.
Thus, the present inventors made every effort to establish a recombinant cell line that could produce MBL in a commercial quantity and at the same time that could produce functional oligomeric form of MBL, which is capable ot activating complement system by binding specifically to MBL binding glycoproteins in the presence of MASPs. As the result of this effort, the present inventors had completed this invention by confirming that active rhMBL suitable for therapeutic use could be produced in massive quantity in the form of multimeric polymer in a CHO cell line transfected with a cloning vector containing a polynucleotide coding sequence for rhMBL.
Description of Drawings
FIG. 1 is a schematic diagram showing the genetic map of pMSG-MBL plasmid
FIG. 2A and FIG. 2B are the pictures of MBL produced from the recombinant CHO cell line, CHO MBL/D1-3, that is transformed with pMSG-MBL. Fig. 2A is the SDS-PAGE analysis pattern, and 2B shows Western Blot analysis pattern using an MBL specific monoclonal antibody.
FIG. 3 is a photograph of electron microscope showing oligomeric forms of MBL. Oligomeric MBL molecules consisting of 2,3,4,5,and 6 trimeric subunits of MBL are clearly seen.
FIG. 4 is a photograph of electron microscope showing the complex of recombinant MBL protein and gold nanoparticle coated with pre S in the presence of calcium ion.
FIG. 5A and FIG. 5B show that rhMBL binds specifically to pre-S, a glycoprotein, or mannan in quantitatively similar activity to the natural MBL,
FIG. 6 shows the relative binding capacity of larger polymer form of MBL and smaller form consisting mainly of monomers and dimmers of trimeric subunits. Smaller forms have little, if any, binding capacity.
FIG. 7 shows the comparison of the C4 activating capacities of larger form and smaller form of rhMBL.
FIG. 8 shows that the rhMBL specifically binds to various microorganisms.
FIG. 9 shows that rhMBL inhibits infection of SARS-CoV into embryonic monkey kidney cells (FRhK-4).
FIG. 10 is a set of phase constrasv microscopic images showing FRhk-4 cells infected with SARS-CoV. Without rhMBL treatment, cells are looking not healthy, whereas with MBL healthy cells are present.
FIG. 11 is a schematic diagram showing the diagnostic kit prepared by using the recombinant human MBL.
Object of the invention
It is the object of the present invention to construct a CHO cell line that is transfected with a vector containing a polynucleotide coding sequence for human MBL for the purpose of producing functional oligotneric form of MBL in massive quantity to be used for commercial purpose.
Further it is the object of present invention to establish a method for purification of rhMBL from the culture media of the recombinant CHO cell line.
Summary of the Invention
The present invention provides an expression vector pMSG-MBL containing the sequence of rhMBL coding region represented in the plasmid map of FIG. 1.
The present invention also provides a host cell line transfected with the expression vector.
The present invention further provides a method for the mass-production of multimeric recombinant human MBL according to the following steps:
(1) preparing host cells transfected with an expression vector containing the
sequence of human MBL coding region;
(2) producing a recombinant human MBL from the transformed cell line with the
expression vector with the sequence of human MBL coding region in a culture system;
(3) purifying the recombinant human MBL prepared in the step (2).
The present invention also provides a detection kit for the recombinant human
MBL comprising i) a solid support with a test line and a control line wherein mouse anti-human mannose binding lectin antibodies are fixed on the test line and anti-mouse IgG antibodies or anti-pre-S antibodies are fixed on the control line; ii) a dye pad adsorbed with a dye conjugate and connected to the bottom of the solid support, wherein if anti-mouse IgG antibodies are fixed on the control line, the dye conjugate is mouse anti-human mannose binding lectin antibodies-gold conjugate and if anti-pre-S antibodies are fixed on the control line, the dye conjugate is pre-S-gold conjugate; and iii) a sample pad connected to the bottom of the dye pad, whereon a sample is loaded.
Detailed Description of the Invention
The present invention provides an expression vector pMSG-MBL containing a polynucleotide coding for rhMBL represented in the plasmid map of FIG. 1, which is sufficient enough to express olygomeric recombinant human MBL that can activate complement system by combining specifically with a MBL binding glycoprotein in the presence of MBL associated seririe proteases.
In a preferred embodiment of the present invention, human MBL cDNA (Gene Bank NM 000242) was inserted in pMSG vector (Accession No: KCCM(Korean culture center of microorganisms) 10202) containing MAR element (nuclear matrix attachment region element) of beta-globia gene, poly-A of SV40 virus and transcription terminator complex of gastrin gene, resulting in the construction of a unique vector named pMSG-MBL.
The present invention also provides host cells transfected with the expression vector containing the polynucleotide coding for rhMBL which is sufficient enough to express the recombinant human MBL, which is capable of activating complement system by binding specifically to a glycoprotein on the surface of microorganisms in the
presence of MBL associated serine proteases.
Host cells used in the present invention are animal cells in general, and preferably selected from a group consisting of CHO (Chinese hamster ovary) cells, hepatocytes, HEK (human embryonic kidney) cells, etc.
In a preferred embodiment of the present invention, a transformant was prepared by introducing a cloning vector pMSG-MBL containing the polynucleotide encoding rhMBL, which is sufficient enough to express a recombinant MBL capable of activating complement system by binding specifically to glycoproteins on the surface of microorganisms in the presence of a MBL associated serine proteases, into a host cell line such as CHO cell, and then, adapting the transfortnants to the condition with MTX (methotrexate), leading to the selection of the transformant which was able to mass-express rhMBL protein in the form of multimeric polymer. The selected transformant was named as MBL Dl-3 (CHO cell line) and deposited at Korean Collection for Type Culture, Daejeoa Korea, on May 16, 2003 (Accession No: KCTC 10472BP).
The recombinant human MBL produced from the transformant described in the present invention is mainly in the form of multimeric forms, which is similar to the natural MBL from human blood. The cell line established in the present invention produced mainly functional multimeric form of MBL and showed high expression level, making it possible to use the transformed CHO cell line for mass-production of functional rhMBL protein.
The present invention further provides a method for the production of a recombinant human MBL, comprising the following steps:
(1) Method providing host cells transfected with a DNA construct including a cloning vector containing the polynucleotide encoding human MBL, which is sufficient enough to express a recombinant human MBL capable of activating complement system
by binding specifically to a glycoprotein on the surface of microorganisms in the presence of MBL associated serine protease.
(2) Method producing recombinant human MBL by expressing the polynucleotide
encoding human MBL in animal cell and from the cell employing a massive cell culture
(3) Method purifying the recombinant MBL produced in the step (2).
Particularly, exploiting the characters of MBL binding to glycoprotein of the step
(1) and the MBL binding glycoprotein is exemplified by glycosylated viral envelope protein containing pre-S of HBV, glycosylated bacterial protein, glycosylated fungal protein, and synthetic glycoprotein.
In a preferred embodiment of the present invention, the producing step of a recombinant MBL of step (2) is characterized by the steps of suspension-culturing the cells in a serum-free/protein-free medium and sub-culturing those cells adapted well to the serum-free/protein-free medium to scale up gradually, resulting in the mass-production of rhMBL protein in the form of multimeric polymer.
In the step (3), a recombinant MBL protein was purified from the culture media of MBL gene transformants by using anion exchange chromatography and MBL binding capacity to pre-S of hepatitis B virus. The purification method of MBL is composed of the following steps: (a) fractionating samples containing multimeric recombinant human MBL by using anion exchange chromatography; (b) preparing column by packing substrate to which hepatitis B virus pre-S was immobilized; (c) capturing the recombinaut MBLs specifically on to the immobilized hepatitis B virus pre-S by passing through the column a sample containing the recombinant MBL proteins in the presence of calcium ion, after equilibrating the column with a calcium ion containing buffer; and (d) eluting the recombinant MBL proteins by adding a buffer solution supplemented
with EDTA or EGTA to the column onto which the recombinant MBL was captured in the step (c).
In the step (a), a material which was generally used for anion exchange chromatography was used, and Q-sepharose was one of such column material used. The sample containing the MBL proteins in the form of monomer or dimer was fractionated in the presence of 150 - 200 mM NaCl, and the other portion containing a high molecular weight MBL protein in the form of copolymer was eluted in the presence of350-400mMNaCl.
In the step (b), a substrate that was generally used for affinity chromatography was used, and sepharose was one of the general substrates.
In the step (c), equilibrium of column was performed by adding a buffer solution or the same solution as used for the affinity binding of a recombinant MBL and hepatitis B virus pre-S. The binding of recombinant MBL to hepatitis B virus pre-S was preferably performed in the presence of calcium ion, and at that tune, the concentration of calcium was preferably 2-20 inM. A sample containing a recombinant MBL protein could be supernatant obtained from MBL producing cell transformant culture medium or MBL solution eluted from MBL fractionation column mentioned above.
In the step (d), the elution of rhMBL protein was performed using EDTA or EGTA buffer solution in the absence of calcium ion. The buffer solution could be the solution containing EDTA or EGTA at the concentration of 2 - 10 mM, for example, distilled water or Tris-Cl buffer, pH 7.4.
The eluent prepared in the step (d) was freeze-dried or dialyzed before freeze-drying, resulting in a purified recombinant MBL protein.
The purification method of rhMBL protein of the present invention is also
applicable for purifying MBL obtained from natural biological samples, in addition to recombinant MBL protein prepared from the transformant. The biological sample can be exemplified by blood, plasma or serum.
The present invention also provides a detection kit for the recombinant human MBL comprising i) a solid support comprising a test line and a control line wherein mouse anti-human mannose binding lectin antibodies are fixed on the test line and anti-mouse IgG antibodies or anti-pre-S antibodies are fixed on the control line; ii) a dye pad adsorbed with a dye conjugate and connected to the bottom of the solid support, wherein if anti-mouse IgG antibodies are fixed on the control line, the dye conjugate is mouse anti-hurnan mannose binding lectin antibodies-gold conjugate and if anti-pre-S antibodies are fixed on the control line, the dye conjugate is pre-S-gold conjugate; and iii) a sample pad connected to the bottom of the dye pad, whereon a sample is loaded.
In the present invention, nitrocellulose membrane, polyvinylidene difluoride (PVDF) and nylon membrane can be used as the solid support. And as a dye pad, polyester, glass fiber, etc can be used, but not limited thereto.
The present invention also provides a preparation method of the detection kit for the recombinant human MBL comprising the following steps:
(1) preparing dye conjugates by binding anti-human mannose binding lectin
antibodies or pre-S protein to gold particles;
(2) adsorbing the dye conjugates into a dye pad;
(3) binding anti-human mannose binding lectin antibodies on a test line of the
solid support and anti-pre-S antibodies on a control line of the solid support if the dye
conjugates are anti-human mannose binding lectin antibodies-gold particles or anti-pre-S
antibodies to the solid support as a control line if the dye conjugates are mouse anti-
human MBL antibodies-gold particles; and
(4) joining the sample pad and the dye pad to the bottom of the solid support.
In the present invention, pre-S proteins or anti-human MBL antibodies can be used as a dye conjugate, but not limited thereto.
The present invention also provides a method for the detection of MBL by using the MBL detection kit, which comprises the following steps:
(1) applying a sample containing MBL to a dye pad connected with the bottom of
the solid support; and
(2) confirming mat antibodies are bound to test line and control line of the solid
The recombinant human MBL of the present invention is similar to the natural form purified from human blood. It is oligomeric forms and active in binding to microorganism thereby activating complement system. And high yield production level (50 fig/million cells/day) ensures commercial production of functional MBL for product development. So produced rhMBL may be effectively used for the treatment of an individual infected with virus, bacteria or fungi. It can also be used for the production of a diagnostic kit for detecting the recombinant human MBL.
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1: Preparation of a MBL gene transformant and expression of a recombinant
Construction of an expression vector
pEZ-MBL 2-5 was constructed by cloning MBL cDNA obtained by PCR from human hepatocyte cDNA library into pEZ vector, and then the nucleotide sequence of MBL cDNA was confirmed to be authentic from the known nucleotide sequence of MBL (Gene Bank NM 000242). PCR was performed by using the pEZ-MBL 2-5 as a template and using a primer set (forward primer 1 and backward primer 2) to amplify about 750 kb sized MBL cDNA. Each primer contains restriction enzyme recognition site and Kozak sequence, so the whole coding region was amplified. Then, pMSG-MBL was prepared by cloning MBL cDNA into pMSG vector (Accession No: KCCM 10202), and then the inserted nucleotide sequence of MBL was confirmed (FIG. 1).
Forward primer 1 (SEQ. ID. No 1) ctagctagcc accatgtccc tgtttccatc actc (34mer) Backward primer 2 (SEQ. ID. No 2) gaagatctca gatagggaac tcacagacg (29mer)
Transformation of host cell with pMSG-MBL
E. co/i was transfected with pMSG-MBL, and the resultant transformant was cultured in 100 M# of LB medium supplemented with 100 \iQ/M of ampicillin (Sigma, USA). The pMSG-MBL DNA was separated by plasmid purification kit (QUIAPREP Plasmid Midi Kit, Quiagen, USA). The purified DNA was digested with restriction
enzyme Sea I to make it linear, which was separated by PCR product purification kit (QIAQuick PCR product purification kit, Quiagen, USA).
After culturing CHO DG44 (dhfr-/dhfr-) host cells in a-MEM medium supplemented with 10% cFBS, the number of cells was counted by hemacytometer. The cells were distributed (2 x 105cells/well) in a-MEM medium supplemented with 10% cFBS, which were cultured in a COz incubator for 24 hours.
2 [ig of pMSG-MBL vector was mixed with 5.3 ui of Dosper™ and 16 ng of pDCHIP vector (plasmid containing DHFR gene, Venolia. L. et al., 1987, Somat. Cell Mol, Genet. 13, 491-501) DNA to induce reaction at room temperature for 45 minutes. The resultant product was added to host cells. The cells were cultured at 37°C for 6 hours, then medium was replaced with fresh a -MEM medium containing 10% cFBS (3 M^/well), followed by further culture. 2-3 days later, when transformed cells were grown enough, trypsin was added and 2 M of a -MEM (w/o) containing 10% dFBS was added to the cells (4 x 105 cells/well), followed by further culture again. Every 2-3 days, medium was replaced with a fresh one, and the condition of cells and the formation of single colony were observed under microscope. 10 days later, adapted early cells were obtained.
After obtaining adapted early cells, the MTX (methotrexate) concentration in mednim was increased gradually to induce amplification of MBL gene inserted in the
transformed cells. The cells were distributed by 4 x 105 cells/well, and then cultured in a medium (a-MEM + 10% dFBS) supplemented with 10 nM MTX until confluence, during which the medium was replaced with a fresh one every 2-3 days. The cells were distributed again by 4 x 105 cells/well, and adapted to a medium supplemented with 100 nM MTX by following the same procedure as used above, and then adapted again to 1 uM MTX condition. During the amplification of the MBL gene, western blot analysis was performed in every stage to observe the changes of expression level of MBL. In this the expression of MBL was increased with the increase of MTX content in medium.
In order to separate a single cell line showing high MBL expression rate, cells cultured in a medium (a-MEM + 10% dFBS) containing 1 uM MTX were distributed to a 96-well plate to make 0.5 cells/well, then cultured in a medium containing 1 uM MTX. After 2 weeks, when the formation of a single colony was confirmed, the cells were transferred to a 24-well plate, which was further cultured until cells were proliferated enough. Some of them were frozen and stored, and some of them were used for the comparison of expressions by Western blot analysis. And, a single cell Dl-3 transformant, which was proved to produce high molecular weight form of MBL in a large quantity, was selected as a cell line of the present invention. The selected transformant was named as MBL Dl-3 (CHO cell line) and deposited at Korean Collection for Type Culture, Daejeon, Korea, on May 16, 2003 (Accession No: KCTC 10472BP). PAGE analysis of the recombinant MBL protein expressed from the transformant was performed under non-denaturing condition and found to be multimeric form of MBL, which is very similar to the characteristics of human originated natural MBL
Quantification of recombinant MBL expressed in a transformant
Based on the standard amount of purified MBL obtained from human serum, the expression level of recombinant MBL protein from transformed CHO MBL/D1-3 cells was estimated. Cells were distributed into T25 flask at a concentration of 5 x 105, and then cultured in a medium (a-MEM(w/o) + 10% dFBS) until 90% confluence. Then, 3 Mfi of medium (a -MEM(w/o) + 5% dFBS) was added thereto and further cultured for 4 days. The culture medium was diluted 10 times, followed by Western blot analysis. The result was compared with the standard amount of natural MBL. From this the expression level of recombinant MBL protein was confirmed to be about 50 ^/106 cells/day, as cultured on a cell culture flask.
Example 2: Method for mass-production of rhMBL protein
Preparation of a cell line adapted for suspension culture in a serum-free medium
Anchorage-dependent MBL expressing cell line was cultured in a-MEM (w/o) medium supplemented with 10% dFBS and 1 uM MTX. After recovering viable cells, they were inoculated into a 250 ml spinner flask containing 100 ml of protein-free HyQ SFM4 CHO medium (Hyclone, USA) supplemented with 0.15% sodium bicarbonate at the inoculum size of 5x10" cells/ml. The culture in the spinner flask was performed at 37°C in a 5% CC>2 incubator, which was stirred at 40 rpm whole time. When the number of cells reached 1.0 - 2.0 x 106 cells/ml, cells were inoculated again into 100 ml HyQ SFM4 CHO medium supplemented with 0.15% sodium bicarbonate with the inoculum size of 5 x 105 cells/ml. Viability of the cells was determined by trypan blue exclusion method. In particular, over 90% viability was confirmed in the late adaptation period.
The cells adapted to serum-free/protein-free medium were cultured until the cell density reached 2.5 x 106 cells/M£. Then, the cells were recovered and re-suspended in a freezing medium. The cells were distributed into cryovials to make the concentration of 2.8 x 107 cells/M#, which was then stored at liquid nitrogen tank.
Development of bioreactor culture by using a cell line adapted for serum-free suspension culture
Cells for suspension culture were inoculated to a 250 M£ spinner flask containing 100 M£ of serum-free/protein-free medium (HyQ SFM4 CHO medium) supplemented with 0.15% sodium bicarbonate with the inoculum size of 5 x 105cells/M£. When the cell number was increased to 1.0 - 2.0 x 106 cells/M, the cells were inoculated to a 500 M£ spinner flask containing 200 M of HyQ SFM4 CHO medium with the inoculum size of 5 x 1()5 cel!s/M£, Then, when the cell number was increased again to 2 x 106 cells/M£, the cells were inoculated to a 1,000 M£ spinner flask containing 400 ml HyQ SFM4 CHO medium with the inoculum size of 5 x 105 cells/M£. This way the cells were cultured scaling up gradually, resulting in the preparation of 1 L of seed culture with the concentration of 2.5 x 106 cell/M£.
The seed cell culture prepared above were inoculated to a 7.5 L (5 L working volume) bioreactor and cultured at 34°C for 5 days (50 rpm, DO 50, pH 7.2 - 7.4).
Example 3: Method for the purification of multimeric recombinant human MBL protein
MBL expressing CHO cell was cultured in HyQ SFM4 CHO medium, and then the culture medium was centrifuged and passed through a 0.45 um membrane filter to remove cells. Anion exchange chromatography and affinity chromatography were
performed to purify recombinant MBL proteins. The quantity of purified proteins was determined by using MBL ELISA kit.
Fractionation of sample containing multimeric polymer by using Q-sepharose column
After optimizing pH and conductivity of supernatant, smaller form of MBL such as monomer and dimer was removed by using a column packed with Q-sepharose, and only MBL in larger forms was collected. The sample containing smaller form of MBL was fractionated by passing through 150 - 200 mM NaCl buffer solution, and the sample containing larger form of MBL was eluted with 350 - 400 mM NaCl buffer solution. This result showed that more than 80% were expressed in high molecular copolymer form of MBL from the cell line that is established in this invention (FIG. 2A).
Preparation of pre-S-sepharose column
Recombinant pre-S protein used in the present invention was obtained by expressing pre-S gene, which is a portion of surface protein of hepatitis B virus, in Sacharomyces c>irevisiae, and then purifying it in the form of highly glycosylated molecules (International Patent Publication No. WO 02/094866).
One gram of sepharose 4B powder activated by CNBr was dissolved in 1 mM HC1, followed by washing several times. In order to use recombinant pre-S protein as a ligand, 6.4 mg of pre-S protein was dissolved in coupling buffer (0.2 M NaHCOs, 0.5 M NaCl and pH 8.3), making the concentration of 0.5 - 10 mg/M£. CNBr activated Sepharose 4B resins were mixed with the pre-S solution and reacted at room temperature for 2 hours. Then, the mixture was transferred to blocking buffer (0.1 M Tris-Cl, pH 8.0), followed by further reaction at room temperature for 2 hours. After washing, pre-
S immobilized to sepharose was confirmed by western blot analysis and found to be almost all pre-S recombinant proteins were immobilized to the sepharose column, which was used for the purification of a recombinant MBL protein. Finally, pre-S-sepharose column for the purification of recombinant human MBL was prepared by packing pre-S-sepharose 4B resin in a column.
Purification of a recombinant human MBL
The column filled with pre-S-sepharose was equilibrated with a binding buffer (20 mM Tris, 150 mM NaCl and lOmM CaCl2, pH 7.6). The column fraction from the Q-Sepharose containing recombinant MBL proteins or a culture media containing MBL was loaded onto the column to adsorb MBL. Then, the column was washed several times with the binding buffer. The eluting buffer (20 mM Tris, 150 mM NaCl and 5mM EDTA, pH 7.6) without Ca2+ was passed through the column to elute recombinant human MBL. The obtained eluent was investigated with SDS-PAGE. As a result, purified recombinant human MBL showing 99.9% purity was obtained (FIG. 2A and FIG. 2B).
Example 4: Verification of a multimeric recombinant human MBL by EM and by MBL
binding activity to the surface of a microorganism
Confirmation of the formation of a muitimeric recombinant human MBL
In order to confirm whether or not a purified recombinant human MBL formed a multimeric polymer in the shape of a flower bouquet, the purified recombinant human MBL was treated with amorphous carbon, and then the shape of the protein was observed under transmission electron microscope (Tecnai 12, FBI, Netherlands) and found the purified recombinant human MBL to be multimeric polymers in the shape of a
bouquet (FIG. 3).
Binding of recombinant human MBLs to pre-S coated gold particles
The binding capacity of recombinant human MBL to the surface of a microorganism was observed by using pre-S, a surface protein of hepatitis B virus. Pre-S proteins were attached to the surface of gold particles which is of 20 - 40 nm size, which was, thus, made as if a microorganism. Recombinant human MBLs were added thereto in the presence of calcium. Then, whether recombinant human MBL was attached to the pre-S proteins was observed under transmission electron microscope Tecnai 12, resulting in the confirmation of a complex formation. As shown in FIG. 4, pre-S proteins coated on the gold particles were surrounded by recombinant human MBLs in the presence of calcium ion. This demonstrates how rhMBL binding to the surface of a microorganism looks like. In actual case, MBL binding to microorganism might mechanically block infectivity of the organism-specially, such as viruses-to the cells, in addition to activating complement system and acting as an opsonin.
Example 3: Confirmation of binding activity of recombinant human MBL
In order to investigate the biological activity of rhMBL, binding activity to pre-S and mannan was assayed in the presence of calcium ion.
Mannan or pre-S protein of hepatitis B virus dissolved in 50 mM carbonate-bicarbonate buffer solution was distributed into a microtiter plate (Nunc Maxisorp Immunoplate) to make 1 }ig per well, which was then incubated for overnight at 4°C. The pre S or mannan coated plate was washed with a washing buffer (20 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Tween-20, pH 7.6) four times, followed by blocking with 0.2% BSA at room temperature for one hour. After washing with the washing
buffer three times, enough MBL was added to each well to make the MBL quantity in each well as indicated in the figure. To do this 1 pg of recombinant human MBL in 1ml of binding buffer (20 mM Tris, 1 M NaCi, 10 mM CaCl2, 0.1% BSA, 0.05% Tween-20, pH 7.6) were made serial dilution to make the necessary amount of MBL in 100 \iA, which was then added onto each well and incubated at room temperature for 2 hours. The plates were washed with the washing buffer 6 times again. Then, mouse monoclonal anti-human MBL antibodies (MBL8F6, Dobeel Corp., Korea) were diluted 10,000 fold and added 100 \l£ onto each well followed by incubating at 37 degree C for 1 hour. Anti-mouse IgG-HRP was diluted 10,000 fold and added lOOul to each well. The plate was incubated at 37 degree C for one hour and then, the color was developed by adding 150 [\£ of TMB solution. After 20 minutes, the reaction was terminated by the addition of 50 \d of 2 M HaSO^ OD^o was measured using ELISA plate reader (Multiskan Ex, Labsystems, USA). FIG. 5A shows the binding of natural MBL purified from human semni to mannan and pre-S protein, and FIG. 5B shows the binding of recombinant human MBL to the pre-S and mannan. In conclusion, recombinant human MBL and the natural MBL for human had similar binding activity to pre-S and mannan, but both of them did not bind to Bovine Serum Albumin (FIG. 5 A and FIG. 5B).
Example $; Comparison of biological activity between high molecular weight multimeric recombinant human MBL and low molecular weight oligomeric recombinant human MQL
The biological activity of the larger and smaller form of recombinant human MBL was investigated in order to confirm which one of recombinant human MBL proteins, larger form or smaller form including monomer and dimer, binds more efficiently to pre-S and to mannan in the presence of calcium ion.
MIJJL binding to glycosylated protein pre-S
Pre-S proteins were coated on a plate (Nunc Maxisorp Immunoplate) under the same condition as described in the Example 5. Then, the larger form or smaller form was dissolved in a binding buffer (20 mM Tris, 1 M NaCl, 10 mM CaCl2, 0.1% BSA, 0.05% Tween-20, pH 7.6) and added onto pre-S coated plate at the different concentrations to make desired amount in each well. And the rest of the binding assays were performed as in the example 5. As described earlier, MBL binding activity to pre-S proteins was much greater with multimeric recombinant human MBL compare to the smaller form containing mainly monomers and dimers (FIG. 6).
Activation of C4 in the complement system
Nunc Maxisorp Immunoplate was coated with 500 ng of pre-S proteins per well, to which different concentrations of recombinant human MBLs were added, allowing MBL binding to pre-S at room temperature for 2 hours. As a source of MASP protein, 100 ul of MBL-free serum diluted 100 fold was added to each well and incubated 90 minutes at room temperature. The plate was washed with a washing buffer 6 times, then 500 ng of C4 was added, followed by further reaction at room temperature for 2 horns. Anti-C4 antibody-HRPs were diluted 2000 fold and 100 ul was added to each well. Then, allowed the reaction to take place at room temperature for one hour. One hundred fifty (l£ of OPD solution was added, and color was development for 20 minutes. Then, C4b deposit was measured after adding 50 \t£ of 3 M HC1 and reading the OD at 00492 using an ELISA plate reader (FIG. 7). For the experiments, both multimeric recombinant human MBLs in larger form provided by the present invention and recombinant human MBLs in smaller form such as monomers or dimers
were used respectively for the comparison of their activities. As shown in FIG. 7, C4 was greatly activated by multirneric recombinant human MBL in larger form but was hardly activated by those proteins in smaller form. Therefore, it was confirmed that recombinant human MBLs in the form of multirneric polymer provided by the present invention have an excellent activity for the C4 activation.
Example 7: Binding of recombinant human MDL to various microorganisms
Eleven strains including both Gram-positive bacteria and Gram-negative bacteria and fungi were cultured in liquid medium appropriate for each of them. To immobilize the cultured bacterial cell, 100 ul of cultured cells were distributed in a plate (Maxisorp Immunoplate, Nunc) to make 1 x 106 cells/well and incubated at 4°C for overnight. Blocking was performed by using 0.2% BSA at 37°C for one hour. Five hundred ug of recombinant human MBL dissolved in lOOul binding buffer (20 mM Tris, 1 M NaCl, 10 mM CaCl2, 0.1% BSA, 0.05% Tween-20, pH 7.6) was added to each well, followed by reaction at 37°C for one hour. Anti-human MBL mouse monoclonal antibody MB1B5 (Dobeel Corp., Korea) was diluted 10,000 fold and added 100 \)£ to each well, followed by reaction at 37°C for one hour. Anti-mouse IgG-HRP was diluted 10,000 fold and added lOOul to each well, followed by reaction at 37°C for one hour. Then, 100 \iA of TMB solution was added thereto and color was development for 30 minutes. The reaction was terminated by adding 50 \l£ of stop solution (H2SO4), and then 00450 was measured by using ELISA plate reader (Multiskan Ex, Labsystems, USA).
The microorganisms used in the present invention showed different binding activity to a recombinant human MBL, and as shown in FIG. 8, C. albicans, H. influenzae ATCC 51907, S. aureus CCARM 3197 and S. aureus ATCC 29213 showed
very high binding activity to a recombinant MBL protein, but S. pyrogenes ATCC 8668, S. aureus CCARM 3114 and E.faecalis ATCC 29212 showed a medium level of binding activity. K. pneumoniae ATCC 10031, S. epidermis ATCC 12228, S. epidermis CCARM 35048 and E. faecium CCARM 5028 had low binding activity to MBL. In conclusion, it was proved that a recombinant human MBL, like a natural MBL, binds to microorganisms, recognizing the pattern of a glycosylated surface protein and binding activity varies with target microorganisms.
Example 8: Inhibition of SARS-CoV infection by recombinant human MBL
FRhk-4 cells, monkey kidney cells, were cultured in MEM. To the culture solution, added was recombinant human MBL, which was then infected with SARS-CoV. The recombinant human MBL was diluted with cell culture media in four-fold serially starting from concentration of 2.5 p.g/M£, and SARS-CoV, primary isolate from patient, was used (Ksiazek TO et al. N. Eng. J. Med 348, 1953-1966, 2003; Peiris et al., Lancet 361, 1319-1325,2003).
In order to confirm whether or not the host cells were infected with SARS-CoV and replicated, quantitative real-time PCR (GeneAmp PCR system, Applied Biosystems, USA) was performed with a set of SARS-CoV specific primers. FRhk-4 cells infected with SARS-CoV and not treated with a recombinant human MBL were used as a control group.
As shown in FIG. 9, the infection with SARS-CoV was inhibited by the treatment of recombinant human MBL in dose-dependent manner. For example, when cells were cultured in the presence of recombinant human MBL by the concentration of 2.5 JiQ/M£, the proliferation of the virus was inhibited greatly (less than 15% proliferation), comparing to a control group. This 15% is most likely from the virus inoculum.
FIG. 10 is a photograph of phase contrast microscope showing FRhk-4 cells infected with SARS-CoV. In FIG. 10 (I), healthy cells were not observed in a control group that was not treated with recombinant human MBL, but in FIG. 10 (2) - (6), healthy cells were observed in testing groups treated with recombinant human MBL. In particular, the number of healthy cells was increased with the increase of the concentration of recombinant human MBL.
Example 9: Quantification of MBL contained in clinical samples
The present inventors developed a method for the quantification of MBL included in clinical samples such as blood, serum, saliva, etc, which is designed to take advantage of binding capacity of pre-S protein of hepatitis B virus to MBL.
Immobilization of pre-S on the surface of gold particle
20-80 nm sized colloidal gold particles were prepared, and pre-S proteins (about 100 ^g/ml) were added thereto under the condition of pH 6.0-8.0, which was stirred at room temperature for reaction. Upon reaction was completed, enough 10% BSA was added to make 0.3% (weight/volume) final concentration, which was stirred at room temperature for 15 minutes for further reaction, resulting in the blocking of the surface. Centrifugation was performed at 12,000 rprn for 15 minutes to separate supernatant. The pellet was suspended in a buffer solution supplemented with 2% BSA. As Pre-s proteins uniformly binds to the surface of colloidal gold-particles, a dye-conjugates has been created that can be used in the preparation of an MBL test kit. This dye-conjugates display red or purple color from the colloidal gold-particles.
Preparation of diagnostic strip
Using an ink-jet printer or a spray type printer, a test line and a control line were drawn on nitrocellulose membrane (S&S, USA) to make an MBL test strip. To create a test line, anti-human MBL mouse monoclonal antibody MB IBS (Dobeel Corp., Korea) was used and pre-S specific monoclonal antibody for the control line. The dye conjugates prepared above were absorbed onto a dye pad which is made of polyester or glass fiber fixed to the lower part of the solid support of the diagnostic strip.
When a drop of a clinical sample including human MBL is dropped on a sample pad which is linked to the dye pad of the diagnostic strip, the sample migrates on nitrocellulose membrane by capillary phenomenon and diffusion. When the sample reaches the test line, MBL-pre-S-gold particle complexes are captured by anti-human MBL mouse monoclonal antibodies on the test line, so that the test line becomes red. And when the sample reaches the control line, both MBL-pre-S-gold particle complexes and pre-S-gold particles are captured by monoclonal antibody against pre-S, so that the control line becomes red. Therefore, when a sample contained MBL, both the test line and the control line will show positive reaction, turning red. On the other hand, when a s?mple did not include MBL, only the control line turned red, showing negative reaction (FIG. 11).
As exemplified hereinbefore, methods for production of multimeric recombinant human mannose binding lectin (rhMBL) in massive quantity presented in this invention is a significant advancement in the development of MBL as a useful commercial product. In particular multimeric form of MBL efficiently binds to microorganisms, thereby it could efficiently inhibit infection of virus, bacteria, or fungi, so that it might be used for the development of a medicine for the prevention and/or the treatment of infection with microorganisms such as virus, bacteria, fungi, in particular SARS-CoV, and the production of diagnostic kit for the detection of human MBL.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
1. A method for the mass-production of multimeric recombinant human MBL comprising the
(1) preparing CHO cell lines transfected with an expression vector pMSG-MBL
illustrated in a plasmid map of FIG. 1 containing a polynucleotide coding for human
mannose binding lectin,
Wherein, the CHO cell lines is the CHO cell line MBL Dl-3 deposited under accession No: KCTC 10472BP;
(2) producing recombinant human MBL by expressing the sequence of human MBL
coding region in an animal cell and from the culture of that cell by:
i. suspension-culturing the cells in a serum-free/protein-free medium; and
ii. sub-culturing those cells adapted well to the serum-free/protein-free medium to scale them up gradually, resulting in the mass-production of rhMBL protein in the form of multimeric polymer; and
(3) purifying recombinant human MBL prepared in the step (2) by:
i. fractionating samples containing multimeric recombinant human MBL by using anion exchange chromatography;
ii. preparing column by packing substrate to which hepatitis B virus pre-S
iii. binding the recombinant human MBL to the hepatitis B virus pre-S
specifically by adding a sample containing the recombinant human MBL to
the column in the presence of calcium ion, after equilibrating the column;
iv. eluting the recombinant human MBLs by adding a buffer solution supplemented with EDTA or EGTA from the column onto which the recombinant human MBL was bound.
|Indian Patent Application Number||3367/DELNP/2007|
|PG Journal Number||50/2010|
|Date of Filing||04-May-2007|
|Name of Patentee||DOBEEL CO., LTD.,|
|Applicant Address||#406 BYUCKSANTECHNOPIA 434-6 SANGDAEWON-DONG JUNGWONG-GU, SEONGNAM-SI, KYONGGI-DO 460-716, REPUBLIC OF KOREA|
|PCT International Classification Number||C07K 1/16|
|PCT International Application Number||PCT/KR2004/002739|
|PCT International Filing date||2004-10-28|