Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-12-23
2001-12-11
Swartz, Rodney P. (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S145100, C424S158100, C435S070100, C435S070210, C435S071100, C436S547000, C436S548000, C530S388100, C530S388230, C530S391100
Reexamination Certificate
active
06328963
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel anti-human MP52 mouse monoclonal antibody. More specifically, this invention relates to an anti-human MP52 mouse monoclonal antibody which is capable of binding to a dimeric human MP52, but not to a monomeric human MP52.
Moreover, this invention relates to a hybridoma capable of producing an anti-human MP52 mouse monoclonal antibody described above and the use thereof.
BACKGROUND OF THE INVENTION
Human MP52 was first isolated for its CDNA as an osteogenetic factor belonging to TGF-&bgr; gene superfamily in 1994 (Biochem. Biophy. Res. Comm., Vol. 204, No. 2, 1994). Human MP52 is considered as a protein having 120 amino acid residues with alanine at the N-terminus, and its amino acid sequence is reported in WO93/16099 and WO95/04819. It is evident from various animal tests that MP52 is involved in osteogenesis similar to other osteogenetic factors. However, there have been left many unknown points in reference to by what MP52 is directly induced in osteogenesis and by what mechanism osteogenesis is performed, and there have been issued only a few reports thereon.
In addition, mouse MP52 is different from human MP52 in only one of the amino acid sequence at the N-terminal site, and thus MP52 is derived from a gene firmly preserved over species so that it is not that easy to obtain an antibody to human MP52 from a mouse, although it was reported in WO93/16099 that a mouse monoclonal antibody became available.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide an anti-human MP52 mouse monoclonal antibody which is capable of binding to a dimeric human MP52, but not to a monomeric human MP52.
There has been attempted to produce human MP52 by means of genetic engineering. In purifying human MP52 produced by incubation of host cells having integrated therein cDNA encoding human MP52, it is found effective to purify the human MP52 using a monoclonal antibody capable of specifically binding to human MP52. More specifically, a monoclonal antibody specific to human MP52 is borne on a carrier, and contacted with a roughly purified MP52 to isolate human MP52 by binding and then human MP52 is separated. In this case, in order to isolate the active dimeric MP52, it is apparently advantageous to employ such a monoclonal antibody that is capable of binding specifically to a dimeric human MP52.
The monoclonal antibody to human MP52 can be used for quantitation of human MP52. The human MP52 quantitation is useful for elucidation of MP52-inducing factors and studies on osteogenesis mechanism. In this case, a dimeric MP52 may be catabolized in a living body to an inactivated monomer or fragments thereof. The active dimeric MP52 solely can be detected using a monoclonal antibody specifically binding to a dimeric MP52.
Moreover, the invention provides an antibody which is capable of binding to a biologically active site of human MP52. That is to say, there is provided an anti-human MP52 mouse monoclonal antibody which may prevent the binding between human MP52 and the receptor thereof. The antibody of the invention specifically recognizes a high-order structure of human MP52 and binds to human MP52 at the active site thereof to inhibit the activity thereof. Since the antibody recognizes an active site of human MP52, it does not recognize any inactive portions even in the dimeric MP52. This is evidently advantageous when applied to its separation or assay.
The mouse monoclonal antibody of this invention can be produced by the following steps in accordance with a well-known process for producing a monoclonal antibody:
(1) sensitizing mice with human MP52 as an immunogen over a prolonged period of time,
(2) performing cell fusion between spleen cells of sensitized mice and mouse myeloma cells,
(3) screening the hybridoma producing a monoclonal antibody to human MP52 from the resulting hybridoma,
(4) cloning the desired antibody-producing hybridoma,
(5) performing large scale culture of the cloned cells to produce an antibody, or transplanting the cloned cells into mice intraperitoneally to produce an antibody, and
(6) separating and purifying the antibody contained in culture supernatant or the ascites.
The above steps will be explained in more details hereinafter.
In the preparation of immunosensitized mice in the step (1), a solution of human MP52 in 10 mM aqueous hydrochloric acid is admixed and emulsified with a complete Freund adjuvant (CFA) and the resultant emulsion is administered to mice intraperitoneally several times at a frequency of 4 times per 4 months. A content of the MP52 is suitably from 10 &mgr;g to 100 &mgr;g. After about one month interval from the administration, a solution of 10-100 &mgr;g of human MP52 emulsified similarly with an incomplete Freund adjuvant (IFA) is administered to the mice intraperitoneally. Desirably, the mice which have been continuously sensitized with human MP52 together with the adjuvants are given subcutaneously, after a further interval of one to two months, 10-100 &mgr;g of human MP52 without any adjuvant, and, a few days before the cell fusion, 10-100 &mgr;g of human MP52 is further administered intravenously to the mice at the tail vein.
Production of human MP52 has been attempted by using an expression vector having integrated therein the cDNA encoding said human MP52 and animal cells such as CHO cells and bacteria such as
Escherichia coli
as a host. The present inventors have attempted to produce a monoclonal antibody using as a sensitizing antigen human MP52 produced in CHO cells (hereinafter referred to as CHO-MP52) [Human MP52-producing animal cell line MC-2 has been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) with the international deposit number FERM BP-5142.] and human MP52 produced by
Escherichia coli
(hereinafter referred to as rhMP52) [An expression vector for human MP52 has been deposited in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) with the international deposit number FERM BP-5499.], respectively. The resulting antibodies were all IgMs having a lower specificity. However, the inventors have succeeded in producing a monoclonal antibody having a higher specificity by using both CHO-MP52 and rhMP52 as a sensitizing antigen.
In the cell fusion of the step (2), the spleen of the sufficiently immunosensitized mouse In the step (1) is first excised and then a suspension of the spleen cells is prepared in a conventional manner. Next, a mixture of the resulting spleen cells and mouse myeloma cells is subjected to cell fusion treatment using warm polyethylene glycol. After the treatment, non-fused cells are removed from the cell mixture using a medium containing fetal calf serum (FCS) and HAT [hypoxanthine (H), aminopterine (A) and thymidine (T)]. The resulting mixture is poured portionwise into a 96-well plate at a low concentration (a concentration at which one clone is capable of being propagated in one well) and after one week a supernatant in the well where propagation has been confirmed is recovered.
The assay of an anti-human MP52 antibody produced in the supernatant of the step (3) may be performed by using a plate with 96 wells coated with human MP52 used as an immunogen according to a conventional ELISA method. In order to select the antibody to human MP52 which is not bound to a monomeric human MP52, but is bound to a dimeric human MP52, two ELISA methods are combined wherein 1) untreated (nonreduced) dimeric human MP52 (D-rhMP52) and 2) reduced monomeric human MP52 (M-rhMP52) are used. In this case, the monomer molecule in a reduced state may easily form the corresponding dimer when coated on the plate and therefore, the monomer to be used for coating is sulfonated in a conventional manner to avoid reconstruction of the dimer molecule. Further, specific reactivity of the antibody with t
Jitsukawa Tomofumi
Kitagawa Hiroshi
Nakagawa Hiraku
Yanagisawa Sachiko
Bierman, Muserlian and Lucas
Hoechst Marion Roussel
Swartz Rodney P.
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