Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
1998-03-18
2001-03-06
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S007210, C435S330000, C435S332000, C435S333000, C435S339000, C435S346000, C435S372000
Reexamination Certificate
active
06197582
ABSTRACT:
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art.
BACKGROUND OF THE INVENTION
The seminal discovery by Kohler and Milstein (Kohler, G. and Milstein, C., 1975) of mouse “hybridomas” capable of secreting specific monoclonal antibodies (mAbs) against predefined antigens ushered in a new era in experimental immunology. Many problems associated with antisera were circumvented. Clonal selection and immortality of hybridoma cell lines assured monoclonality and permanent availability of antibody products. At the clinical level, however, the use of such antibodies is clearly limited by the fact that they are foreign proteins and act as antigens to humans.
Since the report of Kohler and Milstein (Kohler, G. and Milstein, C., 1975), the production of mouse monoclonal antibodies has become routine. However, the application of xenogenic mAbs for in vivo diagnostics and therapy is often associated with undesirable effects such as a human anti-mouse immunoglobulin response. mAbs have great potential as tools for imaging; therapeutic treatment has motivated the search into the means of production of human mAbs (humAbs) (Levy, R., and Miller R A., 1983). However, progress in this area has been hampered by the absence of human myelomas suitable as fusion partners with the characteristics similar to those of mouse myeloma cells (Posner M R, et al., 1983). The use of Epstein-Barr virus (EBV) has proved to be quite efficient for human lymphocyte immortalization (Kozbor D, and Roder J., 1981; Casual O, 1986), but has certain limitations such as low antibody secretion rate, poor clonogenicity of antibody-secreting lines and chromosomal instability requiring frequent subcloning. Undifferentiated human lymphoblastoid cell lines appear more attractive. In contrast to differentiated myeloma cells, these cell lines are readily adapted to culture conditions, though the problems of low yield and unstable secretion remain unresolved (Glassy M C, 1983; Ollson L, et al., 1983). The best potential fusion partners are syngenic myeloma cells with well-developed protein synthesis machinery (Nilsson K. and Ponten J., 1975). However, culturing difficulties explain why few lines have been conditioned for in vitro growth and capability to produce viable hybrids (Goldman-Leikin R E, 1989). Existing myelomas have low fusion yield and slow hybrid growth, although mAb production is relatively stable (Brodin T, 1983). Genetic instability is a major disadvantage of interspecies hybrids. This is the case, for example, when a mouse myeloma is used as the immortalizing partner. Production of mouse-human cell hybrids is not difficult. In vitro these cells have growth characteristics similar to those of conventional mouse-mouse hybridomas (Teng N N H, 1983). However, spontaneous elimination of human chromosomes considerably reduces the probability of stable mAb secretion (Weiss M C, and Green H., 1967). In order to improve growth characteristics and stability of humAb production, heterohybrids between mouse myeloma cells and human lymphocyte (Oestberg L, and Pursch E., 1983) as well as heteromyelomas (Kozbor D, et. al., 1984) are used as the fusion partners.
SUMMARY OF THE INVENTION
The present invention provides a heteromyeloma cell which does not produce any antibody, capable of producing a trioma cell which does not produce any antibody, when fused with a human lymphoid cell, wherein the trioma cell is capable of producing a tetroma cell capable of producing a monoclonal antibody having specific binding affinity for an antigen, when fused with a second human lymphoid cell, the second human lymphoid cell being capable of producing antibody having specific binding affinity for the antigen, with the proviso that the heteromyeloma cell is not B6B11 (ATCC Designation Number HB-12481).
The present invention further provides a trioma cell obtained by fusing a heteromyeloma cell which does not produce any antibody with a human lymphoid cell.
The present invention also provides a tetroma cell capable of producing a monoclonal antibody having specific binding affinity for an antigen, obtained by fusing the described trioma cell which does not produce any antibody with a human lymphoid cell capable of producing antibody having specific binding affinity for the antigen.
The present invention additionally provides a monoclonal antibody produced by the described tetroma.
The present invention further provides a method of generating the described trioma cell comprising: (a) fusing a heteromyeloma cell which does not produce any antibody with a human lymphoid cell thereby forming a trioma cell; (b) incubating the trioma cell formed in step (a) under conditions permissive to the production of antibody by the trioma cell; and (c) selecting a trioma fusion cell that does not produce any antibody.
Still further, the present invention provides a method of generating a tetroma cell comprising: (a) fusing the described trioma cell with a human lymphoid cell, thereby forming a tetroma cell; (b) incubating the tetroma cell formed in step (a) under conditions permissive to the production of antibody by the tetroma cell; and (c) selecting a tetroma cell capable of producing a monoclonal antibody.
The present invention also provides a method of producing a monoclonal antibody comprising (a) fusing a lymphoid cell capable of producing of producing antibody with the described trioma cell, thereby forming a tetroma cell; and (b) incubating the tetroma cell formed in step (a) under conditions permissive to the production of antibody by the tetroma cell, thereby producing the monoclonal antibody.
Also the present invention provides a method of producing a monoclonal antibody specific for an antigen associated with a condition in a subject comprising: (a) fusing a lymphoid cell capable of producing antibody with the described trioma cell, thereby forming a tetroma cell; (b) incubating the tetroma cell formed in step (a) under conditions permissive to the production of antibody by the tetroma cell; (c) selecting a tetroma cell producing a monoclonal antibody; (d) contacting the monoclonal antibody of step (c) with (1) a sample from a subject with the condition or (2) a sample from a subject without the condition under conditions permissive to the formation of a complex between the monoclonal antibody and the sample; (e) detecting the complex formed between the monoclonal antibody and the sample; (f) determining the amount of complex formed in step (e); and (g) comparing the amount of complex determined in step (f) for the sample from the subject with the condition with amount determined in step (f) for the sample from the subject without the condition, a greater amount of complex formation for the sample from the subject with the condition indicating that a monoclonal antibody specific for the antigen specific for the condition is produced.
Additionally, the present invention provides a method of identifying an antigen associated with a condition in a sample comprising: (a) contacting the monoclonal antibody produced by the described method with the sample under conditions permissive to the formation of a complex between the monoclonal antibody and the sample; (b) detecting the complex formed in step (a); and (c) isolating the complex detected in step (b), thereby identifying the antigen associated with the condition in the sample.
The present invention additionally provides a method of diagnosing a condition in a subject comprising: (a) contacting a sample from the subject with a monoclonal antibody produced by the described method under conditions permissive to the formation of a complex between the monoclonal antibody and the sample; and (b) detecting the formation of a complex between the monoclonal antibody and the
Cooper & Dunham LLP
Lee Li
Smith Lynette R. F.
The Trustees of Columbia University in the City of New York
White John P.
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