Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1995-06-02
2001-05-08
Navarro, Albert (Department: 1645)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S159100, C424S160100
Reexamination Certificate
active
06228361
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced by Arabic numerals. Full citations for these references may be found 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 to which this invention pertains.
The routine production of human monoclonal antibodies has been of interest since the construction of murine hybridomas which secrete murine monoclonal antibodies of predetermined specificity were originally described (1).
While murine monoclonal antibodies provide valuable tools for the study of biological processes, major limitations are apparent. First, there are restricted number of antigens recognized by these antibodies (50). For example, antibodies directed to polymorphic determinants of the HLA and DR antigens have been difficult to identify (51). Moreover, it has been almost impossible to identify specific human tumor-associated antigens (51-57). Secondly, the pathogenesis of the autoimmune phenomenon in diseases such as diabetes require that the human autoantibodies be defined (58). Finally, therapeutics using murine monoclonal antibodies are restricted due to the formation of antimurine antibodies by the patients receiving the murine monoclonal antibodies for treatment (59-62). It is therefore likely that human monoclonal antibodies will provide major tools for the study of human neoplasia (9-14), autoimmune diseases (2-8), and infectious diseases (16-20), and will serve as potential therapeutic and diagnostic agents for these and other illnesses.
To date, Epstein-Barr Virus (EBV) transformation of antibody-producing human B cells, selection of myeloma serum proteins, and fusion of both murine and human myeloma cell lines or analogs with antibody-producing cells have served as the only practical methods for obtaining human monoclonal antibodies. These methods, however, lack one or more of the features which have made the routine production of murine monoclonal antibodies useful (21-26). While myeloma serum proteins have been used by some investigators as sources of antibodies, this method is dependent upon the large scale screening of rare patients. Lack of reproducibility and continual production, as well as restricted antigen specificities, limit the applicability of this method. EBV virus transformation of antibody-producing B cells has provided the major source of human monoclonal antibodies reported in the literature. There are numerous inherent and methodological problems associated with the use of EBV transformation as a technique for producing antibodies. First and foremost is the instability of monoclonal antibody production by these cell lines (21). Because they have an extremely poor cloning efficiency and unstable antibody secretion, only a few human monoclonal antibody-secreting cell lines have been maintained and have produced sufficient quantities of antibody for use in subsequent studies (3,11). Moreover, the low frequency and lack of specificity of EBV transformation has necessitated selection methods designed to enhance the recovery and transformation of antibody-secreting B cells (21, 29, 30).
The development of human monoclonal antibodies by fusion of myeloma cell lines or analogs with antibody-producing cells has been slowed by two major factors: 1) lack of an appropriate human fusion partner and 2) insufficiently available antigen-specific, human B cells. The presently available human fusion partners are lacking in important characteristics necessary for the production of monoclonal antibodies, i.e., efficient fusion, easy clonability of cell lines and fusion-resulting hybrids, and continuous secretion of large quantities of antibody by the hybrids. Without these characteristics, which are important features of murine fusion partners, it will be extremely difficult to obtain human monoclonal antibodies to many antigens. Human myeloma or lymphoblastoid cell lines have been used for fusion, but frequently these have either a low fusion efficiency, poor growth and cloning, or unstable secretion by the resulting hybrids (6, 23, 31-33). For example, NSI, a murine myeloma cell line, fuses with an efficiency of 1/10,000 with mouse spleen cells (66). Comparative fusion efficiency of LiCron HMY-2, SK007, UC729-6 or GM 1500 is between 1/500,000 and 1/1,000,000 with human cells (6, 33, 67, 68). In addition, several of these cell lines, including derivatives of UC729-6 and LTR228, fuse poorly with normal peripheral blood mononuclear cells (PBM). High fusion efficiency is particularly important in a human system because of the relative rarity of antibody-producing B cells, even in individuals undergoing programmed immunization. In optimally tetanus immunized volunteers, as few as 1 out of 10,000 circulating B cells secrete anti-tetanus antibody (38). Since B cells represent less than 10% of circulating PBM, large numbers would be needed to obtain a single antibody-secreting hybrid. Direct comparisons of a number of human myeloma cell lines, mouse myeloma cell lines, and human lymphoblastoid cell lines as human fusion partners have generally indicated fusion efficiencies on the order of 1/10
5
-10
6
cells, with poor stability, and secretion between 100 ng and 10 &mgr;gm/ml in routine cultures (6, 23, 31).
As an alternative to presently available human and murine cell lines used as fusion partners, a number of investigators have attempted to construct myeloma Analogs that might be superior for human monoclonal antibody production. Murine hybridization experiments have shown that fusions between B cells with undifferentiated characteristics and B cells with more differentiated characteristics result in the promotion of those differentiated characteristics in the hybrids (43, 44). Thus, Laskow, et al., and others, were able to promote the appearance of phenotypic characteristics of a more differentiated B cell, specifically including intact immunoglobulin production or secretion, by fusing undifferentiated B cells with a myeloma cell line (43-46). In the attempts to-construct a human myeloma analog that would retain the desirable characteristics in the human fusion partner, it was theorized that the appropriate selection of cells for hybridization would result in the sequential improvement of a series of constructed myeloma analogs (25, 26). These human myeloma analogs were constructed by the fusion of a non-secreting human myeloma cell line with a variety of human cells at selected stages of differentiation. In these studies, while fusion efficiency was high and growth characteristics were excellent, stable secretion of monoclonal immunoglobulin was obtained only from fusions with established malignant human cell lines already committed to secretion. Antibody secretion was rapidly lost by the cloned hybridomas. It is possible that the choice of the non-secreting human myeloma cell line as the basis for the series of constructed human myeloma analogs may have had an impact on the ability of subsequently generated human myeloma analogs and hybridomas to support stable antibody production.
As an alternative to analogs formed by the fusion of human myeloma cells with human cells, heterohybridomas have been constructed by the fusion of murine myeloma cells with human cells (34-37). Some investigators, including the present inventor, have constructed human-mouse myeloma analogs by fusing murine myelomas with a variety of human cells. The murine myelomas used for fusion derive principally from the MOPC21 cell line, developed by Potter and associates and adapted to in vitro growth by Horibata and Harris (27, 28, 39). This cell line and derivatives thereof are routinely used in the production of murine monoclonal antibodies as the fusion partner. Teng, et al., fused MOPC21 with the human cell line SK007 (34), Ostberg and Pursch fused it with a human B lymphocyte (37), and Foung, et al. fused normal peripheral blood lymphocytes with a derivative
Cooper & Dunham LLP
Navarro Albert
Roger Williams General Hospital
White John P.
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