Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-09-01
2004-11-30
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S006120, C435S091100, C435S069100, C435S325000, C435S320100, C530S387300
Reexamination Certificate
active
06824989
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of recombinant monoclonal antibodies and their uses in clinical and scientific procedures, including diagnostic procedures, especially where such processes involve the detection of phosphotyrosine-containing proteins.
BACKGROUND OF THE INVENTION
Cellular growth, especially the uncontrolled growth seen in cancers, has, at least partly, been attributed to the functions of protein kinases that add phosphate to the hydroxyl group of tyrosine. In a similar way, oncogenic transformation of cells by entities such as Rous sarcoma virus and murine leukemia virus, leads to a significant increase, by at least an order of magnitude, in the level of phosphotyrosines seen in both virus-encoded proteins and proteins found in the transformed cells. Tyrosine kinases are believed intimately involved in such transformation processes.
Anti-phosphotyrosine monoclonal antibodies specific for proteins containing phosphotyrosine residues have proven valuable in studying such transformation processes, as well as in the detection of phosphotyrosine residues in proteins and detection of proteins containing such moieties. Thus, because oncogenes present in transforming organisms have been found to have natural counterparts, or homologs, within the untransformed cells, tyrosine-kinases have been implicated in normal growth control, possibly including during embryonic development or regeneration. Consequently, the availability of significant quantities of monoclonal antibodies with specificity directed toward such proteins can be of far reaching value, especially where methods are available for the production of such antibodies on a high scale. For example, monoclonal antibodies can facilitate the identification of cellular substrates for tyrosine kinases and make possible affinity purification of their substrates as well as being highly useful in screening for, and detecting, diseases.
Modern technology, such as that involving the use of hybridomas, has made available to researchers and clinicians alike sources of highly specific and potent monoclonal antibodies useful in general diagnostic and clinical procedures, such as where these antibodies are specific for phosphotyrosine-containing proteins and polypeptides. Among the more useful monoclonal antibodies are those derived from the 4G10 hybridoma cell line. The 4G10 monoclonal antibody is described further in Oda et al,
Blood
, Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin, 88(11):4304-13 (Dec. 1, 1996). However, while such antibodies are useful, they are not easy to produce in pure form. For example, the 4G10 cell line is a low producer of the required antibodies and therefor it is difficult, as well as expensive, to procure such antibodies in sufficient quantity to support all the different uses to which they might be put. In addition, the quality of said antibodies from one lot to another may also be less that what is desired by most researchers and clinicians.
More specifically, the purified His tagged recombinant antibody is purer than what can be obtained from the hybridoma source. The hybridoma Protein A purified 4G10 has an as yet unidentified heavy chain doublet whereas the IMAC purified recombinant antibody of the present invention is highly pure with a single heavy chain band and light chain band. Furthermore, the phosphotyrosine binding properties of 4G10 are sensitive to acid exposure and elution from Protein A or G columns with the required low pH conditions leads to partial inactivation and thus a lower specific activity.
The present invention solves such problems by offering a recombinant monoclonal antibody comprising utility and specificity the same or similar to that derived from the 4G10 hybridoma cell line but showing much greater purity with concomitant high specific activity and consistency of performance. Especially useful is the histidine-tagged form of the immunoglobulin of the present invention. The His tag allows for IMAC (immobilized metal affinity chromatography) purification under neutral pH conditions so that the recombinant antibody is never exposed to low pH conditions. Greater purity is, of course, highly useful in clinical applications.
The His tag is also useful in that it facilitates attachment of the recombinant antibody or immunoglobulin in an orientated fashion to Nickel matrices for solid phase applications. Thus, the His tagged recombinant form is particularly useful. The methods disclosed herein thus afford a purer and more uniform product with generally higher specific activity.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to a recombinant monoclonal antibody having specificity directed to proteins and polypeptides comprising phosphotyrosine moieties.
It is therefore an object of the present invention to provide for a recombinant monoclonal antibody having specificity for phosphotyrosine-containing proteins and polypeptides.
It is a further object of the present invention to provide polynucleotides, such as cDNAs, whose nucleotide sequences code for the heavy and light chains of the aforementioned recombinant antibodies and immunoglobulins.
It is a still further object of the present invention to provide recombinant antibodies specific for phosphotyrosine-containing proteins and polypeptides wherein said antibodies are tagged with sequences of amino acids, and other tags, making them easily isolatable as well as affording versatility in using said antibodies for research, diagnostic and clinical purposes.
It is another object of the present invention to provide a method of using the recombinant antibodies disclosed herein for research, diagnostic and clinical uses.
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Kanakura, Y, et al, “Phorbol 12-Myristate 13-Acetate Inhibits Granulocyte-Macrophage Colony Stimulating Factor-Induced Protein Tyrosine Phosphorylation in Human Factor-Dependent Hematopoietic Cell Line”, Journal of Biological Chemistry, vol. 266, No. 1, 1991, pp. 490-495.
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Mayer, A, et al, “Exemplifying guidelines for preparation of recombinant DNA products in phase I trails in cancer: Preparation of a genetically engineered anti-CEA single chain Fv antibody”, European Journal of Cancer, vol. 34, No. 7, Jun. 1998 (1998
Eisinger Dominic
Jelinek Thomas
LaMarche Arthur
Stiles Lynn
Cheu Changhwa J
Grant Alan J.
Nguyen Bao-Thuy L.
Olstein Elliot M.
Upstate Biotechnology, Inc.
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