Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1994-08-15
1998-01-20
Feisee, Lila
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
435330, 435331, 435344, 4241381, C07K 1600, C12P 2108, C12N 500, A61K 39395
Patent
active
057102554
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to monoclonal antibodies directed against the retinoblastoma protein p110.sup.RB. More specifically, it relates to a novel monoclonal antibody having the ability to detect p110.sup.RB that has been previously bound by a naturally occurring or non-naturally occurring ligand.
BACKGROUND OF THE INVENTION
The retinoblastoma gene (RB) is one of the best-studied tumor suppressor genes. Mutations which prevent the normal expression of the retinoblastoma gene have been linked to the pathogenesis of several human malignancies. These include small-cell lung carcinomas, osteosarcomas, breast carcinomas, soft tissue sarcomas, bladder carcinomas, prostate carcinomas and testicular tumors.
p110.sup.RB, the protein product of the RB (retinoblastoma) gene, specifically binds and forms a complex with several DNA tumor vital oncoproteins, including adenovirus E1A, polyomavirus Tag and papillomavirus E7. It is believed that these oncoproteins bind to and inactivate an important function of p110.sup.RB, thereby mimicking the loss of retinoblastoma gene function. Studies have suggested that p110.sup.RB has intrinsic DNA-binding activity and may also interact with cellular transcription factors E2F/DRTF. Improved methods of determining the absence of p110.sup.RB could aid clinicians in their evaluation of patients and their decision as to appropriate treatment. Recently, evidence has become available that the loss of p110.sup.RB expression, as measured by immunohistochemistry, may be predictive of patient prognosis in non-small lung cancer, and in bladder cancer. It is likely that additional studies of this sort will add to the list of cancers in which loss of p110.sup.RB expression will be predictive of the aggressiveness of disease. Because of this, improved methods of immunohistochemistry are required to aid clinicians in their evaluation of patients and their decisions regarding aggressiveness or modality of therapy. In this case, increased intensity of available chemotherapy is one option, another option may be therapies derived from the tumor suppressor gene, RB, or its gene product p110.sup.RB. In the latter case, lack of Rb protein expression would indicate that the patient is a candidate for RB-related therapy.
The currently available antibodies to p110.sup.RB are either polyclonal, and therefore, variable, or monoclonal but inappropriate because of low affinity or because they are targeted to an epitope of p110.sup.RB likely to be masked by cytoplasmic proteins. If such epitopes are masked by cytoplasmic proteins, therefore giving a "false negative" reading with respect to p110.sup.RB expression, then an incorrect therapeutic modality may be chosen. Thus, there exists a need for monoclonal antibodies reactive with p110.sup.RB regardless of associated cytoplasmic proteins. The present invention satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
This invention provides a family of monoclonal antibodies specific for epitopes of p110.sup.RB protein present in the nucleus. These antibodies have superior properties that prove useful for the detection of p110.sup.RB or its complexes with other cellular regulatory proteins in cells and in cell lysates.
This invention also provides hybridoma cell lines that produce such monoclonal antibodies and methods of using these antibodies diagnostically, prognostically and therapeutically.
Further, the invention provides a method for isolating proteins associated with p110.sup.RB proteins.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows an immunoblot analysis of p110.sup.RB expression in various transformed cell lines with 1 .mu.g/ml of 3C8(A), PMG3-245(B) and MuIgG (as negative control, C). The proteins were visualized by NBT/BCIP substrate.
FIG. 2 shows 20 .mu.l of A549 call lysate (started from 1 mg/ml, lane 1) with 2-fold serial dilutions were immunoblotted and detected with antibody 3C8 (2 .mu.g/ml).
FIG. 3 shows ELISA for biotinylated 3C8, 1E5 and PMG-3-245. Various concentrations of biotinyla
REFERENCES:
patent: 4942123 (1990-07-01), Lee et al.
"Monoclonal antibodies reactive with four distinct epitopes of the human retinoblastoma (Rb) protein", Proceedings of the American Association for Cancer Research Annual Meeting, 33:390 (1992).
Shew J. et al., "Antibodies detecting abnormalities of the retinoblastoma susceptibility gene pb110RB in osteosarcomas and sarcomas", Chemical Abstracts, vol. 111, No. 15 (Oct. 9, 1989).
Wen-Hwa L. et al., "The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity", Nature, 329:642-645 (1987).
Shepard H. Michael
Wen Shu Fen
Canji Inc.
Eyler Yvonne
Feisee Lila
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