Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1994-12-08
2001-02-06
Schwadron, Ronald B. (Department: 1644)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S325000, C435S366000, C435S352000, C424S093100, C424S093200, C424S093210, C424S093700
Reexamination Certificate
active
06184032
ABSTRACT:
Throughout this application, various publications are referenced by Arabic numerals within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
BACKGROUND OF THE INVENTION
A procedure which has proven successful in identifying dominant acting oncogenes associated with various human malignancies has been DNA transfection (1-3). By employing DNA Ca
2+
-mediated DNA transfection techniques and various molecular cloning strategies, the genetic elements presumably responsible for malignant conversion of specific human and rodent cells have been identified and cloned (4-21). The primary method used to identify transforming oncogenes has been by Ca
2+
-mediated DNA transfer of sheared HMW-tumor DNA into NIH-3T3 cells followed by the isolation and characterization of DNA from morphologically transformed foci. A limitation of this assay has been that the majority of HMW-DNA from primary and established human tumor isolates, approximately 80%, fail to induce morphological transformation of NIH-3T3 cells.
By using a modification of this procedure, the Ca
2+
-mediated transfer of HMW-DNA plus the neomycin resistance gene into NIH-3T3 cells, selection for neomycin resistant colonies in G418-supplemented medium, injection of pooled neomycin resistant cells into nude mice and the generation and subsequent isolation of tumor cells, it has been possible to identify transforming DNA which either fails to form morphologically transformed foci or is weakly focus-producing when transfected into NIH-3T3 cells (1,12,13).
More recent studies indicate that a specific clone of Fisher rat embryo cells, CREF (14), develops a tumorigenic phenotype in nude mice after being cotransfected with high molecular weight (HMW-prostatic carcinoma DNA or HMW-colon carcinoma DNA in combination with pSV2-Neo plasmid DNA followed by selection for G418 resistant colonies (2). In contrast, both types of HMW-DNAs did not induce foci of morphologically altered CREF cells when assayed using a monolayer culture system (2). In addition, when the same HMW tumor-DNA samples and transfection protocol used with CREF cells were applied to NIH-3T3 cells, no tumors were induced by transfected cells in nude mice (2).
The recent development of hybridoma technology has resulted in the development of specific monoclonal antibodies to various human tumor associated antigens. This approach has greatly facilitated the study of the potential role of tumor associated antigens in expression of the neoplastic state (38). However, the production of monoclonal antibodies with good tumor specificity, especially toward various stages in the development of a malignant neoplasm, remains a laborious and uncertain undertaking, especially when freshly isolated heterogeneous tumor cells are used as immunogens. An innovative recent approach to the generation of monoclonal antibodies with specificity to transforming gene products which may be mediators of the tumor state, has been to utilize transfected heterologous species cells containing and expressing human transforming gene products as immunogens (39-42). Using this approach, monoclonal antibodies have been produced against the neu transforming gene transferred and expressed in NIH-3T3 cells. Similarly, Roth, et al. have used the transfection approach in generating monoclonal antibodies against NIH-3T3 transfectants produced following transfer of HMW-DNA from a human acute lymphocytic leukemia cell line (ALL) and to a c-Ha-ras transformed cell line (40, 41). In addition, Hollingsworth, et al. transfected NIH-3T3 cells with HMW-DNA from a human pancreatic adenocarcinoma cell line to generate monoclonal antibodies (42).
SUMMARY OF THE INVENTION
The present invention provides a general method for identifying genes and producing immunological reagents which encode cell surface antigens of human origin.
Specifically, this invention provides a method for preparing a hybridoma cell line which produces an antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell. The method comprises: (a) cotransfecting an established non-human, non-tumorigenic cell line with DNA isolated from a neoplastic, human cell and DNA encoding a selectable or identifiable trait; (b) selecting transfected cells which express the selectable or identifiable trait; (c) recovering the transfected cells so selected; (d) injecting the transfected cells so recovered into a suitable first murine host; (e) maintaining the resulting first murine host for a period of time effective to induce the injected transfected cells to form a tumor in the first murine host; (f) isolating the resulting tumor from the first murine host; (g) obtaining tumor cells from the tumor so isolated; (h) coating the tumor cells so obtained with an antiserum generated against the established non-human, non-tumorigenic cell line; (i) injecting the antiserum-coated cells into suitable second hosts; (j) screening the resulting second hosts to identify hosts which produce serum reactive with the neoplastic, human cell; (k) removing spleens from the second hosts so identified; (l) preparing from the spleens so removed hybridomas; and (m) recovering therefrom a hybridoma cell line which produces an antibody which specifically recognizes and binds to the cell surface antigen.
This invention also provides a method for producing a monoclonal antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell. This method comprises producing a hybridoma according to the above method and recovering from the hybridoma so produced the monoclonal antibody.
This invention further provides a method for preparing a polyclonal antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell. This method comprises: (a) cotransfecting an established non-human, non-tumorigenic cell line with DNA isolated from a neoplastic, human cell and DNA encoding a selectable or identifiable trait; (b) selecting transfected cells which express the selectable or identifiable trait; (c) recovering the transfected cells so selected; (d) injecting the transfected cells so recovered into a suitable first murine host; (e) maintaining the resulting first murine host for a period of time effective to induce the injected transfected cells to form a tumor in the first murine host; (f) isolating the resulting tumor from the first murine host; (g) obtaining tumor cells from the tumor so isolated; (h) coating the tumor cells so obtained with an antiserum generated against the established non-human, non-tumorigenic cell line; (i) injecting the antiserum-coated cells into suitable second hosts; (j) screening the resulting second hosts to identify hosts which produce serum reactive with the neoplastic, human cell; and (k) recovering from the second hosts so identified the polyclonal antibody.
This invention provides a method of diagnosing in a subject a neoplastic condition which comprises contacting a sample from the subject with a monoclonal antibody labeled with a detectable marker under conditions permitting the antibody to specifically recognize and bind to the cell surface antigen associated with the neoplastic condition, detecting the presence of antibody bound to the antigen, and thereby diagnosing the neoplastic condition.
This invention also provides a method of diagnosing in a subject a neoplastic condition which comprises contacting a sample from the subject with a polyclonal antibody labeled with a detectable marker under conditions permitting the antibody to specifically recognize and bind to the cell surface antigen associated with the neoplastic condition, detecting the presence of antibody bound to
Cooper & Dunham LLP
Schwadron Ronald B.
The Trustees of Columbia University in the City of New York
White John P.
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