Development of DNA probes and immunological reagents of human tumor associated antigens

Abstract

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. This invention also provides a method for preparing a monoclonal and a polyclonal antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell. Using the antibodies produced, this invention further provides a method for diagnosing in a subject a neoplastic condition, a method for treating a neoplastic condition, and a method for imaging a neoplastic, human cell. This invention also provides a method for preparing DNA encoding a cell surface antigen associated with a neoplastic, human cell, DNA probes hybridized with the DNA prepared therein, as well as a method for diagnosing a neoplastic condition using the DNA probes.

What is claimed is:  
     
         1 . 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 which 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.  
   
     
     
         2 . A method of    claim 1   , wherein the established non-human, non-tumorigenic cell line is the CREF-Trans 6 cell line (ATCC Accession No. ______).  
     
     
         3 . A method of    claim 1   , wherein the neoplastic, human cell is a benign cell.  
     
     
         4 . A method of    claim 1   , wherein the neoplastic, human cell is metastatic cell.  
     
     
         5 . A method of    claim 1   , wherein the neoplastic, human cell is a human prostatic carcinoma cell derived from cell line LNCaP.  
     
     
         6 . A method of    claim 1   , wherein the neoplastic, human cell is a human breast carcinoma cell derived from cell line T47D.  
     
     
         7 . A method of    claim 1   , wherein the neoplastic, human cell is a human colorectal carcinoma cell derived from cell line SW480.  
     
     
         8 . A method of    claim 1   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from cell line GBM-18.  
     
     
         9 . A method of    claim 1   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from a primary tumor.  
     
     
         10 . A method of    claim 1   , wherein the DNA encoding the selectable or identifiable trait is plasmid DNA encoding resistance to an antibiotic.  
     
     
         11 . A method of    claim 10   , wherein the plasmid DNA comprises pSV2-Neo.  
     
     
         12 . A method of    claim 11   , wherein the antibiotic is G418.  
     
     
         13 . A method of    claim 1   , wherein the suitable second host is a murine host.  
     
     
         14 . A method of    claim 1   , wherein the suitable second host is a non-human primate host.  
     
     
         15 . A method of    claim 1   , wherein the cell surface antigen is a tumor associated antigen.  
     
     
         16 . A method of    claim 1   , wherein the cell surface antigen is a growth factor receptor.  
     
     
         17 . A method of    claim 1   , wherein the cell surface antigen is a viral-encoded, surface-expressed antigen.  
     
     
         18 . A method of    claim 1   , wherein the cell surface antigen is encoded by an oncogene product.  
     
     
         19 . A method of    claim 1   , wherein the cell surface antigen is a surface epitope.  
     
     
         20 . A method of    claim 1   , wherein the cell surface antigen is a membrane protein which mediates classical multi-drug resistance.  
     
     
         21 . A method of    claim 1   , wherein the cell surface antigen is a membrane protein which mediates atypical multi-drug resistance.  
     
     
         22 . A method of    claim 1   , wherein the cell surface antigen is an antigen which mediates a tumorigenic phenotype.  
     
     
         23 . A method of    claim 1   , wherein the cell surface antigen is an antigen which mediates a metastatic phenotype.  
     
     
         24 . A method of    claim 1   , wherein the cell surface antigen is an antigen which suppresses a tumorigenic phenotype.  
     
     
         25 . A method of    claim 1   , wherein the cell surface antigen is an antigen which suppresses a metastatic phenotype.  
     
     
         26 . A method of    claim 1   , wherein the cell surface antigen is an antigen which is recognized by a specific immunological effector cell.  
     
     
         27 . A method of    claim 1   , wherein the specific immunological effector cell is a T-cell.  
     
     
         28 . A method of    claim 1   , wherein the cell surface antigen is an antigen which is recognized by a non-specific immunological effector cell.  
     
     
         29 . A method of    claim 28   , wherein the non-specific immunological effector cell is a macrophage cell.  
     
     
         30 . A method of    claim 28   , wherein the non-specific immunological effector cell is a natural killer cell.  
     
     
         31 . A hybridoma cell line produced according to the method of    claim 1   .  
     
     
         32 . A method of producing a monoclonal antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell which comprises: 
 (a) producing a hybridoma according to the method of    claim 1   ; and    (b) recovering from the hybridoma so produced the monoclonal antibody.    
     
     
         33 . A monoclonal antibody produced according to the method of    claim 32   .  
     
     
         34 . A method for preparing a polyclonal antibody which specifically recognizes and binds to a cell surface antigen associated with a neoplastic, human cell which 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.    
     
     
         35 . A method of    claim 34   , wherein the established non-human, non-tumorigenic cell line is the CREF-Trans 6 cell line (ATCC Accession No. ______).  
     
     
         36 . A method of    claim 34   , wherein the neoplastic, human cell is a benign cell.  
     
     
         37 . A method of    claim 34   , wherein the neoplastic, human cell is metastatic cell.  
     
     
         38 . A method of    claim 34   , wherein the neoplastic, human cell is a human prostatic carcinoma cell derived from cell line LNCaP.  
     
     
         39 . A method of    claim 34   , wherein the neoplastic, human cell is a human breast carcinoma cell derived from cell line T47D.  
     
     
         40 . A method of    claim 34   , wherein the neoplastic, human cell is a human colorectal carcinoma cell derived from cell line SW480.  
     
     
         41 . A method of    claim 34   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from cell line GBM-18.  
     
     
         42 . A method of    claim 34   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from a primary tumor.  
     
     
         43 . A method of    claim 34   , wherein the DNA encoding the selectable or identifiable trait is plasmid DNA encoding resistance to an antibiotic.  
     
     
         44 . A method of    claim 43   , wherein the plasmid DNA comprises pSV2-Neo.  
     
     
         45 . A method of    claim 44   , wherein the antibiotic is G418.  
     
     
         46 . A method of    claim 34   , wherein the suitable second host is a murine host.  
     
     
         47 . A method of    claim 34   , wherein the suitable second host is a non-human primate host.  
     
     
         48 . A method of    claim 34   , wherein the cell surface antigen is a tumor associated antigen.  
     
     
         49 . A method of    claim 34   , wherein the cell surface antigen is a growth factor receptor.  
     
     
         50 . A method of    claim 34   , wherein the cell surface antigen is a viral-encoded, surface-expressed antigen.  
     
     
         51 . A method of    claim 34   , wherein the cell surface antigen is encoded by an oncogene product.  
     
     
         52 . A method of    claim 34   , wherein the cell surface antigen is a surface epitope.  
     
     
         53 . A method of    claim 34   , wherein the cell surface antigen is a membrane protein which mediates classical multi-drug resistance.  
     
     
         54 . A method of    claim 34   , wherein the cell surface antigen is a membrane protein which mediates atypical multi-drug resistance.  
     
     
         55 . A method of    claim 34   , wherein the cell surface antigen is an antigen which mediates a tumorigenic phenotype.  
     
     
         56 . A method of    claim 34   , wherein the cell surface antigen is an antigen which mediates a metastatic phenotype.  
     
     
         57 . A method of    claim 34   , wherein the cell surface antigen is an antigen which suppresses a tumorigenic phenotype.  
     
     
         58 . A method of    claim 34   , wherein the cell surface antigen is an antigen which suppresses a metastatic phenotype.  
     
     
         59 . A method of    claim 34   , wherein the cell surface antigen is an antigen which is recognized by a specific immunological effector cell.  
     
     
         60 . A method of    claim 59   , wherein the specific immunological effector cell is a T-cell.  
     
     
         61 . A method of    claim 34   , wherein the cell surface antigen is an antigen which is recognized by a non-specific immunological effector cell.  
     
     
         62 . A method of    claim 61   , wherein the non-specific immunological effector cell is a macrophage cell.  
     
     
         63 . A method of    claim 61   , wherein the non-specific immunological effector cell is a natural killer cell.  
     
     
         64 . A polyclonal antibody produced according to the method of    claim 34   .  
     
     
         65 . An antibody of claims  33  or  64  labeled with a detectable marker.  
     
     
         66 . A method of diagnosing in a subject a neoplastic condition which comprises contacting a sample from the subject with the antibody of    claim 65    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.  
     
     
         67 . An antibody of claims  33  or  64  labeled with a therapeutic agent.  
     
     
         68 . A method of treating a neoplastic condition which comprises contacting neoplastic, human cells associated with the neoplastic condition with an antibody of    claim 67    under conditions such that the therapeutic agent selectively inhibits proliferation of the neoplastic, human cells.  
     
     
         69 . An antibody of claims  33  or  64  labeled with an imaging agent.  
     
     
         70 . A method of imaging a neoplastic, human cell which comprises contacting the neoplastic, human cell to be imaged with the antibody of    claim 69    under conditions permitting the antibody to specifically recognize and bind to the cell surface antigen associated with the neoplastic cell and detecting the imaging agent bound thereto, thereby imaging the neoplastic cell.  
     
     
         71 . A method of preparing DNA encoding a cell surface antigen associated with a neoplastic, human cell which comprises: 
 (a) cotransfecting CREF-Trans 6 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; and    (h) recovering DNA encoding the cell surface antigen associated with the neoplastic human cell from the tumor cells so obtained.    
     
     
         72 . A method of    claim 71   , wherein the neoplastic, human cell is a benign cell.  
     
     
         73 . A method of    claim 71   , wherein the neoplastic, human cell is metastatic cell.  
     
     
         74 . A method of    claim 71   , wherein the neoplastic, human cell is a human prostatic carcinoma cell derived from cell line LNCaP.  
     
     
         75 . A method of    claim 71   , wherein the neoplastic, human cell is a human breast carcinoma cell derived from cell line T47D.  
     
     
         76 . A method of    claim 71   , wherein the neoplastic, human cell is a human colorectal carcinoma cell derived from cell line SW480.  
     
     
         77 . A method of    claim 71   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from cell line GBM-18.  
     
     
         78 . A method of    claim 71   , wherein the neoplastic, human cell is a human glioblastoma multiform (stage IV astrocytoma) cell derived from a primary tumor.  
     
     
         79 . A method of    claim 71   , wherein the DNA encoding the selectable or identifiable trait is plasmid DNA encoding resistance to an antibiotic.  
     
     
         80 . A method of    claim 79   , wherein the plasmid DNA comprises pSV2-Neo.  
     
     
         81 . A method of    claim 80   , wherein the antibiotic is G418.  
     
     
         82 . A method of    claim 71   , wherein the cell surface antigen is a tumor associated antigen.  
     
     
         83 . A method of    claim 71   , wherein the cell surface antigen is a growth factor receptor.  
     
     
         84 . A method of    claim 71   , wherein the cell surface antigen is a viral-encoded surface-expressed antigen.  
     
     
         85 . A method of    claim 71   , wherein the cell surface antigen is an oncogene product.  
     
     
         86 . A method of    claim 71   , wherein the cell surface antigen is a surface epitope.  
     
     
         87 . A method of    claim 71   , wherein the cell surface antigen is a membrane protein which mediates classical multi-drug resistance.  
     
     
         88 . A method of    claim 71   , wherein the cell surface antigen is a membrane protein which mediates atypical multi-drug resistance.  
     
     
         89 . A method of    claim 71   , wherein the cell surface antigen is an antigen which mediates a tumorigenic phenotype.  
     
     
         90 . A method of    claim 71   , wherein the cell surface antigen is an antigen which mediates a metastatic phenotype.  
     
     
         91 . A method of    claim 71   , wherein the cell surface antigen is an antigen which suppresses a tumorigenic phenotype.  
     
     
         92 . A method of    claim 71   , wherein the cell surface antigen is an antigen which suppresses a metastatic phenotype.  
     
     
         93 . A method of    claim 71   , wherein the cell surface antigen is an antigen which is recognized by a specific immunological effector cell.  
     
     
         94 . A method of    claim 93   , wherein the specific immunological effector cell is a T-cell.  
     
     
         95 . A method of    claim 71   , wherein the cell surface antigen is an antigen which is recognized by a non-specific immunological effector cell.  
     
     
         96 . A method of    claim 95   , wherein the non-specific immunological effector cell is a macrophage cell.  
     
     
         97 . A method of    claim 95   , wherein the non-specific immunological effector cell is a natural killer cell.  
     
     
         98 . DNA prepared according to the method of    claim 71   .  
     
     
         99 . A DNA probe hybridizable with the DNA of    claim 98   .  
     
     
         100 . A DNA probe of    claim 99    labeled with a detectable marker.  
     
     
         101 . A method of diagnosing in a subject a neoplastic condition which comprises contacting a sample from the subject with the DNA probe of    claim 100    under conditions permitting the DNA probe to hybridize with the DNA associated with the neoplastic condition, detecting the presence of hybridized DNA, and thereby diagnosing the neoplastic condition.