Novel antibody conjugates reactive with human carcinomas

Abstract

The present invention relates to novel antibodies, antibody fragments and antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells. More particularly, the antibodies, conjugates and single-chain immunotoxins of the invention include: a murine monoclonal antibody, BR96; a human/murine chimeric antibody, ChiBR96; a F(ab′) 2 fragment of BR96; ChiBR96-PE, ChiBR96-LysPE40, ChiBR96 F(ab′) 2 -LysPE40 and ChiBR96 Fab′-LysPE40 conjugates and recombinant BR96 sFv-PE40 immunotoxin. These molecules are reactive with a cell membrane antigen on the surface of human carcinomas. The BR96 antibody and its functional equivalents, displays a high degree of selectivity for carcinoma cells and possess the ability to mediate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. In addition, the antibodies of the invention internalize within the carcinoma cells to which they bind and are therefore particularly useful for therapeutic applications, for example, as the antibody component of antibody-drug or antibody-toxin conjugates. The antibodies also have a unique feature in that they are cytotoxic when used in the unmodified form, at specified concentrations.

What is claimed is:  
     
         1 . A monoclonal antibody BR96 produced by hybridoma ATCC HB 10036, or fragments thereof, and functional equivalents thereof having an antigen-binding region that competitively inhibits the immunospecific binding of monoclonal antibody BR96, having specific immunological reactivity with human carcinoma cells, said antibody characterized by being capable of internalizing within the carcinoma cells with which it reacts, mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity, and/or killing of said human carcinoma cells in the absence of host effector cells or complement.  
     
     
         2 . Hybridoma HB 10036 as deposited with the ATCC.  
     
     
         3 . An Fab, F(ab′) 2  or Fv fragment of the antibody of  claim 1 .  
     
     
         4 . An immunoconjugate comprising a molecule containing the antigen-binding region of the BR96 monoclonal antibody joined to a cytotoxic agent.  
     
     
         5 . The immunoconjugate of  claim 4 , wherein the molecule comprises BR96 monoclonal antibody or fragments thereof.  
     
     
         6 . The immunoconjugate of  claim 4 , wherein the molecule comprises chimeric human/murine BR96 antibody or fragments thereof.  
     
     
         7 . The immunoconjugate of  claim 5 , wherein the fragments are selected from the group consisting of Fv, F(ab′) and F(ab′) 2  fragments.  
     
     
         8 . The immunoconjugate of  claim 6 , wherein the fragments are selected from the group consisting of Fv, F(ab)′ and F(ab′) 2  fragments.  
     
     
         9 . A method for selectively killing tumor cells expressing the antigen that immunospecifically binds to BR96 monoclonal antibody comprising reacting the immunoconjugate of  claim 4  with said tumor cells.  
     
     
         10 . A recombinant single-chain immunotoxin molecule comprising a cloned heavy chain Fv portion and a cloned light chain Fv portion of the BR96 monoclonal antibody joined to a cytotoxic agent.  
     
     
         11 . A bispecific antibody with a binding specificity for two different antigens, one of the antigens being that with which the monoclonal antibody of  claim 1  binds.  
     
     
         12 . The bispecific antibody of  claim 11 , wherein one of the antigens comprises a variant of Le y  determinant which includes an epitopic site containing fucose α1-3.  
     
     
         13 . A monoclonal antibody, the antigen-binding region of which competitively inhibits the immunospecific binding of monoclonal antibody BR96 produced by hybridoma HB 10036 to its target antigen.  
     
     
         14 . The antibody of  claim 13 , wherein said antigen comprises a variant of Le y  determinant which includes an epitopic site containing fucose α1-3.  
     
     
         15 . A human/murine recombinant antibody, the antigen-binding region of which competitively inhibits the immunospecific binding of monoclonal antibody BR96 produced by hybridoma HB 10036 to its target antigen.  
     
     
         16 . The antibody of  claim 1 , wherein the antibody is conjugated to a therapeutic agent to form an antibody conjugate.  
     
     
         17 . The antibody of  claim 16 , wherein the therapeutic agent is an anti-tumor drug, a cytotoxin, a radioactive agent, a second antibody or an enzyme.  
     
     
         18 . The antibody of  claim 17 , wherein the cytotoxin is a ribosome binding toxin.  
     
     
         19 . The antibody of  claim 18 , wherein the ribosome binding toxin is ricin A.  
     
     
         20 . A composition comprising a combination of an immunoconjugate comprising the antibody of  claim 1  linked to an enzyme capable of converting a prodrug into a cytotoxic drug, and said prodrug.  
     
     
         21 . A pharmaceutical composition useful in the treatment of carcinomas comprising a pharmaceutically effective amount of the antibody of  claim 1  and an acceptable carrier.  
     
     
         22 . A pharmaceutical composition useful in the treatment of carcinomas comprising a pharmaceutically effective amount of at least one antibody conjugate according to  claim 16  and an acceptable carrier.  
     
     
         23 . A method of treating carcinomas in vivo comprising administering to a patient a pharmaceutically effective amount of a composition containing the antibody of  claim 1 .  
     
     
         24 . A method for determining the presence of carcinoma in human tissue comprising contacting a specimen of said tissue with the antibody of  claim 1  and detecting the binding of said antibody to said tissue.  
     
     
         25 . The method of  claim 24 , wherein said antibody is labeled so as to directly or indirectly produce a detectable signal with a compound selected from the group consisting of a radiolabel, an enzyme, a chromophore and a fluorescer.  
     
     
         26 . A method for imaging carcinoma comprising administering to a patient intravenously the antibody of  claim 1  in an amount effective for detection of the carcinoma, allowing the antibody to bind to carcinoma cells and to localize to the site of carcinoma cells and detecting said antibody bound to the carcinoma cells.  
     
     
         27 . The method of  claim 26 , wherein said antibody is labeled so as to directly or indirectly produce a detectable signal with a label selected from the group consisting of a radiolabel, an enzyme, a chromophore, and a fluorescer.  
     
     
         28 . A monoclonal anti-idiotypic antibody reactive with an idiotope on the antibody of  claim 1 .  
     
     
         29 . A diagnostic kit comprising: 
 a) the antibody of  claim 1;  and    b) a conjugate of a detectable label and a specific binding partner of the antibody of (a) above.    
     
     
         30 . The diagnostic kit of  claim 29 , wherein the label is selected from the group consisting of enzymes, radiolabels, chromophores and fluorescers.  
     
     
         31 . The immunoconjugate of  claim 4 , wherein the cytotoxic agent is selected from a group consisting of antimetabolites, alkylating agents, anthracyclines, antibiotics, anti-mitotic agents, and chemotherapeutic agents.  
     
     
         32 . The immunoconjugate of  claim 4 , wherein the cytotoxic agent is selected from a group consisting of ricin, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, dihydroxy anthracin dione, actinomycin D, 1-dehydrotestosterone, and glucocorticoid.  
     
     
         33 . A method for curing a subject suffering from a cancer, the cancer being characterized as a group of cells having a tumor associated antigen on the cell surface, which method comprises administering to the subject a cancer killing amount of a tumor targeted antibody joined to a cytotoxic agent under conditions which permit the antibody so joined to bind the tumor associated antigen on the cell surface so as to kill the cells so bound thereby curing the subject.  
     
     
         34 . The method of  claim 33 , wherein the tumor targeted antibody is an internalizing antibody.  
     
     
         35 . The method of  claim 33 , wherein the tumor targeted antibody is an internalizing antibody which recognizes and binds to a Le y  determinant.  
     
     
         36 . A method of inhibiting the proliferation of mammalian tumor cells which comprises contacting the mammalian tumor cells with a proliferation inhibiting amount of a tumor targeted antibody joined to doxorubicin so as to inhibit proliferation of the mammalian tumor cells.  
     
     
         37 . The method of  claim 36 , wherein the tumor targeted antibody is the monoclonal antibody BR96 produced by hybridoma ATCC HB 10036.  
     
     
         38 . The method  claim 36 , wherein the tumor targeted antibody is a chimeric antibody ChiBR96 produced by the hybridoma having the identifying characteristics of HB 10460 as deposited with the ATCC.  
     
     
         39 . The method of  claim 36 , wherein the tumor targeted antibody is the bispecific antibody with a binding specificity for two different antigens, one of the antigens being that with which the monoclonal antibody BR96 produced by hybridoma ATCC HB10036 binds.  
     
     
         40 . The method of  claim 36 , wherein the tumor targeted antibody is the monoclonal antibody, the antigen-binding region of which competitively inhibits the immunospecific binding of monoclonal antibody BR96 produced by hybridoma HB 10036 to its target antigen.  
     
     
         41 . The method of  claim 36 , wherein the tumor targeted antibody is the human/murine recombinant antibody, the antigen-binding region of which competitively inhibits the immunospecific binding of monoclonal antibody BR96 produced by hybridoma HB 10036 to its target antigen.  
     
     
         42 . A method for selectively killing tumor cells expressing the antigen that immunospecifically binds to BR96 monoclonal antibody comprising reacting an immunoconjugate comprising a molecule containing the antigen-binding region of the BR96 monoclonal antibody joined to doxorubicin with the tumor cells so as to obtain a BR96/doxorubicin-tumor cell complex thereby permitting the doxorubicin to kill the tumor cells so complexed.  
     
     
         43 . A method of inhibiting the proliferation of mammalian tumor cells which comprises contacting the mammalian tumor cells with a sufficient concentration of an immunoconjugate comprising a molecule containing the antigen-binding region of the BR96 monoclonal antibody joined to doxorubicin so as to obtain a BR96/doxorubicin-tumor cell complex thereby inhibiting proliferation of the mammalian tumor cells so complexed.  
     
     
         44 . A method for treating a subject suffering from a proliferative type disease characterized by cells having the BR96 antigen on the cell surface which comprises administering to the subject an effective amount of an immunoconjugate comprising the antigen-binding region of the BR96 monoclonal antibody joined to doxorubicin such that the immunoconjugate binds the BR96 antigen and kills said cells thereby treating the subject.