Endosialin-binding antibody

Abstract

The present disclosure relates to the generation of an antibody that specifically recognizes and binds Endosialin, a cell surface antigen characteristic of tumor pericytes and cells of tumor stroma. The antibody has the ability to become internalized in Endosialin expressing cells and to block the activation of MAPK in PDGF stimulated human pericytes. The antibody is able to block angiogenesis induced by LGALS3BP, a known Endosialin interactor and to inhibit tumor growth alone and in combination with 1959, a humanized antibody against LGALS3BP in human osteosarcoma xenograft. Furthermore, upon conjugation of the hu manized version of the anti-Endosialin antibody with a duocarmycin derivative, the resulting ADC displays potent and antigen de pendent in vitro tumor cell cytotoxicity and effective antitumor efficacy in vivo. The disclosure is also related to nucleotides encoding the antibodies of the disclosure and cell expressing the antibodies.

1 . An antibody or functional fragment thereof, which is directed against an epitope between amino acids 477-488 of human Endosialin according to SEQ ID No: 1. 
     
     
         2 . The antibody of  claim 1  which comprises
 (i) a heavy chain comprising:
 a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence as shown in SEQ ID No: 2 or a sequence differing in 1 or 2 amino acids therefrom, 
 a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence as shown in SEQ ID No: 3 or a sequence differing in 1 or 2 amino acids therefrom, and/or 
 a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence as shown in SEQ ID No: 4 or a sequence differing in 1 or 2 amino acids therefrom, and/or 
 
 (ii) a light chain comprising:
 a light chain complementarity determining region 1 (CDRL1) having the amino acid sequence as shown in SEQ ID No: 5 or a sequence differing in 1 or 2 amino acids therefrom, 
 a light chain complementarity determining region 2 (CDRL2) having the amino acid sequence as shown in SEQ ID No: 6 or a sequence differing in 1 or 2 amino acids therefrom, and/or 
 a light chain complementarity determining region 3 (CDRL3) having the amino acid sequence as shown in SEQ ID No: 7 or a sequence differing in 1 or 2 amino acids therefrom, 
 
 or an antibody recognizing the same epitope on human FGRF4. 
 
     
     
         3 . The antibody of  claim 1  or  2  which comprises
 (i) a heavy chain comprising a CDRH1 as shown in SEQ ID NO: 2, a CDRH2 as shown in SEQ ID NO: 3 and a CDRH3 as shown in SEQ ID NO: 4, and 
 (ii) a light chain comprising a CDRL1 as shown in SEQ ID NO: 5, a CDRL2 as shown in SEQ ID NO: 6 and a CDRL3 as shown in SEQ ID NO: 7. 
 
     
     
         4 . The antibody of any one of the previous claims comprising:
 a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID No: 8, or an amino acid sequence having a sequence identity of at least 90% thereto, and/or   a light chain variable region comprising an amino acid sequence as shown in SEQ ID No: 9, or an amino acid sequence having a sequence identity of at least 90% thereto.   
     
     
         5 . The antibody of any one of the previous claims, which is a Fab fragment, a Fab′ fragment, a F(ab′) fragment, a Fv-fragment, a diabody, ScFv, SMIP, single chain antibody, affibody, avimer, nanobody and/or a domain antibody. 
     
     
         6 . The antibody of any one of the previous claims, which is of the IgG1-, IgG2-, IgG3-or IgG4-type or an IgM, IgA1, IgA2, IgAsec, IgD or IgE-type antibody. 
     
     
         7 . The antibody of any one of the previous claims, which is a monoclonal antibody, in particular a murine antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, in particular a bispecific antibody, or a fragment thereof. 
     
     
         8 . The antibody of any of the previous claims comprising a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 or an amino acid sequence having a identity of at least 90% thereto, and/or
 a light light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25 or an amino acid sequence having a sequence identity of at least 90% thereto.   
     
     
         9 . The antibody of any one of the previous claims, wherein a labeling group and/or to an effector group, preferably a therapeutic group is coupled to the antibody. 
     
     
         10 . The antibody of  claim 9 , wherein the antibody is linked to a paramagnetic, radioactive or fluorogenic ion that is detectable upon imaging. 
     
     
         11 . The antibody according to  claim 9 , wherein the antibody is linked to an anticellular agent, in particular an anti-mitotic or DNA damaging agent capable of killing or suppressing growth or cell division of endothelial cells. 
     
     
         12 . The antibody of  claim 11 , wherein the anticellular agent comprises a chemotherapeutic agent, radioisotope or cytotoxin. 
     
     
         13 . The antibody of  claim 12 , wherein the anticellular agent comprises an antimetabolite, an anthracycline, a  vinca  alkaloid, an antibiotic, an alkylating agent or a plant-, fungus- or bacteria-derived toxin. 
     
     
         14 . The antibody of  claim 12 , wherein the anticellular agent comprises a DNA damaging agent, in particular a Minor Grove Binder duocarmycin derivative. 
     
     
         15 . The antibody of  claim 12 , wherein the cytotoxin comprises an A chain toxin, a ribosome inactivating protein, a-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or  Pseudomonas  exotoxin. 
     
     
         16 . The antibody of  claim 12 , wherein the cytotoxin comprises deglycosylated ricin A chain. 
     
     
         17 . The antibody of any one of  claims 1 - 16 , wherein the antibody recognizes human Endosialin that is expressed on the cell surfaces of tumor vascular cells to a greater degree than on the surfaces of normal endothelial cells. 
     
     
         18 . The antibody of any one of  claims 1 - 17 , wherein the antibody is a bispecific antibody that recognizes the human tumor-associated antigen LGALS3BP (aka Mac-2 BP or 90K). 
     
     
         19 . An isolated nucleic acid molecule selected from the group consisting of:
 (a) a nucleic acid sequence encoding an antibody, antibody fragment or a derivative thereof of any of  claims 1 - 18 , preferably a nucleic acid sequence as shown in any one of SEQ ID NO: 14 to SEQ ID NO. 25   (b) a nucleic acid sequence complementary to any of the sequences in (a), and   (c) a nucleic acid sequence capable of hybridizing to (a) or (b) under stringent conditions.   
     
     
         20 . A vector comprising the nucleic acid sequence of  claim 19 , preferably an expression vector, wherein the nucleic acid sequence is operably linked to a control sequence. 
     
     
         21 . A host comprising the nucleic acid of  claim 19  or the vector of  claim 20 , which is preferably a human, bacteria, animal, fungal, amphibian or plant cell, or a non-human transgenic animal. 
     
     
         22 . A process of manufacturing a monoclonal antibody according to anyone of  claims 1 - 18  comprising the step of obtaining said antibody from the host of  claim 21 . 
     
     
         23 . A pharmaceutical composition comprising an antibody of anyone of  claims 1 - 18 , a nucleic acid molecule of  claim 19 , a vector of  claim 20 , a host of  claim 21 , or an antibody generated by the process of  claim 22 , optionally in combination with a pharmaceutically acceptable carrier, diluent, and/or excipient. 
     
     
         24 . The pharmaceutical composition according to  claim 23 , comprising a further active agent, such as a further antibody or antibody fragment, in particular an anti-neoplastic agent, preferably selected from the group consisting of antibodies, small molecules, antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule-targeting agents, kinase inhibitors, protein synthesis inhibitors, immuno-therapeutics, hormones or analogs thereof. 
     
     
         25 . A compound selected from the antibody according to any one of  claims 1 - 18 , the nucleic acid molecule of  claim 19 , the vector of  claim 20 , the host of  claim 21  and the pharmaceutical composition according to  claim 24 , for use in the prevention or treatment of a neoplastic disease or cancer. 
     
     
         26 . The compound for the use of  claim 25 , wherein the disease is neuroblastoma, sarcoma (synovial sarcoma, fibrosarcoma, MFH, liposarcom, osteosarcoma), high-grade glioma, brain tumor, carcinoma (bladder, breast, colon, renal, gastric cancer, endometrial cancer, lung cancer, ovarian cancer) and/or a tumor expressing Endosialin in tumor vasculature and stroma and/or in tumor cells. 
     
     
         27 . The compound for the use of  claim 25  or  26 , which is to be administered intravenously, intramuscularly, and/or subcutaneously. 
     
     
         28 . The compound for the use of any one of  claims 25 - 27 , for administration in combination with a further therapeutic composition and/or irradiation.