Bispecific binding agents for modulating biological activity

Abstract

Methods for improving the biological and pharmaceutical properties of bispecific binding agents are described herein where the bispecific binding agents are able to target cells by a high affinity binding domain to a first cell surface marker that does not induce a significant biological effect and a low affinity binding domain that binds specifically to a second cell surface marker, causing a significant and desired biological effect. Compositions of such bispecific binding agents, uses for them, and kits containing them are also provided.

What is claimed is: 
     
         1 . A method of use of a bispecific binding agent in the manufacture of a medicament, the method comprising formulating in a pharmaceutically acceptable carrier a bispecific binding agent having a first and a second binding domain, which agent is capable of modulating biological activity of target cancer cells that have a first and a second cell surface marker on their exterior surfaces, wherein
 (i) the first and second cell surface markers do not share a common natural ligand;   (ii) the first cell surface marker is selected from selected from the group consisting of carcinoembryonic antigen (CEA), ErbB2, EGFR, Lewis Y , MUC-1, EpCAM, CAl25, prostate specific membrane antigen (PSMA), and TAG72;   (iii) the second cell surface marker is a member of the Tumor Necrosis Factor (TNF) receptor superfamily; and,   (iv) the first a binding domain is a high affinity antibody or ligand with a dissociation constant (Kd) for the first cell surface marker of 10 −7  M or less, and the second binding domain is a low affinity antibody or ligand that binds to the TNF receptor superfamily member with a Kd for the TNF receptor superfamily member that is at least 10 times greater than the Kd of the first binding domain for the first cell surface marker;   
       wherein the binding of the first binding domain to the first cell surface marker does not modulate biological activity of the first cell surface marker, but the binding of the second binding domain to the TNF receptor superfamily member modulates biological activity of the TNF receptor superfamily member; and, 
       wherein the modulation of biological activity evokes an observable difference in cell proliferation, cell viability, apoptosis, activation of downstream kinases, transcriptional activation, adhesion to surfaces, or ability to grow colonies in soft agar. 
     
     
         2 . The method of  claim 1  wherein the first binding domain is a single chain Fv or a disulfide stabilized Fv and is directly connected to the second binding domain. 
     
     
         3 . The method of  claim 1  wherein the first binding domain is a single chain Fv or a disulfide stabilized Fv and is connected to the second binding domain via a linker. 
     
     
         4 . The method of  claim 1  wherein the first binding domain is an antibody that is an IgM, IgG, IgD, IgA or IgE, or an antibody fragment that retains antigen recognition and binding capability that is an Fab, Fab′, F(ab′) 2 , or Fv fragment, and is connected to the second binding domain directly or by a linker. 
     
     
         5 . The method of  claim 1  wherein the binding domains are comprised of a covalently linked hetero-tetramer. 
     
     
         6 . The method of  claim 1  wherein the ligand is a natural ligand. 
     
     
         7 . The method of  claim 1  wherein the ligand is a fragment of a natural ligand. 
     
     
         8 . The method of  claim 1  wherein the modulation of the biological activity of the TNF receptor superfamily member induces apoptosis of the target cell. 
     
     
         9 . The method of  claim 1  wherein the first cell surface marker is carcinoembryonic antigen (CEA). 
     
     
         10 . The method of  claim 1  wherein the first cell surface marker is EpCAM. 
     
     
         11 . The method of  claim 1  wherein the second cell surface marker is a TNF receptor superfamily member selected from p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor. 
     
     
         12 . The method of  claim 1  wherein the Kd of the first binding domain for the first cell surface marker is between 10 −8 M and 10 −12  M. 
     
     
         13 . The method of  claim 1  wherein the Kd of the second binding domain to the second cell surface marker is at least 20 times greater than the Kd of the first binding domain to the first cell surface marker. 
     
     
         14 . The method of  claim 1  wherein the Kd of the second binding domain for the second cell surface marker is at least 50 times greater than the Kd of the first binding domain for the first cell surface marker. 
     
     
         15 . The method of  claim 1  wherein the Kd for the second cell surface marker that is at least 100 times greater than the Kd of the first binding domain for the first cell surface marker. 
     
     
         16 . The method of  claim 1  wherein the patient is a human patient and the target cell is a cell of culture of human cells. 
     
     
         17 . The method of  claim 1  wherein the bispecific binding agent is formulated for injection. 
     
     
         18 . The method of  claim 1  wherein the target cells are cancer cells. 
     
     
         19 . The method of  claim 18  wherein the cancer cells are breast, bladder, lung, endometrium, salivary gland, kidney, colon or stomach carcinoma cells. 
     
     
         20 . The method of  claim 18  wherein the cancer cells are ovarian or pancreatic cancer cells.