Multifunctional Heteromultimeric Constructs

Abstract

The present invention is situated in the field of multimers used for targeted therapies. More particularly, the invention relates to methods for preparing multifunctional heteromultimeric protein complexes with a defined ratio of functional components and to multifunctional heteromultimeric protein complexes for directing complement-dependent cytolysis, optionally comprising a scaffold, which display three or more different functional components present in a defined relative ratio, of which one is a tracking component.

1 - 35 . (canceled) 
     
     
         36 . A multifunctional heteromultimeric protein complex, comprising three or more different functional components present in a defined relative ratio;
 wherein the three or more different functional components are each independently selected from a tracking component, a targeting component and an effector component and wherein said functional components comprise said tracking component, one or more of said targeting components and one or more of said effector components;   wherein said functional components are selected from the group comprising synthetic or natural peptides, polypeptides, proteins, peptide analogues, peptidomimetics, antibodies, antibody fragments and derivatives thereof;   wherein said multifunctional heteromultimeric protein complex comprises eight multimerizing scaffold polypeptides, of which seven are a C-terminal part of the alpha-chain of the C4bp multimer and one is a C-terminal part of the beta-chain of the C4bp multimer; and   wherein said tracking component is fused to said C-terminal part of the beta-chain of the C4bp multimer and said one or more targeting components and said one or more effector components are each fused to one or more of said C-terminal part of said alpha chain of the C4bp multimer.   
     
     
         37 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein said tracking component is a tag and a proteolytic cleavage site is present between the tag and the C-terminal part of the beta-chain of the C4bp multimer. 
     
     
         38 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein said tracking component is selected from a fluorescent dye, biotin for staining with labelled avidin or streptavidin conjugate, an enzyme, a substrate, a cofactor, a chemiluminescent group, a chromogenic agent and a colorimetric label or bead. 
     
     
         39 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein said functional components are located upstream and downstream of said C-terminal part of each of said alpha-chain of the C4bp multimer. 
     
     
         40 . The multifunctional heteromultimeric protein complex according to  claim 39  wherein said functional component located upstream of said C-terminal part of the alpha-chain of the C4bp multimer is a targeting component or an effector component and wherein said functional component located downstream of said C-terminal part of the alpha-chain of the C4bp multimer is an effector component. 
     
     
         41 . The multifunctional hetermultimeric protein complex according to  claim 36 , wherein one of the functional components is a monomeric Fc. 
     
     
         42 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein said one or more targeting components allow binding of the multifunctional heteromultimeric protein complex to a target cell and are selected from antibodies, binding fragments thereof, ligands to a target cell receptor and soluble receptors. 
     
     
         43 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein said one or more effector components are factors capable of modulating the complement system and/or factors capable of activating antibody-dependent cell-mediated cytotoxicity (ADCC). 
     
     
         44 . The multifunctional heteromultimeric protein complex according to  claim 43 , comprising at least one effector component which is a factor capable of modulating the complement system selected from factor H-related protein 4 (FHR4) and Properdin. 
     
     
         45 . The multifunctional heteromultimeric protein complex according to  claim 36 , wherein at least one of the targeting components is an antibody directed against a tumor antigen, against a surface marker of erythrocytes or against a pathogen-associated surface marker. 
     
     
         46 . The multifunctional heteromultimeric protein complex according to  claim 45 , wherein at least one of the targeting components is an antibody or fragment thereof directed against tumor-associated antigen HER2/neu or scFv anti-GYPA. 
     
     
         47 . The multifunctional hetermultimeric protein complex according to  claim 46 , wherein the effector component is a monomeric Fc, preferably monomeric Fc of IgG comprising the hinge and the CH2 and CH3 domain.