Conditionally Active Biological Proteins

Abstract

This disclosure relates to a method of generating conditionally active biologic proteins from wild type proteins, in particular therapeutic proteins, which are reversibly or irreversibly inactivated at some physiological conditions. For example, conditionally active biologic proteins are active in tumors, but virtually inactive at other body parts, or conditionally active antibodies capable of crossing blood-brain-barrier.

What is claimed is: 
     
         1 . A method of preparing a conditionally active antibody for crossing the blood-brain barrier, the method comprising the steps of:
 i. evolving DNA which encodes a parent antibody against a blood-brain barrier receptor using one or more evolutionary techniques to create mutant DNAs;   ii. expressing the mutant DNAs to obtain mutant antibodies;   iii. subjecting the mutant antibodies to an assay under a first physiological condition in blood plasma and to an assay under a second physiological condition in brain extracellular fluid; and   iv. selecting the conditionally active antibody from the mutant antibodies which exhibit both (a) an affinity to the blood-brain barrier receptor in the assay under the first physiological condition, and (b) an affinity selected from the group consisting of a decreased affinity to the blood-brain barrier receptor in the assay under the second physiological condition in comparison with the affinity in the assay under the first physiological condition and no affinity to the blood-brain barrier receptor in the assay under the second physiological condition.   
     
     
         2 . The method of  claim 1 , further comprising the step of conjugating the conditionally active antibody to a molecule. 
     
     
         3 . The method of  claim 2 , wherein the conjugating step comprises forming a covalent bond between the conditionally active antibody and the molecule. 
     
     
         4 . The method of  claim 2 , wherein the conjugating step comprises forming a non-covalent bond between the conditionally active antibody and the molecule. 
     
     
         5 . The method of  claim 2 , wherein the molecule is selected from the group consisting of cytokines, interleukins, enzymes, hormones, growth factors, cytotoxic agents, chemotherapy drugs, radioactive particles, antibodies and diagnostic agents. 
     
     
         6 . The method of  claim 2 , wherein the molecule is conjugated to the Fc region of the conditionally active antibody. 
     
     
         7 . The method of  claim 1 , further comprising the step of introducing at least one amino acid substitution in the Fc region of the conditionally active antibody. 
     
     
         8 . The method of  claim 7 , wherein the at least one amino acid substitution is two or more amino acid substitutions. 
     
     
         9 . The method of  claim 1 , further comprising the step of engineering the conditionally active antibody to be multispecific. 
     
     
         10 . The method of  claim 9 , wherein the selecting step selects the conditionally active antibody which also exhibits an affinity to an antigen in addition to an affinity for the blood-brain barrier receptor. 
     
     
         11 . The method of  claim 1 , wherein the blood-brain barrier receptor is selected from the group consisting of a transferrin receptor, an insulin receptor, an insulin-like growth factor receptor, low density lipoprotein receptor-related protein 1, low density lipoprotein receptor-related protein 8, and a heparin-binding epidermal growth factor-like growth factor. 
     
     
         12 . The method of  claim 1 , wherein in the affinity for the blood-brain barrier receptor in the assay under the first physiological condition is measured by the conditionally active antibody's IC50 for inhibiting binding of the blood-brain barrier receptor's natural ligand, and the IC50 of the conditionally active antibody is from about 1 nM to about 100 μM. 
     
     
         13 . The method of  claim 1 , wherein the evolving step comprises mutating a Fc region of the antibody. 
     
     
         14 . The method of  claim 1 , wherein the evolving step comprises a technique selected from PCR, error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis, gene reassembly, gene site saturated mutagenesis, ligase chain reaction, in vitro mutagenesis, ligase chain reaction, oligonucleotide synthesis, and combinations thereof. 
     
     
         15 . The method of  claim 1 , wherein the expression step comprises expressing the mutant DNA in a host cell selected from a bacterial cell, a fungal cell, an insect cell, a mammalian cell, adenoviruses, and a plant cell. 
     
     
         16 . The method of  claim 15 , wherein the host cell is a mammalian cell selected from a Bowes melanoma cell, a COS-7 cell, a C127 cell, a 3T3 cell, a CHO cell, a HeLa cell and a BHK cell. 
     
     
         17 . A conditionally active antibody prepared by the method of  claim 1 , wherein the conditionally active antibody is reversibly inactivated under the second physiological condition. 
     
     
         18 . The conditionally active antibody of  claim 17 , wherein the conditionally active antibody is conjugated to a molecule that is released under the second physiological condition.