Compositions and methods for growth factor modulation

Abstract

Provided herein are proteins, antibodies, assays and methods useful for modulating growth factor levels and/or activities. In some embodiments, such growth factors are members of the TGF-β superfamily of proteins.

1 . An antibody produced by a method comprising the steps of:
 i. expressing proGDF-8 (SEQ ID NO: 5);   ii. forming a growth factor prodomain complex (GPC) by subjecting the expressed proGDF-8 (SEQ ID NO: 5) to enzymatic cleavage with one or more of furin, bone morphogenetic protein-1 (BMP-1), mammalian tolloid protein (mTLD), mammalian tolloid-like 1 (mTLL1), and mammalian tolloid-like 2 (mTLL2);   iii. carrying out solid-phase or solution-phase enrichment with an antibody fragment phage display library, wherein the GPC formed by enzymatic cleavage is used as a target antigen;   iv. selecting phage particles bound to the GPC formed by enzymatic cleavage; and   v. producing recombinant antibodies having complementarity determining region (CDR) amino acid sequences derived from the antibody fragments expressed at the surface of the selected phage particles.   
     
     
         2 . The antibody of  claim 1 , wherein said antibody is human or humanized. 
     
     
         3 . The antibody of  claim 1 , wherein said antibody is subjected to affinity maturation. 
     
     
         4 . The antibody of  claim 1 , wherein said method further comprises:
 vi. screening the recombinant antibodies and selecting those which inhibit release of GDF-8 growth factor from GDF-8 GPCs.   
     
     
         5 . The antibody of  claim 4 , wherein antibodies that inhibit GDF-8 activity are selected. 
     
     
         6 . The antibody of  claim 1 , wherein said method further comprises:
 vi. conducting a negative selection binding assay to exclude antibodies that bind to one or more undesired antigens.   
     
     
         7 . The antibody of  claim 6 , wherein said one or more undesired antigens are selected from the group consisting of GDF-8 prodomain, GDF-8 growth factor, murine proGDF-8, proGDF-11, proTGF-β1, and a protein with an amino acid sequence selected from the group consisting of SEQ ID NOs: 207-230. 
     
     
         8 . The antibody of  claim 7 , wherein said antibody is human or humanized. 
     
     
         9 . The antibody of  claim 7 , wherein said antibody is subjected to affinity maturation. 
     
     
         10 . The antibody of  claim 1 , wherein phage particle binding is determined using one or more binding assays selected from the group consisting of enzyme-linked immunosorbent assays, surface plasmon resonance assays, and flow cytometry assays. 
     
     
         11 . The antibody of  claim 1 , wherein said enzymatic cleavage comprises sequential enzymatic cleavage with at least two enzymes. 
     
     
         12 . The antibody of  claim 11 , wherein said antibody is human or humanized. 
     
     
         13 . The antibody of  claim 11 , wherein said antibody is subjected to affinity maturation. 
     
     
         14 . The antibody of  claim 1 , wherein at least one CDR amino acid sequence has at least one amino acid deletion, addition, or substitution relative to an amino acid sequence of said antibody fragments expressed at the surface of the selected phage particles. 
     
     
         15 . The antibody of  claim 1  comprising an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgGA2, IgD, IgE, and IgM. 
     
     
         16 . The antibody of  claim 1  comprising a bispecific antibody.