Compositions and methods for growth factor modulation
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. A method of making a pharmaceutical composition comprising an antibody, or an antigen-binding fragment thereof, that binds to pro/latent GDF8 and inhibits GDF8 signaling, the method comprising the steps of:
(i) providing a panel of antibodies, or antigen-binding fragments thereof;
(ii) screening for an antibody, or an antigen-binding fragment thereof, that binds to pro/latent GDF8 but does not bind to mature GDF8, wherein pro/latent GDF8 comprises a complex comprising a latency associated peptide (LAP) domain associated with mature GDF8;
(iii) screening the antibody, or the antigen-binding fragment thereof, that binds to pro/latent GDF8 but does not bind to mature GDF8 in step (ii) for inhibitors of GDF8 signaling; and
(iv) formulating the antibody, or the antigen-binding fragment thereof, that inhibits GDF8 signaling in step (iii) into a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.
2. The method of claim 1 , wherein the panel of antibodies is an antibody library.
3. The method of claim 2 , wherein the antibody library is a phage display library.
4. The method of claim 1 , further comprising a step of subjecting the antibody, or the antigen-binding fragment thereof, to humanization and/or affinity maturation.
5. The method of claim 1 , wherein step (iii) comprises screening for an antibody, or an antigen-binding fragment thereof, that inhibits release of mature GDF8 growth factor from the pro/latent GDF8.
6. The method of claim 1 , wherein step (ii) comprises conducting a negative selection to exclude an antibody, or an antigen-binding fragment thereof, that binds to an antigen selected from GDF8 prodomain, mature GDF8 growth factor, murine proGDF8, proGDF11, proTGFβ1, and a protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 207-230.
7. The method of claim 1 , wherein step (ii) comprises a binding assay selected from an enzyme-linked immunosorbent assay, a surface plasmon resonance assay, and a flow cytometry assay.
8. The method of claim 1 , further comprising a step of introducing at least one amino acid substitution to the antibody, or the antigen-binding fragment thereof, wherein the antibody, or the antigen-binding fragment thereof, comprises at least one CDR amino acid sequence with at least one amino acid deletion, addition, or substitution relative to a corresponding CDR amino acid sequence in the antibody or the antigen-binding fragment resulting from step (ii) or step (iii).
9. The method of claim 1 , wherein the antibody, or the antigen-binding fragment thereof, is an isotype selected from IgG1, IgG2, IgG3, IgG4, IgA, IgA2, IgD, IgE, or IgM.
10. The method of claim 1 , further comprising a step of incorporating a second antibody or antigen-binding fragment thereof, wherein the resultant antibody, or the antigen-binding fragment thereof, is a bispecific antibody, or an antigen-binding fragment thereof.
11. The method of claim 1 , further comprising a step of immunizing a host with an antigen comprising the pro/latent GDF8.
12. The method of claim 1 , wherein the antibody, or the antigen-binding fragment thereof, is a fully human or humanized antibody, or an antigen-binding fragment thereof.
13. The method of claim 1 , wherein the antibody, or the antigen-binding fragment thereof, is a stabilizing antibody, or an antigen-binding fragment thereof.
14. The method of claim 1 , wherein the antibody, or the antigen-binding fragment thereof, inhibits GDF8 signaling in step (iii) by:
inhibiting cleavage of a GDF8 prodomain;
inhibiting release of mature GDF8 growth factor from pro/latent GDF8; and/or
inhibiting activation of pro/latent GDF8 to mature GDF8.
15. The method of claim 14 , wherein the cleavage, release, and/or activation comprises cleavage of a GDF8 prodomain by one or more enzymes.
16. The method of claim 15 , wherein the one or more enzymes comprise a BMP/tolloid proteinase.
17. The method of claim 15 , wherein the one or more enzymes are selected from the group consisting of: furin, bone morphogenetic protein-1 (BMP-1), mammalian tolloid protein (mTLD), mammalian tolloid-like 1 (mTLL1), and mammalian tolloid-like 2 (mTLL2).
18. The method of claim 15 , wherein the activation comprises sequential enzymatic cleavage with at least two enzymes.
19. The method of claim 15 , wherein the cleavage of the GDF8 prodomain comprises BMP/tolloid proteolytic cleavage between residues Arg 75 and Asp 76 of the GDF8 prodomain.
20. The method of claim 1 , wherein step (iii) comprises identifying an antibody, or an antigen-binding fragment thereof, that inhibits BMP/tolloid proteolytic cleavage between residues Arg 75 and Asp 76 of a GDF8 prodomain.
21. The method of claim 1 , further comprising the steps of contacting the antibody, or the antigen-binding fragment thereof, resulting from step (ii) or step (iii) with pro/latent GDF8 and a BMP/tolloid proteinase; and confirming that the antibody, or the antigen-binding fragment thereof, blocks cleavage of the pro/latent GDF8 by the BMP/tolloid proteinase.
22. The method of claim 21 , wherein the BMP/tolloid proteinase is selected from the group consisting of: furin, bone morphogenetic protein-1 (BMP-1), mammalian tolloid protein (mTLD), mammalian tolloid-like 1 (mTLL1), and mammalian tolloid-like 2 (mTLL2).
23. A method of screening for an antibody, or an antigen-binding fragment thereof, that binds to pro/latent GDF8 and inhibits GDF8 signaling, the method comprising the steps of:
(i) providing a panel of antibodies;
(ii) screening for an antibody, or an antigen-binding fragment thereof, that binds to pro/latent GDF8 but does not bind to mature GDF8, wherein pro/latent GDF8 comprises a complex comprising a latency associated peptide (LAP) domain associated with mature GDF8; and
(iii) screening the antibody, or the antigen-binding fragment thereof, that binds to pro/latent GDF8 but does not bind to mature GDF8 in step (ii) for inhibition of GDF8 signaling, wherein the antibody, or the antigen-binding fragment thereof, inhibits GDF8 signaling by:
inhibiting cleavage of a GDF8 prodomain;
inhibiting release of mature GDF8 growth factor from pro/latent GDF8; and/or
inhibiting activation of pro/latent GDF8 to mature GDF8.
24. The method of claim 23 , further comprising a step of formulating the antibody, or the antigen-binding fragment thereof, that inhibits GDF8 signaling in step (iii) into a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.