Methods for making and using anti-myostatin antibodies
Aspects of the present disclosure relate to antibodies that specifically bind proMyostatin and/or latent Myostatin and uses thereof.
1. A method of making a pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin, binds to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH, and inhibits myostatin signaling after binding, the method comprising the steps of:
(i) providing a panel of antibodies or antigen-binding fragments thereof;
(ii) screening for an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin as compared to mature myostatin and/or another member of the transforming growth factor beta family;
(iii) assaying the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin for binding to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH;
(iv) screening the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin for inhibition of myostatin signaling; and
(v) formulating the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin, binds to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH, and inhibits myostatin signaling from steps (ii)-(iv) into a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient,
wherein, optionally, the method further comprises modifying the antibody or antigen-binding fragment with at least one amino acid deletion, addition, or substitution.
2. The method of claim 1 , wherein the antibody or antigen-binding fragment is a fully human antibody or a humanized antibody.
3. The method of claim 1 , wherein the antibody or antigen-binding fragment is a diabody, a chimeric antibody, a Fab fragment, a F(ab′)2 fragment, or an Fv fragment.
4. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises one or more framework regions having a human germline sequence.
5. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises at least one complementarity determining region (CDR) or framework region amino acid sequence having at least one amino acid deletion, addition, or substitution relative to a corresponding CDR or framework region amino acid sequence in the antibody or antigen-binding fragment resulting from any one of steps (ii)-(iv).
6. The method of claim 1 , wherein modifying the antibody or antigen-binding fragment comprises humanizing the antibody or antigen-binding fragment.
7. The method of claim 1 , wherein the antibody or antigen-binding fragment is modified in between any one of steps (ii)-(iv).
8. The method of claim 1 , wherein the antibody or antigen-binding fragment comprises a heavy chain constant domain selected from an IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE.
9. The method of claim 1 , wherein the antibody or antigen-binding fragment binds to pro/latent myostatin with at least 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1,000-fold higher affinity than to mature myostatin and/or another member of the transforming growth factor beta family.
10. The method of claim 1 , wherein the antibody or antigen-binding fragment is a sweeping antibody, or antigen-binding fragment thereof.
11. The method of claim 1 , wherein the acidic pH ranges from 4.0 to 6.5 and the neutral or physiological pH ranges from 7.0 to 7.4.
12. The method of claim 11 , wherein the antibody or antigen-binding fragment binds to pro/latent myostatin at a pH ranging from 4.0 to 6.5 with an equilibrium dissociation constant (Kd) that is at least 2-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold greater than at a pH ranging from 7.0 to 7.4.
13. The method of claim 1 , wherein the antibody or antigen-binding fragment inhibits myostatin signaling by:
inhibiting cleavage of a myostatin prodomain;
inhibiting release of mature myostatin growth factor from pro/latent
myostatin; and/or
inhibiting activation of pro/latent myostatin to mature myostatin.
14. The method of claim 13 , wherein the cleavage, release, and/or activation comprises BMP/tolloid proteolytic cleavage between residues Arg 75 and Asp 76 of the myostatin prodomain.
15. The method of claim 13 , wherein the antibody or antigen-binding fragment inhibits proteolytic activation of pro/latent myostatin to mature myostatin by a BMP/tolloid protease.
16. The method of claim 15 , wherein the antibody or antigen-binding fragment inhibits proteolytic activation with an IC50 of less than 1 μM.
17. The method of claim 1 , wherein screening the antibody or antigen-binding fragment for inhibition of myostatin signaling comprises contacting the antibody or antigen-binding fragment with pro/latent myostatin and a BMP/tolloid protease; and confirming that the antibody or antigen-binding fragment blocks cleavage of the pro/latent myostatin by the BMP/tolloid protease.
18. The method of claim 17 , wherein the BMP/tolloid protease is furin, bone morphogenetic protein-1 (BMP-1), mammalian tolloid protein (mTLD), mammalian tolloid-like 1 (mTLL 1), or mammalian tolloid-like 2 (mTLL2).
19. A method of screening for an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin, binds to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH, and inhibits myostatin signaling after binding, the method comprising the steps of:
(i) providing a panel of antibodies or antigen-binding fragments thereof;
(ii) screening for an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin as compared to mature myostatin and/or another member of the transforming growth factor beta family;
(iii) assaying the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin for binding to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH; and
(iv) screening the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin for inhibition of myostatin signaling,
wherein, optionally, the method further comprises modifying the antibody or antigen-binding fragment with at least one amino acid deletion, addition, or substitution.
20. The method of claim 19 , further comprising a step of formulating the antibody or antigen-binding fragment into a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.
21. A method of screening for an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin and competes for binding to pro/latent myostatin with a reference antibody, the method comprising the steps of:
(i) providing a panel of antibodies or antigen-binding fragments thereof;
(ii) screening for an antibody, or antigen-binding fragment thereof, that specifically binds to pro/latent myostatin as compared to mature myostatin and/or another member of the transforming growth factor beta family; and
(iii) screening the antibody or antigen-binding fragment that specifically binds to pro/latent myostatin for competition with a reference antibody for binding to pro/latent myostatin, wherein the reference antibody comprises three heavy chain CDRs comprising amino acid sequences of SEQ ID NO: 1 (CDRH1), SEQ ID NO: 4 (CDRH2), and SEQ ID NO: 10 (CDRH3), and three light chain CDRs comprising amino acid sequences of SEQ ID NO: 12 (CDRL1), SEQ ID NO: 18 (CDRL2), and SEQ ID NO: 22 (CDRL3), as defined by the Kabat numbering system; or three heavy chain CDRs comprising amino acid sequences of SEQ ID NO: 2 (CDRH1), SEQ ID NO: 5 (CDRH2), and SEQ ID NO: 10 (CDRH3), and three light chain CDRs comprising amino acid sequences of SEQ ID NO: 13 (CDRL1), SEQ ID NO: 19 (CDRL2), and SEQ ID NO: 22 (CDRL3), as defined by the IMGT numbering system.
22. The method of claim 21 , further comprising a step of screening the antibody or antigen-binding fragment that competes for binding to pro/latent myostatin with a reference antibody for inhibition of myostatin signaling.
23. The method of claim 22 , further comprising a step of formulating the antibody or antigen-binding fragment that inhibits myostatin signaling into a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.
24. The method of claim 22 , wherein the inhibition of myostatin signaling is:
inhibiting cleavage of a myostatin prodomain;
inhibiting release of mature myostatin growth factor from pro/latent myostatin; and/or
inhibiting activation of pro/latent myostatin to mature myostatin.
25. The method of claim 22 , wherein the inhibition of myostatin signaling is inhibition of proteolytic activation of pro/latent myostatin to mature myostatin by a BMP/tolloid protease.
26. The method of claim 21 , further comprising a step of screening the antibody or antigen-binding fragment resulting from step (ii) or step (iii) for binding to pro/latent myostatin with a decreased affinity at an acidic pH as compared to a neutral or physiological pH.