Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Abstract

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted antimyostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

Structural basis for potency differences between GDF8 and GDF11

“…This conformation shares a similar exaggerated “open” conformation to the crystal structure of GDF8 bound to a neutralizing antibody (Fig. 5a, bottom) [61]. However, the wrist region in our apo-GDF11 structure is also disordered and not resolved in the crystal structure, similar to apo-GDF8 (Fig.…”

Tolloid cleavage activates latent GDF8 by priming the pro‐complex for dissociation

“…The apo form (pdb: 5JI1) (Walker et al , ) adopts an open conformation (N). In contrast, the GDF8 growth factor adopts a closed conformation when in complex with the antagonist FSL3 (O) (pdb: 3sek) (Cash et al , ) and yet another conformation when bound to Fab (P) (pdb: 5f3b) (Apgar et al , ). Source data are available online for this figure.…”

“…If the GF in the pro‐activin A complex had a closed conformation, as seen in some activin GF complex structures with inhibitors and receptors, the pro‐complex would have to assume a markedly more obtuse V‐angle (Hinck et al , ; Wang et al , ). Notably, the apo GDF8 GF dimer structure adopts an open GF conformation (Fig N; Walker et al , ) with a more acute V‐angle than in the pro‐complex (Fig M), whereas crystal structures of the GDF8 GF in complex with antagonists reveal a closed conformation and a complex with Fab reveals yet another conformation (Fig O and P; Apgar et al , ; Hinck et al , ). Our HDX results showed no changes in GDF8 GF regions that alter in conformation between the open and closed GF conformations, including the GF α3′‐helix (Hinck et al , ), and thus provided no evidence for a change in overall GF conformation associated with TLL2 cleavage.…”

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Domagrozumab (PF‐06252616), an Antimyostatin Monoclonal Antibody, in Healthy Subjects

“…The peak and trough steady‐state exposures of MYO‐029 in patients at biweekly intravenous doses of 10 mg/kg MYO‐029 were predicted to achieve only 50% and 10% of the maximum effect seen in monkeys, respectively . Domagrozumab differentiates from MYO‐029 in that, compared to MYO‐029, domagrozumab demonstrated a higher binding affinity to myostatin (∼4‐fold lower binding affinity by Biacore), and, importantly, its mouse surrogate mRK35 significantly increased the maximum tetanic force, cross‐sectional area, and mass of the extensor digitorum longus muscle in mice, whereas MYO‐029 did not increase any of these parameters . Also, this first‐in‐human study provides evidence of target engagement by domagrozumab, not seen previously with MYO‐029.…”

“…Domagrozumab (PF‐06252616) is a humanized IgG1 monoclonal antibody that is able to neutralize myostatin by binding to the mature myostatin dimer to block the interaction with activin‐type IIB receptor, thereby inhibiting the initiation of the Smad2/3 signaling pathway . This article discusses the findings from the first‐in‐human study of domagrozumab (ClinicalTrials.gov identifier # NCT01616277).…”