Anton Simeonov Ph.D. National Institute of Health

Small Fusion Protein Creates Long-Acting Protein Therapeutics

Unlike small molecule drugs, where multiple analogues are typically synthesized during lead optimization to find the one with improved pharmacokinetic properties, large molecules are far less amenable to modifications. Many therapeutic proteins suffer from short plasma half-lives and, as a consequence, require frequent injections to be therapeutically effective, which in turn can adversely affect patient compliance and quality of life. Here, Peter Schultz's team provides a platform to produce antibody-fusion molecules with dramatically improved PK properties. As a starting point, the team used a previously discovered bovine antibody with a very long half-life in which the Ig domain and a disulfide cross-linked “knob domain” of heavy-chain complementarity-determining region 3 (CDR3H) were found to be spatially separated by a rigid, solvent-exposed, antiparallel β-strand stalk. The team also developed a humanized version of the antibody and showed that appending the rigid stalk motif with smaller-sized protein domains, including protein therapeutics, does not alter the overall PK properties of the antibody significantly. In one example, the authors coupled the long-lived bovine antibody to human growth hormone (hGH), a 22 kDa protein whose recombinant form has been used for the treatment of growth hormone deficiency but where treatment has required daily injection due to short half-life (Figure 1). The team then used the lessons learned from the analyses of the long-lived bovine antibody to design a fusion of hGH, along with the knob domain, with Herceptin, a therapeutic human antibody with good PK properties (Figure 2). In both hGH fusions, one with the bovine antibody and the other with the rigid stalk motif-modified Herceptin, dramatic lengthening of the hGH half-life was achieved along with retention of biological activity. Lastly, the same approach was applied to create improved-PK version of human leptin, representing another short-lived hormone with a central role in the homeostasis of body weight. The approach presented in the paper appears to be general enough to be transferred to other therapeutic proteins, with one remaining risk factor being the immunogenicity of these new fusions.

 (A) X-ray crystal structures of bovine antibody BLV1H12 Fab fragment [Protein Data Bank (PDB) ID code 4K3D] and human growth hormone (PDB ID code 1HGU). The N and C termini of the four-helix–bundle protein are circled in blue. (B) A schematic representation of hGH–BLV1H12 fusion. The knob domain of BLV1H12 is replaced by hGH. (C) SDS/PAGE analysis of hGH–BLV1H12 fusion. (D) hGH–BLV1H12 fusion protein stimulates proliferation of rat NB2-11 cells in a dose dependent manner. Cells were treated with various concentrations of hGH and hGH–BLV1H12 fusion proteins and cell viability was quantified using PrestoBlue reagent; assays were performed in triplicate. (E) hGH-dependent Ba/F3-hGHR cells proliferate in a dose-dependent manner upon treatment with hGH–BLV1H12. Cells were treated with various concentrations of hGH and hGH–BLV1H12 fusion proteins, and cell viability was quantified using PrestoBlue reagent; assays were performed in triplicate. RFU, relative fluorescence units. (F) hGH–BLV1H12 fusion protein stimulates STAT5 phosphorylation in IM9 cells. Serum-starved IM9 cells were treated with various concentrations of hGH and hGH–BLV1H12 for 10 min, and phosphorylation of STAT5 was quantified by flow cytometry analysis. Assays were performed in duplicate. Error bars represent the standard deviation.


Figure 1.

(A) X-ray crystal structure of humanized antibody Herceptin Fab fragment (PDB ID code 1N8Z). CDR loops used for hGH fusion are marked and highlighted in yellow, red, and green for CDR2H, CDR3H, and CDR3L, respectively. (B) A schematic representation of the hGH-CDR3H-coil-Herceptin fusion. The N and C termini of hGH are fused to Herceptin CDR3H with a coiled-coil linker (H2N-GGSGAKLAALKAKLAALK-COOH and H2N-ELAALEAELAALEAGGSG-COOH). CDR loops used for hGH fusion are highlighted in yellow, red, and green for CDR2H, CDR3H, and CDR3L, respectively. Fusions for CDR2H and CDR3L are assembled similarly onto the corresponding loops. (C) Pharmacokinetics of hGH-CDR3H-coil-Herceptin in rat by i.v. injection. A single dose (2 mg/kg) of Genotropin (rhGH) and hGH-CDR3H-coil-Herceptin was administered by i.v. injection into Sprague–Dawley rats (n=3). The estimated terminal half-lives after i.v. injection are 34 min and 47 h for Genotropin and hGH-coil-Herceptin, respectively. (D) Pharmacodynamic study of hGH-coil-Herceptin in the hypophysectomized rat model. Animals matched by initial weights and pretreatment growth rates were sorted and randomized to receive s.c. injections according to one of the following protocols: daily injection of vehicle or Genotropin (0.1 mg/kg=5 nmol/kg); a biweekly administration of vehicle, Herceptin (0.8 mg/kg=5 nmol/kg); or increasing doses of the hGH–Herceptin fusion (1 mg/kg=5 nmol/kg, 3.5 mg/kg=17.5 nmol/kg, 7 mg/kg=35 nmol/kg, and 10 mg/kg=50 nmol/kg). Error bars represent the standard deviation.


Figure 2.

* Abstract from PNAS 2015; Vol. 12:1356–1361

On the basis of the 3D structure of a bovine antibody with a well folded, ultralong complementarity-determining region (CDR), we have developed a versatile approach for generating human or humanized antibody agonists with excellent pharmacological properties. Using human growth hormone (hGH) and human leptin (hLeptin) as model proteins, we have demonstrated that functional human antibody CDR fusions can be efficiently engineered by grafting the native hormones into different CDRs of the humanized antibody Herceptin. The resulting Herceptin CDR fusion proteins were expressed in good yields in mammalian cells and retain comparable in vitro biological activity to the native hormones. Pharmacological studies in rodents indicated a 20- to 100-fold increase in plasma circulating half-life for these antibody agonists and significantly extended in vivo activities in the GH-deficient rat model and leptin-deficient obese mouse model for the hGH and hLeptin antibody fusions, respectively. These results illustrate the utility of antibody CDR fusions as a general and versatile strategy for generating long-acting protein therapeutics.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in PNAS titled  "Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists" authored by Liu T, Zhang Y, Liu Y, Wang Y, Jia H, Kang M, Luo X, Caballero D, Gonzalez J, Sherwood L, Nunez V, Wang D, Woods A, Schultz PG, Wang F.