Soluble engineered monomeric Fc
Inventors
Dimitrov, Dimiter S. • Ying, Tianlei
Assignees
US Department of Health and Human Services
Publication Number
US-9676857-B2
Publication Date
2017-06-13
Expiration Date
2033-03-14
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Abstract
Fc domains and CH3 domains are disclosed that bind the neonatal Fc (FcRn) receptor and are at least 99% monomeric. Monomeric Fc domain molecules and CH3 domain molecules are disclosed herein that include a monomeric Fc domain or a monomeric CH3 domain and an effector molecule. In some embodiments, the monomeric Fc or monomeric CH3 domain include amino acid substitutions and/or CDR insertions in the beta strands such that they specifically bind an antigen. Methods for using these monomeric Fc domains, monomeric CH3 domains, monomeric Fc domain molecules and CH3 domain molecules are also provided.
Core Innovation
The invention discloses Fc domains and CH3 domains that are at least 99% monomeric and bind the neonatal Fc receptor (FcRn). These monomeric Fc domains include mutations in the wild-type CH3 domain and optionally include amino acid substitutions or complementarity determining region (CDR) insertions in the beta strands to confer specific antigen binding capabilities. The monomeric Fc domains and CH3 domains can be fused to heterologous proteins such as variable domains, cytokines, or toxins to form fusion proteins, and nucleic acid sequences encoding these are also provided.
The problem being solved is that full-size antibodies have poor tissue penetration, particularly in solid tumors, and limited access to certain antigenic surfaces due to steric hindrance. Smaller antibody fragments developed previously have limited therapeutic use due to their reduced half-lives compared to full-size IgGs. There is a need for molecules that are small, specifically bind antigens, and retain a long serum half-life.
The invention addresses this need by engineering monomeric Fc domains approximately 27 kD in size that retain pH-dependent FcRn binding, enabling extended half-life while improving tissue penetration and stability. These monomeric Fc domains serve as scaffolds suitable for antigen-binding molecule construction through mutations and CDR grafting, overcoming previous limitations of antibody fragments.
Claims Coverage
The patent presents eight main inventive features within its independent claims related to isolated monomeric Fc polypeptides, isolated CH3 domains, and fusion proteins incorporating these domains.
Isolated monomeric Fc polypeptides with specified sequences
An isolated monomeric Fc polypeptide comprising CH2 and CH3 domains, wherein the Fc comprises one of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 45, 46, 47, or 48.
Isolated monomeric Fc polypeptides consisting of specified sequences
An isolated monomeric Fc polypeptide consisting of one of the amino acid sequences set forth in SEQ ID NOs: 3, 4, 5, 6, 45, 46, 47, or 48.
Isolated CH3 domain segments from monomeric Fc polypeptides
An isolated CH3 domain comprising amino acids 113-217 of the sequences set forth as SEQ ID NOs: 3, 4, 5, 6, 45, 46, 47, or 48.
Fusion proteins comprising CH3 domain and heterologous proteins
An isolated fusion protein comprising the isolated CH3 domain and a heterologous protein.
Fusion proteins with heavy and light chain variable domains binding antigens
The fusion protein heterologous protein comprises a heavy chain variable domain and a light chain variable domain specifically binding an antigen.
Fusion proteins targeting pathogen antigens
The heterologous protein is an antigen from a pathogen, such as a virus or bacterium.
Fusion proteins targeting HIV virus
The pathogen is human immunodeficiency virus (HIV).
Fusion proteins comprising cancer antigens or toxins or cytokines
The fusion protein contains a cancer antigen, toxin, or proteins like cytokines, soluble receptors, growth factors, human interferon, erythropoietin, soluble TNF receptor, CTLA-4, IL-4 receptor, Factor IX, or labels as the heterologous protein.
The claims cover isolated monomeric Fc polypeptides defined by specific amino acid sequences, isolated CH3 domains derived from such polypeptides, and their fusion proteins with various heterologous proteins, including antigen binding domains, pathogen antigens, toxins, cytokines, and cancer antigens, highlighting the invention of stable monomeric Fc formats with therapeutic and diagnostic fusion capabilities.
Stated Advantages
The monomeric Fc domains are small in size (~27 kD), stable, soluble, and maintain specific binding to FcRn, imparting an extended half-life comparable to full-size IgG antibodies.
Monomeric Fc domains can be efficiently produced in bacteria with high yields.
Fusion proteins using monomeric Fc domains enable smaller molecular weight therapeutics with prolonged half-life and improved tissue penetration compared to full-size antibodies or dimeric Fc fusions.
The exposed surface in monomeric Fc domains allows for engineering antigen binding through point mutations and CDR grafting without requiring variable domains.
The monomeric Fc domains retain pH-dependent FcRn binding, ensuring preservation of IgG-like recycling and stability in vivo.
Documented Applications
Therapeutic use in treating infections caused by pathogens including viruses (e.g., HIV), bacteria, fungi, and parasites.
Treatment of various cancers, including solid tumors (sarcoma, carcinoma) and hematologic malignancies (leukemia, lymphoma, multiple myeloma).
Treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, Crohn's disease, psoriasis, type 1 diabetes, and systemic lupus erythematosus.
Use in diagnostic applications for detecting antigens by employing antigen binding monomeric Fc or CH3 domain molecules in immunoassays like ELISA, Western blot, FACS, and immunohistochemistry.
Use as scaffolds to generate libraries of antigen binding monomeric Fc domains or CH3 domains for screening diverse antigen specific binders.
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