Engineered antibody constant domain molecules
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-9527903-B2
Publication Date
2016-12-27
Expiration Date
2029-01-30
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Abstract
Described herein are engineered antibody constant domain molecules, such as CH2 or CH3 domain molecules, comprising at least one mutation, or comprising at least one complementarity determining region (CDR), or a functional fragment thereof, engrafted in a loop region of the CH2 domain. The CH2 domain molecules described herein are small, stable, soluble, exhibit little to no toxicity and are capable of binding antigen.
Core Innovation
The invention concerns engineered antibody constant domain molecules, specifically small, stable, and soluble CH2 or CH3 domain molecules derived from immunoglobulin constant domains that are mutated or engrafted with complementarity determining regions (CDRs) from heterologous immunoglobulin variable domains. These engineered domains are capable of binding antigen with little to no toxicity and have molecular weights less than about 15 kD.
The problem being addressed relates to the limitations of conventional antibodies and their fragments in therapeutic and diagnostic applications. Traditional antibodies are large, restricting tissue penetration and epitope access. Smaller fragments like Fab or single chain variable fragments (scFv) are still relatively large. A need exists for very small antigen-binding molecules that maintain specificity and stability while offering better tissue penetration and epitope accessibility.
The invention provides engineered CH2 or CH3 domains from IgG, IgA, IgD, IgE, or IgM immunoglobulins wherein loop regions are mutated and/or replaced with CDRs or functional fragments thereof from heterologous immunoglobulin variable domains to confer antigen binding. These engineered domains may also have mutations to increase stability, such as introducing disulfide bonds, and may have N-terminal and/or C-terminal truncations. Methods for identifying, generating libraries, expressing, and using these engineered domains for specific antigen binding are provided.
Claims Coverage
The patent includes several independent claims focusing on isolated human immunoglobulin CH2 domains of IgG with specific truncations and mutations that increase stability through disulfide bond formation.
Human immunoglobulin CH2 domain with terminal truncations
An isolated human IgG CH2 domain comprising an N-terminal truncation of seven amino acids and a C-terminal truncation of one to four amino acids, resulting in a molecule under about 15 kD.
Engineered disulfide bond formation to increase stability
A human IgG CH2 domain with an N-terminal truncation of seven amino acids and two amino acid substitutions—either L12 to C12 or V10 to C10 in the N-terminal A strand, and K104 to C104 in the C-terminal G strand—where the introduced cysteines form a disulfide bond enhancing domain stability.
Combination of truncations and cysteine mutations in polypeptides
Polypeptides comprising the human IgG CH2 domain with the described truncations and cysteine substitutions conferring a molecular weight less than about 15 kD and increased stability through a disulfide bond.
The independent claims cover isolated human IgG CH2 domains with specific N- and C-terminal truncations and cysteine substitutions in the A and G strands to form stabilizing disulfide bonds resulting in small, stable antibody constant domain molecules under about 15 kD.
Stated Advantages
The CH2 and CH3 domain molecules are small, allowing greater epitope access and better tissue penetration compared to larger antibodies or their fragments.
These engineered domains are stable, soluble, and exhibit little to no toxicity.
The engineered domains can specifically bind antigen, enabling their use in diagnostics and therapeutics.
Mutations such as engineered disulfide bonds increase stability and improve folding and solubility.
The molecules can be used as scaffolds for further modification to generate libraries of antigen-binding molecules with diverse specificity.
Documented Applications
Variable domain substitute molecules for diagnostic and therapeutic use, including treatment of HIV infection, cancer, autoimmune and inflammatory disorders.
Use as antigen-binding molecules with improved tissue penetration and epitope access for any diagnostic or therapeutic application involving antibodies or their fragments.
Creation of phage display libraries for identifying antigen-specific binder molecules.
Conjugation to effector molecules such as toxins, enzymes, or detectable labels for targeted therapeutic or diagnostic applications.
Use in neutralizing HIV-1 through engineered CH2 molecules binding HIV envelope glycoproteins.
Use in detection methods including immunoassays, fluorescence activated cell sorting, and cell isolation techniques.
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