Engineered antibody constant domain molecules
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-8580927-B2
Publication Date
2013-11-12
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
This invention describes engineered antibody constant domain molecules comprising a CH2 or CH3 domain from immunoglobulins such as IgG, IgA, IgD, (for CH2) or IgE, IgM (for CH3). These domain molecules have been modified by introducing at least one mutation and/or by engrafting complementarity determining regions (CDRs) or their functional fragments from heterologous immunoglobulin variable domains into the loop regions of the CH2 or CH3 domains. The resulting molecules are small (less than about 15 kD), stable, soluble, exhibit little to no toxicity, and are capable of specifically binding to antigens.
The problem addressed by this invention is the limitation of conventional antibodies and antibody fragments in therapeutic and diagnostic use due to their relatively large size, which can hinder tissue penetration and access to epitopes. Natural antibodies have variable and constant regions with typical molecular weights of approximately 150 kD for IgG, or 25-100 kD for antibody fragments such as Fab and scFv. However, smaller antigen binding molecules are desired that retain specificity and affinity but offer improved tissue penetration and epitope access for diverse applications.
This invention overcomes these limitations by engineering constant domain molecules (CH2 or CH3) that are much smaller than conventional antibodies or fragments, can be stabilized by mutations such as non-native disulfide bonds, and can be endowed with antigen specificity by replacing or mutating loop regions with CDR sequences. The engineered molecules do not include variable domains for antigen binding but achieve binding specificity through engrafted CDRs or functional fragments, enabling generation of highly specific, small antigen binding polypeptides for therapeutic and diagnostic use.
Claims Coverage
The patent contains one independent claim detailing a polypeptide molecule with specific structural modifications related to immunoglobulin CH2 or CH3 domains that specifically bind antigen, alongside various dependent claims describing additional features and applications.
Polypeptide comprising modified human immunoglobulin CH2 or CH3 domain
A polypeptide comprising a human immunoglobulin CH2 domain (IgG, IgA or IgD) or CH3 domain (IgE or IgM) with an N-terminal truncation of 7 amino acids, having a mutated Loop 1 and molecular weight less than about 15 kD, which specifically binds an antigen.
Additional mutated loops in CH2 or CH3 domain
The polypeptide may include mutations in other loops such as Loop 2, Loop 3, Loop A-B, Loop C-D, Loop E-F, or combinations thereof.
Polypeptide molecular weight range
The polypeptide has a molecular weight ranging between about 12 kD and about 14 kD.
C-terminal truncation in CH2 or CH3 domain
The polypeptide may include a C-terminal truncation of about 1 to about 4 amino acids in the CH2 or CH3 domain.
Binding capabilities to Fc receptor or complement proteins
The CH2 or CH3 domain polypeptide is capable of binding at least one Fc receptor, complement protein, or both.
Antigen specificity toward pathogen-derived and cancer antigens
The polypeptide binds to antigens from pathogens including viruses (notably HIV) or bacteria, as well as cancer-specific or tumor-associated antigens related to various cancers such as leukemia, lymphoma, melanoma, breast, lung, prostate, colon, and renal cell carcinoma.
Conjugation with effector molecules or labels
A composition comprising the polypeptide conjugated to an effector molecule or a detectable label, optionally with a pharmaceutically acceptable carrier.
Introduction of a non-native disulfide bond for enhanced stability
Inclusion of two amino acid substitutions replacing original residues with cysteines that form a disulfide bond, with at least one substitution in the N-terminal A strand and the other in the C-terminal G strand, enhancing molecule stability.
Specific cysteine substitutions for disulfide bond formation
Particularly, substitutions L12 to C12 and K104 to C104, or V10 to C10 and K104 to C104 (numbered according to SEQ ID NO: 5) in the CH2 domain of IgG.
Randomized mutations using specific residues in loops
Loop 1 and Loop 3 mutations comprise random substitutions of alanine, tyrosine, aspartic acid or serine residues, potentially replacing all residues and optionally including an extra glycine at the C-terminus of each loop for increased flexibility.
The claims cover engineered human immunoglobulin CH2 or CH3 domain polypeptides with specific N- and/or C-terminal truncations, loop mutations or CDR engraftments to confer antigen specificity, with enhanced stability by engineered disulfide bonds, targeting diverse antigens including pathogens and cancers, and compositions including conjugates for therapeutic or diagnostic uses.
Stated Advantages
The engineered CH2 and CH3 domain molecules are small in size (less than about 15 kD), allowing greater epitope access and better tissue penetration than conventional antibodies or fragments.
These molecules exhibit high stability and solubility, including mutants with engineered disulfide bonds that significantly increase thermal stability.
They have minimal to no toxicity, which is advantageous for therapeutic applications.
They are capable of specific antigen binding in the absence of variable domains, enabling creation of small, antigen-specific binding molecules.
Their design permits functionalization by conjugation with effector molecules or detectable labels for therapeutic, diagnostic, or detection purposes.
Documented Applications
Therapeutic use in treating infectious diseases including viral infections such as HIV and bacterial, fungal, parasitic infections.
Treatment of various cancers, including leukemias, lymphomas, multiple myeloma, melanoma, breast, lung, prostate, colon, and renal cell carcinoma by targeting tumor-associated antigens.
Use in autoimmune or inflammatory disorders where binding to relevant antigens such as TNF-α is desirable.
Diagnostic applications including immunoassays (ELISA, Western blot, immunoprecipitation), fluorescence activated cell sorting (FACS), and other antigen detection assays.
Construction of phage-displayed libraries and methods for identifying CH2 or CH3 domain molecules that specifically bind target antigens for therapeutic or diagnostic development.
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