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Publication Number
US-12173054-B2
Publication Date
2024-12-24
Expiration Date
Abstract
Described herein is a polypeptide comprising an immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein: (a) at least one lysine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue, and/or (b) at least one arginine residue in CDR1, CDR2 and/or CDR3 has been substituted with at least one histidine residue. The polypeptides of this disclosure possess increased intestinal stability relative to a corresponding polypeptide not having the histidine substitutions.
Core Innovation
The invention relates to immunoglobulin chain variable-domain polypeptides (ICVDs), including VHH, with increased intestinal stability. The approach substitutes at least one lysine and/or arginine residue with histidine in CDR1, CDR2 and/or CDR3, and the description focuses on HIS-substituted VHH variants in CDR2 and/or CDR3. The binding regions are defined using complementarity determining regions (CDRs) and framework regions (FRs), and the substituted sequences are produced as variant immunoglobulin chain variable-domain polypeptides.
The problem addressed is that progenitor VHHs have insufficient intestinal stability for administration, even when they preserve binding specificity. The solution is to select HIS-substituted VHHs that maintain the binding specificity of the progenitor VHH while improving intestinal stability after exposure in intestinal tract models. Intestinal stability increase is evaluated using a Standard Human Fecal Supernatant Intestinal Tract Model, and potency is constrained by EC50 changes measured using an ELISA-based assay.
The document further describes producing the selected HIS-substituted VHH in yeast and selecting variants that meet quantitative stability and potency constraints. It includes oral-use constructs and pharmaceutical compositions, and it describes multivalent constructs using linkers such as (G4S)x, including bivalent and multivalent formats. Binding targets and example ICVDs are provided for categories such as TNF-alpha, IL-6R, and C. difficile toxins, with examples reporting improved intestinal stability with substantially maintained potency when specific lysine-to-histidine and arginine-to-histidine substitutions are introduced at defined CDR positions.
Claims Coverage
One independent claim is identified, covering a method that generates and selects a HIS-substituted VHH variant with increased intestinal stability upon administration while maintaining binding specificity and controlling EC50 change, and with the variant produced in yeast.
His-substituted CDR2/CDR3 VHH generation
Providing a progenitor VHH having an amino acid sequence comprising three complementarity determining regions (CDR1-CDR3) that binds a target, and substituting an arginine residue or a lysine residue with a histidine residue in CDR2 or CDR3 of the amino acid sequence to produce a HIS-substituted VHH.
Yeast production of HIS-substituted VHH
Producing the HIS-substituted VHH comprising a HIS-substituted amino acid sequence in yeast.
Selection preserving specificity with increased intestinal stability
Selecting from the HIS-substituted VHH comprising a HIS-substituted amino acid sequence a His-substituted VHH that maintains a binding specificity of the progenitor VHH and has increased intestinal stability relative to the progenitor VHH, thereby obtaining a selected HIS-substituted VHH.
Quantitative intestinal stability threshold and potency control
Requiring that the intestinal stability of the selected HIS-substituted VHH is increased by at least 5% relative to the intestinal stability of the progenitor VHH as determined by a Standard Human Fecal Supernatant Intestinal Tract Model, and requiring that the EC50 of the selected HIS-substituted VHH is increased by no more than 400% relative to the EC50 of the progenitor VHH by an ELISA-based assay.
The independent claim covers a yeast-produced HIS-substituted VHH selected for preserved binding specificity and improved intestinal stability, while also imposing quantitative constraints on intestinal stability increase (≥5% in the stated model) and an upper bound on EC50 increase (≤400% in an ELISA-based assay).
Stated Advantages
Increased intestinal stability of the selected HIS-substituted VHH relative to the progenitor VHH by at least 5% as determined by a Standard Human Fecal Supernatant Intestinal Tract Model.
Maintains binding specificity of the progenitor VHH while increasing intestinal stability.
Potency is controlled, with EC50 increased by no more than 400% relative to the progenitor VHH by an ELISA-based assay.
Documented Applications
Oral-use constructs and pharmaceutical compositions that include HIS-substituted ICVD/VHH variants with increased intestinal stability.
Targeted binding use cases described by target categories including TNF-alpha, IL-6R, and C. difficile toxins (C. difficile toxin A and C. difficile toxin B).
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