Transgenic non-human animals producing modified heavy chain-only antibodies
Inventors
van Schooten, Wim • Trinklein, Nathan • Aldred, Shelley Force • Bruggemann, Marianne • Osborn, Mike
Assignees
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Publication Number
US-12495776-B2
Publication Date
2025-12-16
Expiration Date
Abstract
Human or chimeric heavy chain-only antibodies are provided, in the native amino acid residue at the first position of the fourth framework region (FR4) of said HCAb is substituted by a different amino acid residue that is capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position.
Core Innovation
The invention relates to heavy chain-only antibodies (HCAbs) and transgenic non-human animals engineered to express modified HCAbs. In these constructs, the native FR4 first-position residue (Kabat position 101) is replaced with a different amino acid residue, including a tryptophan to arginine substitution (W→R), or other polar/positively charged substitutions. This replacement is described as disrupting a surface-exposed hydrophobic patch comprising or associated with the native residue.
The disclosed modifications are directed to reducing aggregation and light chain association in heavy chain-only formats. The described outcome includes inhibiting association with free lambda light chain protein. The document also describes comparing different FR4 residue substitutions in terms of their impact on aggregation propensity and light chain association, including lambda association.
The invention further provides recombinant heavy-chain-only immunoglobulin loci and transgenes arranged for heavy chain-only expression in non-human animals. The constructs are described as including human or chimeric HCAbs, and recombinant heavy-chain-only Ig loci that lack functional light chain loci, along with specified constant region gene segment composition that lacks Cμ coding functionality in the disclosed embodiments. The document also describes engineered immunoglobulin loci and transgenic animals generated to express the modified HCAbs.
Claims Coverage
Two independent claims are identified. The claims center on a FR4 first-position substitution that disrupts a surface-exposed hydrophobic patch and a heavy chain-only constant region gene segment architecture lacking Cμ coding region with specified Cγ, Cε, and Cα elements.
Recombinant heavy chain-only Ig locus with FR4 hydrophobic-patch disrupting first-position substitution
A recombinant heavy chain-only immunoglobulin locus comprising human V, D, and J gene segments that encode a heavy chain variable region wherein at least one human J segment comprises a codon encoding a different amino acid residue at the first position of FR4 capable of disrupting a surface-exposed hydrophobic patch comprising or associated with the native amino acid residue at that position.
Constant effector region architecture lacking Cμ and comprising specified Cγ, Cε and Cα elements
A recombinant heavy chain-only immunoglobulin locus in which the constant effector region lacks a Cμ coding region, comprises a Cγ coding region with Cγ32a, Cγ31, and Cγ32b coding regions each lacking a CH1 exon, and comprises Cε and Cα coding regions each comprising a CH1 exon.
Transgenic non-human animal lacking functional light chain genes and expressing a heavy chain-only locus
A transgenic non-human animal that does not express any functional immunoglobulin light chain genes and comprises a heterologous heavy chain-only Ig locus in which a VH domain includes the native amino acid residue at the first position of FR4 substituted by a different amino acid residue capable of disrupting a surface-exposed hydrophobic patch.
Rearranged affinity matured heavy chain-only gene locus producing HCAb
A transgenic non-human animal in which gene segments recombine to produce a rearranged affinity matured heavy chain-only gene locus encoding a heavy chain-only antibody (HCAb), where the constant effector region lacks a Cμ coding region and includes specified Cγ, Cε, and Cα coding regions.
The claims combine a FR4 first-position substitution that disrupts a surface-exposed hydrophobic patch with a heavy chain-only constant effector region gene segment configuration lacking Cμ coding and specifying Cγ elements lacking CH1 exon and Cε and Cα elements comprising CH1 exon. In the animal claim, this is coupled to an animal lacking functional immunoglobulin light chain genes and expressing a rearranged affinity-matured heavy chain-only locus that encodes an HCAb.
Stated Advantages
Reduces aggregation.
Reduces light chain association, including association with free lambda protein.
Documented Applications
CAR constructs and multivalent binding proteins are described for targeting applications using the modified VH domains.
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