Calbindin and BH3 domain chimeric proteins and methods for use
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Publication Number
US-11459364-B2
Publication Date
2022-10-04
Expiration Date
Abstract
Therapeutic proteins for inducing apoptosis in cells and method for using such proteins are described. The protein, e.g., a chimeric protein, has a host peptide fused to a more stable BH3 domain peptide. The host peptide may be Calbindin D9k, a N-terminal or C-terminal half-domain of Calmodulin, Parvalbimin (“Pa”), an amino acid sequence variant thereof, or a modified variant thereof. The BH3 domain may be Bim, Bid, Bad, Bik, Bmf, Hrk, Puma, or Noxia. Moreover, various conjugates may be attached to the chimeric protein, including a folate and polyethylene glycol.
Core Innovation
The invention relates to therapeutic chimeric apoptosis-inducing proteins that combine a stable human host peptide with a BH3 domain from BH3-only proteins. The chimeric proteins are capable of inducing apoptosis in cells by grafting the BH3 domain into the host peptide, with examples of host peptides including Calbindin D9k, calmodulin halves, and Parvalbumin, and BH3 domains taken from BH3-only proteins including Bim, Bid, Bad, Puma, and Noxia.
A stated structural basis of the invention is that a C-terminal helix of the first peptide is replaced with the BH3 domain grafted into the first peptide. The document further describes sequence variants consistent with the chimeric design, including SEQ ID NOs 1-10, and reports apoptosis induction across tested combinations of BH3 domains and host peptides.
The document also describes optional targeting and pharmacokinetic-related modifications for the chimeric apoptosis-inducing proteins. Targeting moieties include folate conjugation using disulfide (S-S) linkages or maleimide linkages with PEG spacers, and optional half-life extension is described via PEGylation and multi-arm PEGylation with varying folate stoichiometry. The document additionally describes combination effects with radiation and oxaliplatin and apoptosis induction in activated macrophages.
Claims Coverage
The independent claim recites one core chimeric protein architecture, with dependent claims refining it by specifying provided sequence identifiers, adding optional targeting moieties, including folate and linkage chemistry, and adding optional polyethylene glycol modification for half-life extension. Across the independent claim and dependent coverage, the inventive focus includes the grafted BH3 domain architecture and optional conjugate-based targeting and modification.
Calbindin D9k with replaced C-terminal helix grafted BH3 domain
A chimeric protein capable of inducing apoptosis in cells, comprising a first peptide that is Calbindin D9k; a second peptide that is a BH domain of a BH3-only protein selected from Bim, Bid, Bad, Puma, or Noxia; wherein a C-terminal helix of the first peptide is replaced with the BH3 domain that is grafted into the first peptide.
Chimeric protein amino acid sequence selected from SEQ ID NOs
A chimeric protein, wherein an amino acid sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
Targeting moiety conjugated to the chimeric protein
A chimeric protein, further including a targeting moiety conjugated to it.
Folate targeting moiety
A chimeric protein, wherein the targeting moiety is folate.
Folate attached via disulfide linkage
A chimeric protein where folate is attached to the chimeric protein via a disulfide linkage.
Polyethylene glycol as a modifying group for the chimeric protein
A chimeric protein where the modifying group has polyethylene glycol.
The claim set centers on a Calbindin D9k-based chimeric apoptosis-inducing protein in which a C-terminal helix is replaced by a grafted BH3 domain from specified BH3-only proteins. Dependent coverage narrows the protein to provided sequence identifiers, and further adds optional conjugated targeting moieties, including folate and disulfide linkage chemistry, and optional polyethylene glycol modification for half-life-related modification.
Stated Advantages
Capable of inducing apoptosis in cells.
Documented Applications
Therapeutic use for cancer and inflammatory disease, including apoptosis induction in activated macrophages and tumor inhibition in xenograft tumor contexts, as described.
Combination described with radiation therapy and oxaliplatin, as described.
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