Intracellular delivery compositions
Inventors
SHILOVITZKY, Orit • CHEN ZELTSBURG, Lilach • UZAN GUETA, Rozi Ravit • Lewkowicz, Ayala • Gottfried, Yossi • FAYGENBOIM-ORNAI, Rotem • HAKIM, MOTTI • Sapir, Yair • Mandel, Ilana • Ben-Moshe, Tehila • Shulman, Avidor
Assignees
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Publication Number
US-12544451-B2
Publication Date
2026-02-10
Expiration Date
Abstract
Protein conjugates comprising a protein carrier comprising polyethylenimine, a payload that interacts with an intracellular target and a linker are provided. Pharmaceutical composition comprising the protein conjugates as well as methods of using and producing the protein conjugates are also provided.
Core Innovation
The disclosure provides substantially blood-stable, cell-penetrating protein conjugates constructed from human serum albumin (HSA), polyethyleneimine (PEI) moieties, and a single domain antibody. In these conjugates, the HSA is covalently bound to 3 to 90 PEI moieties, and the HSA comprises the amino acid sequence of SEQ ID NO: 1 or a fragment comprising Cysteine 34 (C34) and at least 400 amino acids in length. The conjugates are characterized by an HSA fragment isoelectric point at most 7 and by a positive zeta potential of at least 8 mV.
A covalent linker is bound to the HSA to connect the HSA to the single domain antibody via a covalent bond, and the disclosure emphasizes linker stability in human blood, plasma, and serum. The linkers include bio-cleavable bond structures such as a disulfide bond, and the conjugates are described as blood-stable with less than 25% cleavage in blood after 24 hours in certain embodiments. The disclosure also describes cytoplasmic dissociation of the carrier from the payload and endosome escape.
The disclosure further introduces kit and method concepts for producing protein conjugates in which covalent attachment of first and second moieties to a protein carrier and payload is performed using coupling chemistry. A selection concept is provided in which linker stability is determined in human blood, plasma, and serum, and a linker-stable conjugate is selected for binding or modulating intracellular targets, optionally using detectable tags. Broad formulation and administration context is provided for pharmaceutical compositions that include pharmaceutically acceptable carriers, excipients, and adjuvants, and may involve systemic or local administration.
Claims Coverage
The provided independent claim set includes one independent claim. It specifies a constrained HSA-PEI architecture, a single domain antibody, and a covalent linker connecting the two, with additional property constraints tied to zeta potential and HSA sequence or fragment characteristics.
HSA-pei protein conjugate with constrained HSA fragment and positive zeta potential
A protein conjugate comprising human serum albumin (HSA) covalently bound to 3 to 90 polyethyleneimine (PEI) moieties, wherein the HSA comprises the amino acid sequence of SEQ ID NO: 1 or a fragment comprising Cysteine 34 (C34) and having at least 400 amino acids, wherein the HSA fragment has an isoelectric point at most 7, and wherein the HSA covalently bound to the PEI moieties has a positive zeta potential at least 8 mV.
Single domain antibody payload covalently connected to HSA via a covalent linker
A protein conjugate further comprising a single domain antibody and a linker bound to said HSA to said single domain antibody via a covalent bond.
Across the independent claim, the inventive subject matter centers on combining a constrained HSA-PEI structure that yields positive zeta potential with a single domain antibody connected through a covalent linker.
Stated Advantages
Blood-stable conjugates, including characterization such as less than 25% cleavage in blood after 24 hours in certain embodiments.
Cell-penetrating behavior.
Endosome escape with cytoplasmic dissociation of the carrier from the payload.
Linker stability in human blood, plasma, and serum for selecting linker-stable conjugates.
Intracellular target binding.
Detectable tags may be supported for intracellular applications.
Improved membrane crossing and endosomal escape associated with PEI moieties.
Cytosolic dispersibility associated with disulfide-containing linkers.
Single-domain antibodies avoid cytosolic disulfide reduction issues.
Documented Applications
Binding or modulating intracellular targets using the produced linker-stable protein conjugates, optionally including detectable tags.
Intracellular-target binding and intracellular delivery, including endosome escape and cytoplasmic dissociation.
Functional efficacy such as apoptosis via BRAF inhibition, cell-cycle arrest via E7 inhibition, and KRAS inhibition using a DARPin.
Biodistribution and pharmacokinetics discussions in connection with linker stability and plasma or serum stability, including mouse plasma, human plasma, and monkey serum stability.
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