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WO-2026092673-A1 - SCREENING AND USE OF ALBUMIN MUTANT

WO2026092673A1WO 2026092673 A1WO2026092673 A1WO 2026092673A1WO-2026092673-A1

Abstract

The present invention relates to a mutant of an albumin domain III (DIII), or a portion or derivative thereof, and an albumin or a portion or derivative thereof, a fusion protein, and a complex comprising the mutant. The present invention further relates to a use of the mutant, fusion protein, and complex in increasing the affinity of a molecule for FcRn, or in extending the half-life of the molecule.

Inventors

  • TIAN, RUI
  • YANG, Shengren
  • LIU, YANLIN

Assignees

  • 翔安创新实验室
  • 厦门大学

Dates

Publication Date
20260507
Application Date
20251031
Priority Date
20241031

Claims (20)

  1. A mutant of albumin third domain (DIII) or a portion thereof (e.g., DIIIb) or a derivative thereof, which, compared with native albumin DIII or a portion thereof, contains the following mutations: amino acid insertions, substitutions, deletions and/or mutations at one or more (e.g., 90-100, 80-90, 70-80, 60-70, 50-60, 40-50, 30-40, 20-30, 15-20, 10-15, 5-10, 1-5) positions in native albumin corresponding to positions 497 to 585 of SEQ ID NO:1; Preferably, the mutant, compared with natural albumin DIII or a portion thereof, contains the following mutation: the natural albumin has an amino acid mutation at one or more (e.g., 40-50, 30-40, 20-30, 15-20, 10-15, 5-10, 1-5) positions corresponding to positions 500 to 573 of SEQ ID NO:1.
  2. The mutant of claim 1 has one or more of the following characteristics: (1) The mutant has a higher FcRn affinity than natural albumin or DIII; (2) Compared with natural albumin DIII, the mutant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid mutations. (3) The position of the amino acid mutation contained in the mutant is selected from one or more of the following positions: positions 498, 500, 505, 508, 509, 510, 512, 523, 524, 527, 528, 531, 547 and 573 in natural albumin corresponding to SEQ ID NO:1; (4) The amino acid mutation is a conserved mutation; (5) The DIII portion comprises a fragment of natural albumin corresponding to amino acids 467 to 585 of SEQ ID NO:1; Preferably, the mutant contains amino acid mutations at positions 523 and 573; Preferably, the mutant contains amino acid mutations at positions 505, 523, 547, and 573; Preferably, the mutant contains amino acid mutations at positions 500, 505, 523, 524, 527, 528, 531, 547, and 573.
  3. The mutant of claim 1 or 2, wherein the mutant has one or more features selected from the following: (1) The amino acid at position 500 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L or D; (2) The amino acid at position 505 of the mutant corresponding to SEQ ID NO:1 in natural albumin is Q, N or T; (3) The amino acid at position 523 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L or M; (4) The amino acid at position 524 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L; (5) The amino acid K at position 527 of natural albumin corresponding to SEQ ID NO:1 in the mutant is K; (6) The amino acid at position 528 of the natural albumin corresponding to SEQ ID NO:1 in the mutant is H or Y; (7) The amino acid at position 531 of the mutant in natural albumin corresponding to SEQ ID NO:1 is L; (8) The amino acid at position 547 of the natural albumin corresponding to SEQ ID NO:1 in the mutant is A or C; (9) The amino acid at position 509 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L; (10) The amino acid at position 510 of the mutant corresponding to SEQ ID NO:1 in natural albumin is R or N; (11) The amino acid at position 498 of the mutant in natural albumin corresponding to SEQ ID NO:1 is E; (12) The amino acid at position 512 of the mutant corresponding to SEQ ID NO:1 in natural albumin is G; (13) The amino acid at position 573 of natural albumin corresponding to SEQ ID NO:1 in the mutant is any amino acid other than K (e.g., P); (14) The amino acid L at position 508 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L.
  4. The mutant of claim 3, wherein the mutant has one or more of the following characteristics: (1) The amino acid at position 500 is mutated from K to L or D; (2) The amino acid at position 505 is mutated from E to Q, N or T; (3) The amino acid at position 523 is mutated from I to L or M; (4) The amino acid at position 524 is mutated from K to L; (5) The amino acid at position 527 is mutated from T to K; (6) The amino acid at position 528 is mutated from A to H or Y; (7) The amino acid at position 531 is mutated from E to L; (8) The amino acid at position 547 is mutated from V to A or C; (9) The amino acid at position 509 is mutated from F to L; (10) The amino acid at position 510 is mutated from H to R or N; (11) The amino acid at position 498 is mutated from V to E; (12) The amino acid at position 512 is mutated from D to G; (13) The amino acid at position 573 is mutated from K to any amino acid other than K (e.g., P); (14) The amino acid at position 508 is mutated from T to L; Preferably, the mutant contains the mutations I523G and K573P; Preferably, the mutant contains mutations of E505Q, I523G, V547A, and K573P; Preferably, the mutant includes mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P.
  5. The mutant according to any one of claims 1-4, wherein the natural albumin is derived from natural serum albumin of mammals; Preferably, the mammal is selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs, and pigs; Preferably, the natural albumin is derived from natural human serum albumin; Preferably, the natural albumin comprises, or is composed of, sequences selected from, the following: (i) The sequence shown in SEQ ID NO:1; (ii) A sequence having one or more amino acid substitutions, deletions and/or additions (e.g., substitutions, deletions and/or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 1; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 1; Preferably, the natural albumin DIII comprises, or is composed of, sequences selected from, the following: (i) The sequence shown in SEQ ID NO:2; (ii) A sequence having one or more amino acid substitutions, deletions and/or additions (e.g., substitutions, deletions and/or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 2; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 2; Preferably, the mutant comprises a sequence as shown in any one of SEQ ID NO: 12-16, 21-46, 49-53.
  6. An albumin or a portion thereof or a derivative thereof, comprising the mutant according to any one of claims 1-5; Preferably, the albumin or a portion thereof or a derivative thereof further comprises: a first domain (DI) or a portion thereof or a derivative thereof or a mutant thereof, and/or, a second domain (DII) or a portion thereof or a derivative thereof or a mutant thereof; Preferably, the DI and DII are each independently derived from mammalian natural serum albumin; Preferably, the mammal is selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs, and pigs; Preferably, the natural albumin is as defined in claim 5.
  7. A fusion protein comprising the mutant of any one of claims 1-5 or the albumin of claim 6 or a portion thereof or a derivative thereof, and additional polypeptides or proteins; Preferably, the additional polypeptide or protein is optionally linked via a linker to the N-terminus or C-terminus of the mutant or the albumin or a portion thereof or a derivative thereof; Preferably, the connector has a sequence as shown in SEQ ID NO: 10 or 11; Preferably, the additional polypeptide or protein is selected from tags, signal peptides or guide peptides, detectable markers (e.g., luciferase (fluc), green fluorescent protein (GFP)), or any combination thereof; Preferably, the label is selected from HA, Myc, His, GST, or any combination thereof; Preferably, the label has a sequence as shown in any one of SEQ ID NO:6-9.
  8. The fusion protein of claim 7, wherein the additional polypeptide is selected from RGD peptide, octreotate, vascular endothelial growth factor (VEGF), OVA peptide, nucleic acid binding polypeptide or protein (such as zinc finger protein ZF9), nuclear localization signal peptide (NLS), nuclease, or any combination thereof. Preferably, the peptide can be expressed by fusion at the N or C terminus, for example: peptide-DIII-peptide. The peptide can also be expressed in the middle of tandem DIII, for example: -(peptide-DIII)n-, where n is an independent positive integer (e.g., 1, 2, 3, 4, 5 or greater). Preferably, the RGD peptide comprises a sequence as shown in (GGGGS) m (RGD) n , wherein m and n are each independently positive integers (e.g., 1, 2, 3, 4, 5 or greater); preferably, the RGD peptide has a sequence as shown in SEQ ID NO:20; Preferably, the zinc finger protein comprises the sequence (GGGGS) m (ZF9) n , wherein m and n are each independently positive integers (e.g., 1, 2, 3, 4, 5 or greater); Preferably, the nucleic acid binding protein or polypeptide can be fused with the NLS and other nuclear localization signal peptides in different ways (e.g., ZF9-NLS, NLS-ZF9); Preferably, the nucleic acid binding protein or polypeptide can be fused with the NLS and other nuclear localization signal peptides in different ways (e.g., (ZF9)m-(NLS)n, (NLS)m-(ZF9)n); wherein m and n are each independently positive integers (e.g., 1, 2, 3, 4, 5 or greater); Preferably, the NLS and other nuclear localization signal peptides can be fused at any position on the N or C terminus of the nucleic acid binding protein, or simultaneously at the N and C terminus (e.g., NLS-ZF9-NLS).
  9. The fusion protein of claim 7 or 8, wherein the fusion protein comprises one or more (e.g., 2, 3, 4, 5) mutants of any one of claims 1-5 or albumin or a portion thereof or a derivative thereof of claim 6; optionally, the fusion protein further comprises one or more linkers or the polypeptide (e.g., 2, 3, 4, 5).
  10. The fusion protein of claim 9, wherein the fusion protein has one or more features selected from the following: (1) The fusion protein comprises multiple mutants or multiple albumins or portions thereof or derivatives thereof, and they are linked together by the polypeptide; (2) The fusion protein comprises one of the mutants or the albumin or a portion thereof or a derivative thereof, and one or two of the polypeptides; (3) The fusion protein has a sequence as shown in SEQ ID NO:17 or 18; (4) The fusion protein comprises two of the mutants or the albumin or a portion thereof or a derivative thereof, and two or three of the polypeptides; (5) The fusion protein has the sequence shown in SEQ ID NO:19; (6) The fusion protein comprises multiple mutants or multiple albumins or portions thereof or derivatives thereof, and they are connected by the linker; (7) The fusion protein has a sequence as shown in SEQ ID NO:5 or 47.
  11. A nucleic acid molecule encoding a mutant as described in any one of claims 1-5, or albumin or a portion thereof or a derivative thereof as described in claim 6, or a fusion protein as described in any one of claims 7-10.
  12. A vector comprising the nucleic acid molecule of claim 11; preferably, the vector is an expression vector; Preferably, the vector is a vector of eukaryotic bacteria (e.g., pPIC9K).
  13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12; Preferably, the cells are eukaryotic cells or prokaryotic cells; Preferably, the eukaryotic cell is a yeast cell (e.g., Saccharomyces cerevisiae, Pichia pastoris); Preferably, the prokaryotic cells are Escherichia coli cells, Bacillus subtilis cells, or any combination thereof; Preferably, the cells are mammalian cells (e.g., 293T cells).
  14. A method for screening mutants according to any one of claims 1-5, albumin or a portion thereof or a derivative thereof according to claim 6, or fusion proteins according to any one of claims 7-10, the method comprising: contacting a candidate mutant, or albumin or a portion thereof or a derivative thereof, or fusion protein with FcRn, and detecting their affinity for FcRn; Preferably, a mutant library containing candidate mutants is constructed using deep learning methods, the candidate mutants are obtained using the host cell described in claim 10, they are contacted with FcRn, and their affinity for FcRn is detected.
  15. A complex comprising a mutant according to any one of claims 1-5, or albumin or a portion thereof or a derivative thereof according to claim 6, or a fusion protein according to any one of claims 7-10, and a molecule (e.g., a dye molecule) bound thereto. Preferably, the dye molecule is a cyanine dye molecule.
  16. A delivery composition comprising: (1) The mutant according to any one of claims 1-5, or the albumin or a portion thereof or a derivative thereof according to claim 6, or the fusion protein according to any one of claims 7-10, or the nucleic acid molecule according to claim 11, or the vector according to claim 12, or the host cell according to claim 13, and (2) An immunogen (e.g., an immunogenic polypeptide, a nucleic acid encoding the polypeptide), or a macromolecular or small molecule drug (e.g., a targeted small molecule drug); preferably, the immunogen is another polypeptide as described in claim 8; Preferably, component (1) serves as a delivery carrier for component (2).
  17. The delivery composition of claim 16, wherein the immunogen is capable of inducing an immune response in a subject; Preferably, the immunogen is derived from influenza virus, coronavirus, hepatitis B virus, or any combination thereof; Preferably, the macromolecular or small molecule drug is a drug that targets tumor cells; Preferably, the component (2) is delivered to the mucosal surface of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa, vaginal and cervical mucosa); Preferably, the delivery composition is delivered via intranasal or inhalation; Preferably, the delivery composition is delivered via the oral or gastrointestinal mucosal epithelium; Preferably, the delivery composition further comprises one or more mucosal adhesives to enhance the residence time of component (2) on the mucosal surface of the subject; Preferably, the delivery composition can be prepared as a complex using biomodification, physical mixing and/or chemical linking methods and drugs or imaging probes.
  18. A pharmaceutical composition comprising one or more of the following: (1) The mutant according to any one of claims 1-5; (2) The albumin or a portion thereof or a derivative thereof as claimed in claim 6; (3) The fusion protein according to any one of claims 7-10; (4) The nucleic acid molecule according to claim 11; (5) The carrier according to claim 12; (6) The host cell according to claim 13; (7) The complex according to claim 15; Preferably, the pharmaceutical composition further comprises an immunogen (e.g., an immunogenic polypeptide, a nucleic acid encoding the polypeptide), or a macromolecular or small molecule drug (e.g., a targeted small molecule drug). Preferably, the pharmaceutical composition has one or more of the following characteristics: (1) The pharmaceutical composition is a drug that is targeted for release into the gastrointestinal tract or a drug that is released in a controlled manner into the gastrointestinal tract; (2) The pharmaceutical composition further comprises a pharmaceutically acceptable carrier; (3) The pharmaceutical composition is in the form of pills, powders, capsules, tablets (e.g., effervescent tablets), film-coated tablets, orally soluble granules, liquids, suppositories or enemas; (4) The administration method of the pharmaceutical composition is selected from oral, intravenous, transmucosal, sublingual, nasal, intrathecal, bronchial, rectal, percutaneous, inhalation or parenteral; (5) The pharmaceutical composition is applied to the mucosal surface of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa).
  19. The use of the mutant of any one of claims 1-5, or the albumin or a portion thereof or a derivative thereof of claim 6, or the fusion protein of any one of claims 7-10, or the nucleic acid molecule of claim 11, or the vector of claim 12, or the host cell of claim 13, or the complex of claim 15, or the delivery composition of claim 16 or 17, or the pharmaceutical composition of claim 18, for increasing the affinity of the molecule for FcRn or prolonging the half-life of the molecule; Preferably, the molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof; Preferably, the molecule is a drug that targets tumor cells; Preferably, the tumor is selected from renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-cell rich lymphoma, central nervous system (CNS) tumors, spinal axis tumors, and brainstem glioma.
  20. Use in the preparation of a pharmaceutical composition of the mutant of any one of claims 1-5, or the albumin or a portion thereof or a derivative thereof of claim 6, or the fusion protein of any one of claims 7-10, or the nucleic acid molecule of claim 11, or the vector of claim 12, or the host cell of claim 13, or the complex of claim 15, or the delivery composition of claim 16 or 17; Preferably, the pharmaceutical composition further comprises an active molecule; Preferably, the active molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof; Preferably, the mutant, the albumin or a portion thereof or a derivative thereof, the fusion protein, the nucleic acid molecule, the vector, the host cell, or the complex is used to increase the half-life of the pharmaceutical composition.

Description

Screening of albumin mutants and their applications Technical Field This application relates to a mutant of the albumin third domain (DIII) or a portion thereof or a derivative thereof, and albumin or a portion thereof or a derivative thereof comprising said mutant, fusion proteins and complexes. This application also relates to the use of said mutants, fusion proteins and complexes in improving transmucosal delivery or extending molecular half-life. Background Technology "Drug transmucosal delivery" and "long-term in vivo circulation of drug molecules" have always been two core challenges in the field of drug delivery. On the one hand, for "drug transmucosal delivery," the mucosal system typically has multiple barriers to protect the body from the risk of exposure from direct contact with the external environment. Therefore, drug passage is often hindered. On the other hand, small molecule drugs and peptides, which account for 80-90% of all drug molecules, have short in vivo half-lives and require repeated administration to maintain effective concentrations, often leading to liver and kidney toxicity. Therefore, improving "drug transmucosal delivery efficiency" and "long-term in vivo circulation time of drug molecules" is a major scientific issue in drug delivery and even the biopharmaceutical field. A classic approach to increasing the in vivo half-life of small molecules is usually to conjugate them with macromolecules to increase their hydration kinetic radius, thereby prolonging their circulation time in the body. Conjugation with endogenous macromolecules (such as antibodies and albumin) is an effective strategy. Human serum albumin (HSA) is a non-glycosylated, globular, water-soluble protein with a molecular weight of 66.5 kilodaltons (kDa), encoded by the ALB gene and composed of 585 amino acids. HSA is the most abundant protein in the human circulatory system, accounting for half of serum proteins, with a concentration between 30-50 mg/ml. It is mainly synthesized in the liver and has a long circulating half-life of approximately 19 days. Albumin is heart-shaped and consists of three highly homologous domains, named D1, D1I, and D1III. Each domain is composed of two subunits, A and B. In the human body, albumin performs many important functions, including maintaining osmotic pressure and plasma pH, and transporting and distributing various endogenous and exogenous molecules. These properties enable HSAs to play a crucial role in the pharmacology of many drugs, from lipophilic small molecule drugs to nanoparticles, peptides, and other biologics, significantly enhancing their biological half-life after binding. HSA crystal structures have revealed the hydrophobic drug and fatty acid binding sites, and the pharmacokinetic mechanisms influenced by HSAs have been extensively reviewed. To date, various drugs and imaging probes have been designed to bind to HSAs to improve their half-life and drug delivery efficiency, some of which have received clinical approval from the U.S. Food and Drug Administration (FDA). For many small molecule drugs, binding to HSAs can serve as a strategy to enhance therapeutic efficacy while reducing systemic toxicity. The chemotherapy nanodrug paclitaxel (nab-paclitaxel, Abraxane), bound to an HSA, is used to treat various types of metastatic cancer and received FDA approval in 2005. Similarly, fatty acid peptide conjugates such as insectin detemir (Levemir) and liraglutide (Victoza) were approved in 2005 and 2010, respectively, for the treatment of diabetes. Semagrutide (Ozenpic) increases the HSA binding affinity of liraglutide by approximately 6 times and exhibits a relatively prolonged circulating half-life. It was approved by the FDA in 2017 for the treatment of type 2 diabetes. More recently, rapamycin (a sirolimus albumin-bound particle suspension for injection, Fyarro), an albumin-based mammalian mTOR kinase inhibitor, was approved by the FDA in 2021 for the treatment of perivascular epithelioid carcinomas. Meanwhile, in the field of biomedical imaging, various HSA-conjugated probes are being developed and applied. Although most of these imaging probes have only undergone preclinical trials, some have already completed clinical trials or received regulatory approval. In the field of optical imaging, representative examples include the near-infrared fluorescence imaging probe indocyanine green (ICG), which can reversibly bind to albumin and has been approved by the FDA for use in tissue perfusion and lymph node assessment. Furthermore, Phase I and II clinical trials of its application in photodynamic therapy and intraoperative tumor margin and tissue perfusion assessment are underway. Another example is Evans Blue T-1824, which also reversibly binds to albumin and is undergoing pre-clinical trials for mileage assay imaging and bright-field imaging. The MRI imaging probe Gadofosveset trisodium ether... ABLAVAR (ium) is an albumin-reversibly binding gadolinium chelator, a