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CN-121975000-A - Screening of albumin mutant and application thereof

CN121975000ACN 121975000 ACN121975000 ACN 121975000ACN-121975000-A

Abstract

The present application relates to a mutant of albumin third Domain (DIII) or a part thereof or a derivative thereof, and albumin or a part thereof or a derivative thereof, fusion protein and complex comprising said mutant. The application also relates to the use of said mutants, fusion proteins and complexes for increasing the affinity of a molecule for FcRn, or for extending the half-life of a molecule.

Inventors

  • TIAN RUI
  • Yang shengren
  • LIU YANLIN

Assignees

  • 翔安创新实验室
  • 厦门大学

Dates

Publication Date
20260505
Application Date
20251031
Priority Date
20241031

Claims (20)

  1. 1. A mutant of albumin third domain (dii) or a portion thereof (e.g., dii b) or a derivative thereof comprising a mutation wherein the amino acid at the position 1 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) corresponding to positions 497 to 585 of SEQ ID No. 1 in native albumin has an amino acid insertion, substitution, deletion and/or mutation as compared to native albumin dii or a portion thereof; Preferably, the mutant comprises a mutation in the natural albumin wherein the amino acid at positions 1 or more (e.g., 40-50, 30-40, 20-30, 15-20, 10-15, 5-10, 1-5) corresponding to positions 500 to 573 of SEQ ID NO: 1, has an amino acid compared to the natural albumin DIII or a portion thereof.
  2. 2. The mutant of claim 1 having one or more characteristics selected from the group consisting of: (1) The mutant has a higher affinity for FcRn than native albumin or DIII; (2) The mutant comprises 1,2,3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more amino acid mutations compared to native albumin DIII; (3) The mutant comprises an amino acid mutation at one or more positions selected from the group consisting of positions 498, 500, 505, 508, 509, 510, 512, 523, 524, 527, 528, 531, 547, and 573 of native albumin corresponding to SEQ ID NO. 1; (4) The amino acid mutation is a conservative mutation; (5) The part of the DIII comprises a fragment of natural albumin corresponding to amino acids 467 to 585 of SEQ ID No. 1; Preferably, the mutants comprise amino acid mutations at positions 523 and 573; Preferably, the mutants comprise amino acid mutations at positions 505, 523, 547 and 573; Preferably, the mutants comprise amino acid mutations at positions 500, 505, 523, 524, 527, 528, 531, 547 and 573.
  3. 3. The mutant according to claim 1 or 2, which has one or more characteristics selected from the group consisting of: (1) The mutant has L or D as the amino acid at 500 th position of natural albumin corresponding to SEQ ID NO. 1; (2) The mutant has Q, N or T amino acid at position 505 of natural albumin corresponding to SEQ ID NO. 1; (3) The mutant has L or M as amino acid at 523 position of natural albumin corresponding to SEQ ID NO. 1; (4) The mutant has L as the amino acid at 524 position of natural albumin corresponding to SEQ ID NO. 1; (5) The mutant has K as the amino acid at position 527 of the natural albumin corresponding to SEQ ID NO. 1; (6) The mutant has H or Y as amino acid at 528 position of natural albumin corresponding to SEQ ID NO. 1; (7) The mutant has L as the amino acid in the 531 rd position of natural albumin corresponding to SEQ ID NO. 1; (8) The mutant has an A or C amino acid at the 547 position of the natural albumin corresponding to SEQ ID NO. 1; (9) The mutant has L as the amino acid in 509 position of natural albumin corresponding to SEQ ID NO. 1; (10) The mutant has R or N as the amino acid at the 510 th position of the natural albumin corresponding to SEQ ID NO. 1; (11) The mutant has E as the amino acid in the 498 th position of natural albumin corresponding to SEQ ID NO. 1; (12) The mutant has G as the amino acid in the 512 th position of natural albumin corresponding to SEQ ID NO. 1; (13) The mutant has any amino acid other than K (e.g., P) at the position 573 of the native albumin corresponding to SEQ ID NO. 1; (14) The mutant has L as the amino acid at 508 th position of natural albumin corresponding to SEQ ID No. 1.
  4. 4. A mutant according to claim 3, which has one or more characteristics selected from the group consisting of: (1) The amino acid at the 500 th position is mutated from K to L or D; (2) The amino acid at the 505 th position is mutated from E to Q, N or T; (3) The amino acid at the 523 rd position is mutated from I to L or M; (4) The amino acid at the 524 th position is mutated from K to L; (5) The amino acid at position 527 is mutated from T to K; (6) The amino acid at the 528 th position is mutated from A to H or Y; (7) The amino acid at the 531 th position 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 the 509 th position is mutated from F to L; (10) The amino acid at the 510 th position is mutated from H to R or N; (11) The amino acid at the 498 position is mutated from V to E; (12) The amino acid at the 512 th position 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 mutants comprise mutations I523G and K573P; preferably, the mutants comprise mutations E505Q, I523G, V547A and K573P; Preferably, the mutants comprise mutations K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A and K573P.
  5. 5. The mutant according to any one of claims 1 to 4, wherein the native albumin is derived from a mammalian native serum albumin; preferably, the mammal is selected from the group consisting of 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 native albumin comprises or consists of a sequence selected from the group consisting of seq id no: (i) A sequence shown in SEQ ID NO. 1; (ii) A sequence having one or more amino acid substitutions, deletions and/or additions (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 base substitutions, deletions and/or additions) as compared to the sequence set forth 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 to the sequence set forth in SEQ ID NO. 1; preferably, the native albumin DIII comprises or consists of a sequence selected from the group consisting of seq id nos: (i) A sequence shown in SEQ ID NO. 2; (ii) A sequence having one or more amino acid substitutions, deletions and/or additions (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 base substitutions, deletions and/or additions) as compared to the sequence set forth 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 to the sequence set forth in SEQ ID NO. 2; Preferably, the mutant comprises a sequence as set forth in any one of SEQ ID NOs 12-16, 21-46, 49-53.
  6. 6. An albumin or a part or derivative thereof comprising the mutant of any one of claims 1-5; Preferably, the albumin or a part or derivative thereof further comprises a first Domain (DI) or a part or derivative or mutant thereof and/or a second Domain (DII) or a part or derivative or mutant thereof; preferably, the DI and DII are each independently derived from serum albumin native to the mammal; preferably, the mammal is selected from the group consisting of 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. 7. A fusion protein comprising the mutant of any one of claims 1-5 or the albumin of claim 6 or a portion or derivative thereof, and an additional polypeptide or protein; preferably, the further polypeptide or protein is linked to the N-terminus or C-terminus of the mutant or the albumin or a portion or derivative thereof, optionally via a linker; preferably, the linker has a sequence as shown in SEQ ID NO. 10 or 11; Preferably, the additional polypeptide or protein is selected from a tag, a signal peptide or leader peptide, a detectable label (e.g., luciferase (fluc), green Fluorescent Protein (GFP)), or any combination thereof; preferably, the tag is selected from HA, myc, his, GST, or any combination thereof; Preferably, the tag has a sequence as set forth in any one of SEQ ID NOS.6-9.
  8. 8. The fusion protein of claim 7, wherein the additional polypeptide is selected from the group consisting of RGD peptide, octreotide (Octreotate), vascular Endothelial Growth Factor (VEGF), OVA peptide, nucleic acid binding polypeptide or protein (e.g., zinc finger protein ZF 9), nuclear localization signal peptide (NLS), nuclease, or any combination thereof; Preferably, the polypeptide (peptide) is expressed in fusion at the N-or C-terminus, e.g., peptide-DIII-peptide, and the polypeptide is also expressed in tandem DIII, e.g., - (peptide-DIII) N-, each N being independently a positive integer (e.g., 1,2,3,4,5 or more); Preferably, the RGD peptide comprises a sequence as shown in (GGGGS) m (RGD) n , wherein m and n are each independently a positive integer (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 a sequence shown as (GGGGS) m (ZF9) n , wherein m and n are each independently a positive integer (e.g., 1,2,3,4,5 or greater); Preferably, the nucleic acid binding protein or polypeptide can be expressed in fusion with the NLS and other nuclear localization signal peptides in different ways (e.g., ZF9-NLS, NLS-ZF 9); Preferably, the nucleic acid binding protein or polypeptide can be expressed in a different manner with the NLS and other nuclear localization signal peptides (e.g., (ZF 9) m- (NLS) n, (NLS) m- (ZF 9) n), where 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 may be fused at the N-or C-terminus, respectively, at any position of the nucleic acid binding protein or at the N, C-terminus simultaneously (e.g., NLS-ZF 9-NLS).
  9. 9. The fusion protein of claim 7 or 8, wherein the fusion protein comprises 1 or more (e.g., 2, 3, 4, 5) mutants of any of claims 1-5 or albumin or a portion or derivative thereof of claim 6, and optionally the fusion protein further comprises 1 or more linkers or the polypeptide (e.g., 2, 3, 4, 5).
  10. 10. The fusion protein of claim 9, wherein the fusion protein has one or more characteristics selected from the group consisting of: (1) The fusion protein comprises a plurality of mutants or a plurality of albumin or a part or derivative thereof, and the albumin or the part or derivative thereof are connected through the polypeptide; (2) The fusion protein comprises 1 of the mutants or the albumin or a part or derivative thereof, and 1 or 2 of the polypeptides; (3) The fusion protein has a sequence shown as SEQ ID NO. 17 or 18; (4) The fusion protein comprises 2 of the mutants or the albumin or a part or derivative thereof, and 2 or 3 of the polypeptides; (5) The fusion protein has a sequence shown as SEQ ID NO. 19; (6) The fusion protein comprises a plurality of mutants or a plurality of albumin or a part or derivative thereof, and the albumin or the part or derivative thereof are connected through the connector; (7) The fusion protein has a sequence shown as SEQ ID NO. 5 or 47.
  11. 11. A nucleic acid molecule encoding the mutant of any one of claims 1-5 or the albumin or a portion or derivative thereof of claim 6 or the fusion protein of any one of claims 7-10.
  12. 12. A vector comprising the nucleic acid molecule of claim 11, preferably the vector is an expression vector; preferably, the vector is a eukaryotic bacterial vector (e.g., pPIC 9K).
  13. 13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12; preferably, the cell is a eukaryotic cell or a prokaryotic cell; Preferably, the eukaryotic cell is a yeast cell (e.g., saccharomyces cerevisiae, pichia pastoris); Preferably, the prokaryotic cell is an E.coli cell, a B.subtilis cell, or any combination thereof; Preferably, the cell is a mammalian cell (e.g., 293T cell).
  14. 14. A method of screening for the mutant of any one of claims 1 to 5 or the albumin or part thereof or derivative thereof of claim 6 or the fusion protein of any one of claims 7 to 10, the method comprising contacting the candidate mutant, or albumin or part thereof or derivative thereof, or the fusion protein with FcRn and detecting their affinity for FcRn; Preferably, a library of mutants comprising candidate mutants is constructed by a deep learning method, the candidate mutants are obtained using the host cell of claim 10, contacted with FcRn and tested for affinity with FcRn.
  15. 15. A complex comprising the mutant of any one of claims 1-5 or the albumin or a part or derivative thereof of claim 6 or the fusion protein of 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. 16. A delivery composition comprising: (1) A mutant according to any one of claims 1 to 5 or an albumin or part or derivative thereof according to claim 6 or a fusion protein according to any one of claims 7 to 10 or a nucleic acid molecule according to claim 11 or a vector according to claim 12 or a host cell according to claim 13, and (2) An immunogen (e.g., a polypeptide having immunogenicity, a nucleic acid encoding the polypeptide), or a large or small molecule drug (e.g., a small molecule drug having targeting), preferably the immunogen is an additional polypeptide as set forth in claim 8; Preferably, the component (1) is used as a delivery vehicle for the component (2).
  17. 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 targeting tumor cells; Preferably, the component (2) is delivered to a mucosal surface of a 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 mucoadhesives to enhance the residence time of component (2) at the mucosal surface of the subject; Preferably, the delivery composition may be prepared as a complex using biological modification, physical mixing, and/or chemical linking methods and drug or imaging probes.
  18. 18. A pharmaceutical composition comprising one or more selected from the group consisting of: (1) The mutant according to any one of claims 1 to 5; (2) The albumin or portion or derivative thereof of claim 6; (3) The fusion protein of any one of claims 7-10; (4) The nucleic acid molecule of claim 11; (5) The vector of claim 12; (6) The host cell of claim 13; (7) The complex of claim 15; Preferably, the pharmaceutical composition further comprises an immunogen (e.g., a polypeptide having immunogenicity, a nucleic acid encoding the polypeptide), or a macromolecule or small molecule drug (e.g., a small molecule drug having targeting); Preferably, the pharmaceutical composition has one or more features selected from the group consisting of: (1) The pharmaceutical composition is a drug targeted to gastrointestinal release or a drug controlled to release in the gastrointestinal tract; (2) The pharmaceutical composition further comprises a pharmaceutically acceptable carrier; (3) The pharmaceutical composition is in the form of a pill, powder, capsule, tablet (e.g., effervescent tablet), caplet, mouth-soluble granule, liquid, suppository, or enema; (4) The mode of administration of the pharmaceutical composition is selected from oral, intravenous, transmucosal delivery, sublingual, nasal, intrathecal, bronchial, rectal, transdermal, inhalation or parenteral; (5) The pharmaceutical composition is administered to a mucosal surface (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa) of a subject.
  19. 19. Use of the mutant of any one of claims 1-5, or the albumin or a portion or 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 a molecule for FcRn, or for increasing the half-life of a molecule; preferably, the molecule is selected from a small molecule drug, a large molecule drug (e.g., nanoparticle, peptide, protein), or any combination thereof; preferably, the molecule is a drug targeting tumor cells; Preferably, the tumor is selected from the group consisting of renal cell carcinoma, colorectal carcinoma, ovarian carcinoma, breast carcinoma, pancreatic carcinoma, gastric carcinoma, bladder carcinoma, esophageal carcinoma, mesothelioma, melanoma, head and neck carcinoma, thyroid carcinoma, sarcoma, prostate carcinoma, glioblastoma, cervical carcinoma, thymus carcinoma, leukemia, lymphoma, myeloma, primary mediastinal large B-cell lymphoma, T-cell/tissue cell enriched B-cell lymphoma, central Nervous System (CNS) tumor, spinal axis tumor, brain stem glioma.
  20. 20. Use of the mutant of any one of claims 1-5, or the albumin or a portion or 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, in the preparation of a pharmaceutical composition; preferably, the pharmaceutical composition further comprises an active molecule; preferably, the active molecule is selected from a small molecule drug, a large molecule drug (e.g., nanoparticle, peptide, protein), or any combination thereof; preferably, the mutant, the albumin or a portion or 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 mutant and application thereof Technical Field The present application relates to a mutant of albumin third Domain (DIII) or a part thereof or a derivative thereof, and albumin or a part thereof or a derivative thereof, fusion protein and complex comprising said mutant. The application also relates to the use of said mutants, fusion proteins and complexes for improving transmucosal delivery capacity, or for extending the half-life of molecules. Background Both "transmucosal delivery of drugs" and "long-acting circulation of drug molecules in vivo" have been two central difficulties in the field of drug delivery. On the one hand, for "drug transmucosal delivery", mucosal systems often have a layer-by-layer barrier to protect the exposure risk created by direct contact of the human body with the outside world. On the other hand, small molecule drugs and polypeptides, which account for 80-90% of all drug molecules, have a short half-life in vivo and require repeated and multiple administrations to maintain effective concentrations of the drug, which often causes hepatotoxicity and renal toxicity. Therefore, how to improve the 'drug transmucosal delivery efficiency' and the 'long-acting circulation time in drug molecules' is a significant key scientific problem in the field of drug delivery and even biological medicine. The classical pathway that generally increases the half-life of small molecules in vivo is to couple macromolecular substances, increasing their kinetic radius of hydration and thus prolonging their duration of circulation in vivo. Among them, coupling of endogenous macromolecular substances (e.g., antibodies, albumin) and the like is an effective strategy. Human Serum Albumin (HSA) is a non-glycosylated, globular water-soluble protein with a molecular mass of 66.5 kilodaltons (kD), encoded by the ALB gene, consisting of 585 amino acids. HSA is the most abundant protein in the human circulatory system, occupies half of serum protein, is synthesized mainly in the liver at a concentration of 30-50 mg/ml, and has a long-lasting circulatory half-life of about 19 days. Albumin-like, heart-shaped, consists of three highly homologous domains, each designated DI, DII, DIII, each consisting of two subunits, A, B subunits. Albumin plays a number of important roles in the human body, including maintaining osmotic pressure, plasma pH, transporting and partitioning a variety of endogenous and exogenous molecules, and the like. The above properties make HSA play an important role in the pharmacology of many drugs, from lipophilic small molecule drugs to nanoparticles, peptides and other biologicals, and the biological half-life is significantly increased after binding to HSA. The HSA crystal structure reveals hydrophobic drug and fatty acid binding sites, and the pharmacokinetic mechanisms of HSA effects are also widely documented. Various drugs and imaging probes have been designed to bind HSA to improve its half-life and drug delivery efficiency, and some have been approved for clinical use by the U.S. Food and Drug Administration (FDA). For many small molecule drugs, binding to HSA can be a strategy to improve therapeutic efficacy while reducing systemic toxicity. Chemotherapeutic nanodrug HSA-conjugated paclitaxel (abaxane) was used to treat various types of metastatic cancers and was approved by the FDA in 2005. As another example, fatty acid peptide coupled drugs, ins detemir (Levemir) and Liraglutide (Victoza), were approved for the treatment of diabetes in 2005 and 2010, respectively. Semagrutide (Ozenpic) increases HSA binding affinity of Liraglutide by about 6-fold, exhibits a relatively prolonged circulatory half-life, and was approved by the us FDA for the treatment of type 2 diabetes in 2017. In the last two years, albumin formulations of mammalian target of rapamycin (mTOR) kinase inhibitors, rapamycin (sirolimus albumin binding particle suspensions for injection, fyarro) were FDA approved in 2021 for the treatment of perivascular epithelial-like cell neoplasms (PE carpinomas). Meanwhile, in the field of biomedical imaging, various HSA coupled probes are developed and applied at present, and although most related imaging probes are only tested before clinical treatment, some of the probes have been subjected to clinical tests or are approved by regulatory authorities. In the field of optical imaging, for example, indocyanine green (ICG), a representative near infrared fluorescence imaging probe, can be reversibly combined with albumin, is currently approved by FDA for tissue perfusion, lymph node assessment and other aspects, and is also respectively in progress in photodynamic therapy and in-operation clinical trials of tumor tissue margin and tissue perfusion assessment at I, II stage, such as Evans Blue T-1824, and is also reversibly combined with albumin for vascular permeability (MILES ASSAY) imaging, A preclinical test for bright field imaging is also underway,