Screening and use of albumin mutant

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.

Description

Screening of albumin mutants and their applications Technical Field

[0001] 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

[0002] "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.

[0003] 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.

[0004] These properties make HSAs play an important role in the pharmacology of many drugs, from lipophilic small molecule drugs to nanoparticles, peptides, and other biologics, significantly improving their biological half-life after binding to HSAs. 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 be a strategy to improve therapeutic efficacy while reducing systemic toxicity. The chemotherapy nanodrug nab-paclitaxel (Abraxane), bound to an HSA, is used to treat various types of metastatic cancer and was approved by the FDA in 2005. Similarly, fatty acid peptide conjugates such as Insulin 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 miles assay imaging and bright-field imaging. The MRI imaging probe Gadofosveset trisodium (ABLAVAR) is an albumin-reversibly binding gadolinium chelator and has been approved by the FDA for use in magnetic resonance angiography; its Phase I clinical trial for tumor and lymph node staging imaging is also underway. The PET probe, radiolabeled somatostatin 177Lu-DOTA-EB-TATE, has entered Phase I clinical trials for targeted radionuclide therapy and imaging of neuroendocrine tumors. The SPECT probe Albamin iodine... I-131serum (MEGATOPE), iodinated albumin, has been approved by the FDA for use in blood pooling and assessment of plasma volume and cardiac output. Albumin has gained increasing popularity as a drug carrier in recent years, and some albumin biologics have been widely approved and used clinically, benefiting numerous patients.

[0005] However, smaller molecules are generally easier to deliver across mucosa, which seems contradictory to increasing their hydration radius to achieve long-lasting circulation. Furthermore, the in vivo half-life and pharmacokinetic characteristics after transmucosal delivery remain challenging problems in the field of drug delivery. Summary of the Invention

[0006] This invention provides a novel strategy to simultaneously improve transmucosal delivery efficiency and in vivo drug half-life. The invention relates to a third domain (DIII) mutant (DIIIv) of albumin, or a mutant of its portion or derivatives (DIIIvs), for conjugation / binding / fusion of small molecule drugs, peptide drugs, protein drugs, and nucleic acid drugs. The mutant DIIIv and its derivative DIIIvs exhibit higher affinity for the neonatal Fc receptor (FcRn) compared to natural albumin. The mutants demonstrate good stability, resistance to lysosomal degradation, and suitability for mucosal delivery of drugs / imaging molecules.

[0007] mutant

[0008] In a first aspect, this application provides a mutant (DIIIv / DIIIvs) of the third domain (DIII) of albumin or a portion thereof (e.g., DIIIb) or a derivative thereof, which, compared with the natural albumin DIII or a portion thereof, contains the following mutations: the amino acids 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 the natural albumin corresponding to positions 497 to 585 of SEQ ID NO:1 have amino acid insertions, substitutions, deletions and / or mutations.

[0009] In some embodiments, the mutant, compared to native albumin DIII or a portion thereof, contains the following mutation: the native 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.

[0010] In some embodiments, the DIII portion comprises a fragment of natural albumin corresponding to amino acids 467 through 585 of SEQ ID NO:1.

[0011] In some embodiments, the mutant exhibits a higher FcRn affinity compared to native albumin or DIII. Those skilled in the art understand suitable methods for determining whether the mutant's affinity for FcRn is higher or lower than that of native albumin. An exemplary approach is to determine and compare the binding constant Kd. Therefore, according to the invention, mutants with Kd lower than that of native albumin or its DIII are considered to have a longer plasma half-life than native albumin or its DIII, and mutants with Kd higher than that of native albumin or its DIII are considered to have a shorter plasma half-life than native albumin or its DIII.

[0012] In some embodiments, the mutant contains 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.

[0013] In some embodiments, the mutant contains an amino acid mutation at a position 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 native albumin corresponding to SEQ ID NO:1.

[0014] In some embodiments, the mutant contains amino acid mutations at positions 523 and 573.

[0015] In some embodiments, the mutant contains amino acid mutations at positions 505, 523, 547, and 573.

[0016] In some embodiments, the mutant contains amino acid mutations at positions 500, 505, 523, 524, 527, 528, 531, 547, and 573.

[0017] In some implementations, the amino acid mutation is a conserved mutation.

[0018] The term "conservative mutation" refers to the interchangeability of amino acid residues within a specific group.

[0019] In some embodiments, amino acids can be classified into the following groups: basic amino acids (e.g., arginine, lysine, histidine), acidic amino acids (e.g., glutamic acid, aspartic acid), polar amino acids (e.g., glutamine and asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, valine), aromatic amino acids (e.g., phenylalanine, tryptophan, and tyrosine), and small amino acids (e.g., glycine, alanine, serine, threonine, methionine).

[0020] In some embodiments, the mutant has an amino acid, L or D, at position 500 of natural albumin corresponding to SEQ ID NO:1.

[0021] In some embodiments, the mutant has an amino acid at position 505 of SEQ ID NO:1 in natural albumin that is Q, N, or T.

[0022] In some embodiments, the mutant has an amino acid, L or M, at position 523 of native albumin corresponding to SEQ ID NO:1.

[0023] In some embodiments, the mutant has an amino acid L at position 524 of natural albumin corresponding to SEQ ID NO:1.

[0024] In some embodiments, the mutant has amino acid K at position 527 of natural albumin, corresponding to SEQ ID NO:1.

[0025] In some embodiments, the mutant has an amino acid, H or Y, at position 528 of natural albumin corresponding to SEQ ID NO:1.

[0026] In some embodiments, the mutant has an amino acid L at position 531 of natural albumin corresponding to SEQ ID NO:1.

[0027] In some embodiments, the mutant has an amino acid, A or C, at position 547 of native albumin corresponding to SEQ ID NO:1.

[0028] In some embodiments, the mutant has an amino acid L at position 509 of SEQ ID NO:1 in natural albumin.

[0029] In some embodiments, the mutant has an amino acid, R or N, at position 510 of natural albumin corresponding to SEQ ID NO:1.

[0030] In some embodiments, the mutant has amino acid E at position 498 of SEQ ID NO:1 in natural albumin.

[0031] In some embodiments, the mutant has amino acid G at position 512 of natural albumin corresponding to SEQ ID NO:1.

[0032] In some embodiments, the mutant has an amino acid other than K (e.g., P) at position 573 of natural albumin corresponding to SEQ ID NO:1.

[0033] In some embodiments, the mutant has an amino acid L at position 508 of natural albumin corresponding to SEQ ID NO:1.

[0034] In some embodiments, the amino acid at the 500th position is mutated from K to L or D.

[0035] In some embodiments, the amino acid at position 505 is mutated from E to Q, N, or T.

[0036] In some embodiments, the amino acid at position 523 is mutated from I to L or M.

[0037] In some implementations, the amino acid at position 524 is mutated from K to L.

[0038] In some implementations, the amino acid at position 527 is mutated from T to K.

[0039] In some implementations, the amino acid at position 528 is mutated from A to H or Y.

[0040] In some embodiments, the amino acid at position 531 is mutated from E to L.

[0041] In some implementations, the amino acid at position 547 is mutated from V to A or C.

[0042] In some implementations, the amino acid at position 509 is mutated from F to L.

[0043] In some embodiments, the amino acid at position 510 is mutated from H to R or N.

[0044] In some implementations, the amino acid at position 498 is mutated from V to E.

[0045] In some implementations, the amino acid at position 512 is mutated from D to G.

[0046] In some embodiments, the amino acid at position 573 is mutated from K to any amino acid other than K (e.g., P).

[0047] In some implementations, the amino acid at position 508 is mutated from T to L.

[0048] In some implementations, the mutant contains the mutations I523G and K573P.

[0049] In some embodiments, the mutant contains mutations such as E505Q, I523G, V547A, and K573P.

[0050] In some embodiments, the mutant includes mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P.

[0051] In some embodiments, the mutant contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, V547C, and K573P. In some embodiments, the mutant contains mutations such as E505N, T508L, F509L, H510R, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the mutant contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505T,I523L,T527K,A528H,E531L,K573P. In some embodiments, the mutant contains the mutations K500L,E505T,I523L,T527K,A528H,E531L,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505Q,I523L,K524L,T527K,A528Y,V547A,K573P. In some embodiments, the mutant contains the mutations E505Q,I523L,T527K,A528H,V547A,K573P. In some embodiments, the mutant contains the mutations K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the mutant contains the mutations V498E, K500L, E505Q, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the mutant contains the mutations E505T, I523L, T527K, A528Y, and K573P. In some embodiments, the mutant contains the mutations E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the mutant contains the mutations K500L, E505T, I523L, T527K, A528Y, E531L, and K573P. In some embodiments, the mutant contains the mutations K500L, E505Q, T508L, F509L, H510R, D512G, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the mutant contains the mutations K500D, E505T, I523L, T527K, A528H, and K573P.In some embodiments, the mutant contains the mutations V498E,K500L,E505T,I523L,T527K,A528Y,E531L,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505T,I523L,T527K,A528Y,K573P. In some embodiments, the mutant contains the mutations K500L,E505N,I523L,K524L,T527K,A528H,E531L,V547A,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505N,I523L,T527K,A528H,E531L,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505T,A511T,I523L,T527K,A528H,E531L,K573P. In some embodiments, the mutant contains the mutations E505N,I523L,K524L,T527K,A528H,V547A,K573P. In some embodiments, the mutant contains the mutations E505Q,I523M,K524L,T527K,A528H,E531L,V547A,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505Q,I523L,K524L,T527K,A528H,E531L,V547A,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505T,I523L,T527K,A528H,K573P. In some embodiments, the mutant contains the mutations V498E,K500L,E505Q,F507V,T508K,F509L,H510N,D512G,I523L,K524L,T527K,A528H,E531L,K573P. In some embodiments, the mutant contains the mutation K573P. In some embodiments, the mutant contains mutations such as E505Q, T527M, K573P. In some embodiments, the mutant contains mutations such as E505Q, I523G, T527M, V547A, K573P.

[0052] In some embodiments, the natural albumin is derived from the natural serum albumin of mammals.

[0053] In some embodiments, the mammal is selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs, and pigs.

[0054] In some embodiments, the natural albumin is derived from natural human serum albumin.

[0055] In some embodiments, the natural albumin comprises, or consists of, sequences selected from, the following:

[0056] (i) The sequence shown in SEQ ID NO:1;

[0057] (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;

[0058] (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.

[0059] In some embodiments, the natural albumin DIII comprises, or consists of, sequences selected from, the following:

[0060] (i) The sequence shown in SEQ ID NO:2;

[0061] (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;

[0062] (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.

[0063] In some embodiments, the natural albumin DIIIb comprises, or consists of, sequences selected from, the following:

[0064] (i) The sequence shown in SEQ ID NO:4;

[0065] (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: 4;

[0066] (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: 4.

[0067] In some embodiments, the mutant comprises a sequence as shown in any one of SEQ ID NO:12-16, 21-46, or 49-53.

[0068] Albumin or a portion thereof or a derivative thereof

[0069] In a second aspect, this application provides an albumin or a portion thereof or a derivative thereof comprising the mutant described in the first aspect.

[0070] In some embodiments, 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.

[0071] In some implementations, the DI and DII are each independently derived from mammalian natural serum albumin.

[0072] In some embodiments, the mammal is selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs, and pigs.

[0073] In some embodiments, the natural albumin is as defined in the first aspect.

[0074] Fusion protein

[0075] In a third aspect, this application provides a fusion protein comprising the mutant described in the first aspect or the albumin or a portion thereof or a derivative thereof described in the second aspect, as well as additional polypeptides or proteins.

[0076] In some embodiments, 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.

[0077] In some embodiments, the connector has a sequence as shown in SEQ ID NO:10 or 11.

[0078] In some embodiments, 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.

[0079] In some implementations, the label is selected from HA, Myc, His, GST, or any combination thereof.

[0080] In some embodiments, the tag has a sequence as shown in any one of SEQ ID NO:6-9.

[0081] In some embodiments, 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.

[0082] In some embodiments, the peptide may be expressed fused to the N or C terminus, for example: peptide-DIII-peptide. The peptide may also be expressed in the middle of a tandem DIII, for example: -(peptide-DIII)n-, where n is an independent positive integer (e.g., 1, 2, 3, 4, 5 or greater).

[0083] In some embodiments, the RGD peptide comprises, for example, (GGGGS). m (RGD) n The sequence shown is where m and n are each an independent positive integer (e.g., 1, 2, 3, 4, 5 or greater).

[0084] In some embodiments, the RGD peptide has a sequence as shown in SEQ ID NO:20.

[0085] In some embodiments, the zinc finger protein comprises (GGGGS). m (ZF9) n The sequence shown is where m and n are each an independent positive integer (e.g., 1, 2, 3, 4, 5 or greater).

[0086] In some embodiments, the nucleic acid binding protein or polypeptide may be fused with the NLS and other nuclear localization signal peptides in different ways (e.g., ZF9-NLS, NLS-ZF9).

[0087] In some embodiments, the nucleic acid binding protein or polypeptide may 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).

[0088] In some embodiments, the NLS and other nuclear localization signal peptides may 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).

[0089] In some embodiments, the fusion protein comprises one or more (e.g., two, three, four, five) 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., two, three, four, five).

[0090] In some embodiments, the fusion protein comprises multiple mutants or multiple albumins or portions thereof or derivatives thereof, and they are linked together by the polypeptide.

[0091] In some embodiments, the fusion protein comprises one of the mutants or albumin or a portion thereof or a derivative thereof, and one or two of the polypeptides.

[0092] In some embodiments, the fusion protein has a sequence as shown in SEQ ID NO:17 or 18.

[0093] In some embodiments, 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.

[0094] In some embodiments, the fusion protein has a sequence as shown in SEQ ID NO:19.

[0095] In some embodiments, the fusion protein comprises multiple mutants or multiple albumins or portions thereof or derivatives thereof, and they are linked together by the linker.

[0096] In some embodiments, the fusion protein has a sequence as shown in SEQ ID NO:5 or 47.

[0097] Nucleic acid molecules, vectors, and host cells

[0098] In a fourth aspect, this application provides a nucleic acid molecule that encodes the mutant described in the first aspect, or the albumin described in the second aspect, or a portion thereof or a derivative thereof, or the fusion protein described in the third aspect.

[0099] In a fifth aspect, this application provides a carrier comprising the nucleic acid molecules described in the fourth aspect.

[0100] In some implementations, the vector is an expression vector.

[0101] In some embodiments, the vector is a vector of eukaryotic bacteria (e.g., Pichia pastoris).

[0102] The techniques used to prepare the mutants or fusion proteins of this application are conventional in the art, for example, those disclosed in WO 2009019314 (included by reference). In addition, albumin has been successfully expressed as a recombinant protein in a range of hosts, including fungi (e.g., Aspergillus (WO06066595), Klebsiella pneumoniae (Fleer 1991, Bio / technology 9, 968-975), Pichia pastoris (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) and yeasts (Sleep 1990, Bio / technology 8, 42-46)), bacteria (Pandjaitab 2000, J. Allergy Clin. Immunol 105, 279-285), animals (Barash 1993, Transgenic Research 2, 266-276) and plants (e.g., potato and tobacco (Sijmons 1990, Bio / technology 8, 217 and Farran 2002, Transgenic Research 11, 337-346)). In principle, any host cell capable of producing a suitable amount of polypeptide can be used to prepare the mutant or fusion protein of this application.

[0103] Therefore, in a sixth aspect, this application provides a host cell comprising the nucleic acid molecule described in the fourth aspect or the vector described in the fifth aspect.

[0104] In some embodiments, the cells are eukaryotic or prokaryotic cells.

[0105] In some embodiments, the eukaryotic cells are yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris).

[0106] In some embodiments, the prokaryotic cells are Escherichia coli cells, Bacillus subtilis cells, or any combination thereof.

[0107] In some embodiments, the cells are mammalian cells (e.g., 293T cells).

[0108] In a seventh aspect, this application provides a method for screening the mutants described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or fusion proteins described in the third aspect, 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.

[0109] In some implementations, a mutant library containing candidate mutants is constructed using deep learning methods, the candidate mutants are obtained using the host cells described in the sixth aspect, they are contacted with FcRn, and their affinity for FcRn is detected.

[0110] complex

[0111] In an eighth aspect, this application provides a complex comprising the mutant described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or the fusion protein described in the third aspect, and a molecule (e.g., a dye molecule) bound thereto.

[0112] In some embodiments, the dye molecule is a cyanine dye molecule.

[0113] Delivery composition

[0114] The mutants or fusion proteins of this invention can be coupled to immunogens (e.g., antigens) using techniques known in the art. Furthermore, this invention is not limited to using specific immunogens. Any immunogen can be used, including but not limited to immunogens derived from microorganisms (e.g., pathogenic microorganisms), tumors (e.g., for cancer vaccines), and so on.

[0115] Therefore, in a ninth aspect, this application provides a delivery composition comprising:

[0116] (1) The mutant described in the first aspect, or the albumin or a portion thereof or a derivative thereof described in the second aspect, or the fusion protein described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the vector described in the fifth aspect, or the host cell described in the sixth aspect, and,

[0117] (2) 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).

[0118] In some embodiments, the immunogen is another polypeptide as described above.

[0119] In some embodiments, the component (1) serves as a delivery carrier for component (2).

[0120] In some implementations, the immunogen is able to induce an immune response in the subject.

[0121] In some embodiments, the immunogen is derived from influenza virus, coronavirus, hepatitis B virus, or any combination thereof.

[0122] In some embodiments, the macromolecular or small molecule drug is a drug that targets tumor cells.

[0123] In some embodiments, 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).

[0124] In some embodiments, the delivery composition is delivered via intranasal or inhalation.

[0125] In some embodiments, the delivery composition is delivered via the oral or gastrointestinal mucosal epithelium.

[0126] In some embodiments, 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.

[0127] In some embodiments, the delivery composition may be prepared as a complex using biomodification, physical mixing, and / or chemical linking methods with a drug or imaging probe.

[0128] In some embodiments, the specific selection of the mucosal adhesive may be referenced, for example, U.S. Patent Application No. 20050281843, which is incorporated herein by reference in its entirety. Mucosal adhesives include (but are not limited to) polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbriae, and carboxymethyl cellulose.

[0129] In some implementations, the use of mucosal adhesives can also enhance the immune response of the subject. In some implementations, the use of mucosal adhesives results in a longer duration of exposure to immunogens and / or an increased amount of immunogens experienced by the subject compared to the absence of mucosal adhesives.

[0130] Pharmaceutical Composition

[0131] In a tenth aspect, this application provides a pharmaceutical composition comprising one or more of the following:

[0132] (1) The mutant described in the first aspect;

[0133] (2) Albumin or a portion thereof or a derivative thereof as described in the second aspect;

[0134] (3) The fusion protein described in the third aspect;

[0135] (4) The nucleic acid molecules described in the fourth aspect;

[0136] (5) The carrier described in the fifth aspect;

[0137] (6) The host cell described in the sixth aspect;

[0138] (7) The complex described in aspect eight;

[0139] In some embodiments, 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).

[0140] In some embodiments, the pharmaceutical composition is a drug that targets gastrointestinal release or a drug that is released in a controlled manner in the gastrointestinal tract.

[0141] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0142] In some embodiments, 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.

[0143] In some embodiments, the pharmaceutical composition is administered via a method selected from oral, intravenous, transmucosal, sublingual, nasal, intrathecal, bronchial, rectal, percutaneous, inhalation, or parenteral administration.

[0144] In some embodiments, the pharmaceutical composition is applied to the mucosal surface of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa).

[0145] In some embodiments, the pharmaceutical compositions of the present invention are administered via mucosal delivery. Specific methods of administration can be found, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995, concerning mucosal delivery techniques, including intranasal mucosal delivery techniques.

[0146] In some embodiments, the pharmaceutical compositions of the present invention are administered via the dermis or transdermis. For specific administration methods, see, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995.

[0147] use

[0148] In another aspect, this application provides the use of the mutant described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or fusion protein described in the third aspect, or nucleic acid molecule described in the fourth aspect, or vector described in the fifth aspect, or host cell described in the sixth aspect, or complex described in the eighth aspect, or delivery composition described in the ninth aspect, or pharmaceutical composition described in the tenth aspect, for increasing the affinity of the molecule for FcRn, or prolonging the half-life of the molecule.

[0149] In some embodiments, the molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof.

[0150] In some embodiments, the molecule is a drug that targets tumor cells.

[0151] In some embodiments, 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.

[0152] In another aspect, this application provides the use of the mutant described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or fusion protein described in the third aspect, or nucleic acid molecule described in the fourth aspect, or vector described in the fifth aspect, or host cell described in the sixth aspect, or complex described in the eighth aspect, or delivery composition described in the ninth aspect, in the preparation of a pharmaceutical composition.

[0153] In some embodiments, the pharmaceutical composition further comprises an active molecule.

[0154] In some embodiments, the active molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof.

[0155] In some embodiments, 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.

[0156] method

[0157] On the other hand, this application provides a method for increasing the affinity of a molecule for FcRn or prolonging the half-life of a molecule, the method comprising:

[0158] The mutant described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or the fusion protein described in the third aspect, or the nucleic acid molecule described in the fourth aspect, or the vector described in the fifth aspect, or the host cell described in the sixth aspect, or the complex described in the eighth aspect, or the delivery composition described in the ninth aspect, is fused, conjugated or covalently bound to the molecule.

[0159] In some embodiments, the molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof.

[0160] In another aspect, this application provides a method for preparing the complex described in the eighth aspect, the method comprising: reacting the mutant or the albumin or a portion thereof or a derivative thereof or the fusion protein with the cyanine dye molecule at room temperature or under heating conditions.

[0161] In another aspect, this application provides a method for targeting cells, the method comprising contacting the complex described in the eighth aspect with the cells; optionally, after contact, irradiating the cells with a laser to obtain an image of the cells.

[0162] In some embodiments, the complex targets the cell by binding to cell surface molecules, cell surface proteins, or cell surface receptors expressed on the cell.

[0163] In some embodiments, the cells are selected from stem cells, proliferating cells, cells in the process of proliferation, inflammatory cells, negatively regulating immune cells, cells infected by pathogens, neurons, adipocytes, or lipid cells.

[0164] In some implementations, the cells are tumor cells.

[0165] In some embodiments, 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.

[0166] In some implementations, the cells are present in tissues or living organisms.

[0167] On the other hand, this application provides the use of the mutant described in the first aspect, or albumin or a portion thereof or a derivative thereof described in the second aspect, or fusion protein described in the third aspect, or nucleic acid molecule described in the fourth aspect, or vector described in the fifth aspect, or host cell described in the sixth aspect, or complex described in the eighth aspect, in the preparation of a kit for diagnosing whether a subject is infected with a pathogen or has a disease caused by the pathogen infection (e.g., a respiratory disease).

[0168] In some embodiments, the method of administration of the kit is selected from oral, sublingual, nasal, intrathecal, bronchial, rectal, transdermal, inhalation, parenteral, or any combination thereof.

[0169] In some embodiments, the complex targets the cell by binding to cell surface molecules, cell surface proteins, or cell surface receptors expressed on the cell.

[0170] In some embodiments, the cells are selected from stem cells, proliferating cells, cells in the process of proliferation, inflammatory cells, negatively regulating immune cells, cells infected by pathogens, neurons, adipocytes, or lipid cells.

[0171] In some implementations, the cells are tumor cells.

[0172] In some embodiments, 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.

[0173] In some implementations, the cells are present in tissues or living organisms.

[0174] In another aspect, this application provides an imaging method comprising using the complex described in the eighth aspect as an imaging developer.

[0175] In some implementations, the imaging is near-infrared II fluorescence imaging.

[0176] In some embodiments, the imaging method is fluorescence imaging of cells, tissues, or living organisms.

[0177] Terminology Definition

[0178] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0179] As used herein, the term "albumin" refers to a protein whose three-dimensional structure is substantially the same as that of HSA. Examples of albumin proteins according to the invention include (but are not limited to) human serum albumin, primate serum albumin (e.g., chimpanzee serum albumin, gorilla serum albumin), rodent serum albumin (e.g., rabbit serum albumin, mouse albumin, and rat serum albumin), bovine serum albumin, horse serum albumin, donkey serum albumin, hamster serum albumin, goat serum albumin, sheep serum albumin, canine serum albumin, guinea pig serum albumin, chicken serum albumin, and porcine serum albumin.

[0180] As used herein, the terms "albumin third domain" and "DIII" have the same meaning and are used interchangeably. The serum albumin molecule consists of three homologous domains (DI, DII, DIII), with DIII being the C-terminal domain. In some embodiments, the position of DIII corresponds to amino acids 381 through 585 of SEQ ID NO:1.

[0181] Furthermore, the term "DIIIa" refers to the N-terminal subdomain of DIII, and the term "DIIIb" refers to the C-terminal subdomain of DIII. In some embodiments, the position of DIIIb corresponds to amino acids 467 to 585 of SEQ ID NO:1.

[0182] As used herein, the term "fragment of albumin" refers to an albumin portion that retains the ability to bind FcRn. The fragment may consist of a continuous sequence derived from the same albumin, or a fragment comprising two or more sequences derived from different albumins. In some embodiments, the fragment is at least 20 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 75 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, or at least 500 amino acid residues in length. In some embodiments, the fragment comprises or consists of at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of albumin or the albumin third domain. In some exemplary embodiments, the first domain of albumin is a domain consisting of amino acids 1-194 (±1-15) of the amino acid residues shown in SEQ ID NO: 1, or a domain with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with such amino acids. In some exemplary embodiments, the second domain of albumin is a domain consisting of amino acids 192-387 (±1-15) of the amino acid residues shown in SEQ ID NO: 1, or a domain with at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with such amino acids. For a more detailed classification of the first, second, and third domains of albumin, please refer to the descriptions by Dockal et al. (The Journal of Biological Chemistry, 1999, Vol. 274(41): 29303-29310) or Kjeldsen et al. (Protein Expression and Purification, 1998, Vol 13: 163-169).

[0183] As used herein, the terms "variant" or "mutant," when referring to a polypeptide / protein, are amino acid sequences that differ from another related polypeptide / protein by one or more amino acids. Furthermore, amino acid mutations are named in this document using the following convention: original amino acid, position, substituted amino acid. Therefore, a lysine at position 573 substituted with proline is referred to as "K573P".

[0184] As used herein, the term "conserved mutation" refers to an amino acid substitution within a specific group of amino acids that generally does not substantially affect protein function. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic-hydroxy side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; non-natural amino acids such as p-aminobenzoic acid; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. Therefore, in some embodiments, conserved mutations include, but are not limited to, mutations between the following amino acids: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0185] As used herein, the terms “wild” and “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, polypeptides, or proteins, they mean that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing. As used herein, natural human serum albumin refers to naturally occurring, biologically active human serum albumin. Those skilled in the art can readily obtain the amino acid sequence of human serum albumin from various public databases (e.g., the GenBank database). For example, the amino acid sequence of natural human serum albumin may be as shown in SEQ ID NO: 1. The amino acid sequence of wild-type human serum albumin can be readily obtained from various public databases (e.g., the GenBank database). In some embodiments, the GenBank database number for human serum albumin is AEE60908.1.

[0186] Those skilled in the art will understand that natural human serum albumin can have multiple versions, which have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), as well as substantially the same biological function, but may still have minor differences in their amino acid sequences. Therefore, in this application, natural human serum albumin is not limited to the protein shown in SEQ ID NO: 1, but is intended to cover all known natural human serum albumin.

[0187] Therefore, in this application, the term "natural human serum albumin" should include, for example, the natural human serum albumin shown in SEQ ID NO: 1 and its naturally occurring variants. Furthermore, when describing the amino acid positions of natural albumin, it includes not only the specific amino acid position in SEQ ID NO: 1, but also the amino acid position in its natural variant that corresponds to the specific amino acid position. For example, the expression "the 500th amino acid residue in natural albumin" includes the 500th amino acid residue of SEQ ID NO: 1 and the corresponding amino acid position in its natural variant. According to this application, the expression "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when performing optimal sequence alignment, i.e., when the sequences are aligned to obtain the highest percentage identity. Similarly, the expression "the position corresponding to the 500th position of SEQ ID NO: 1" refers to the amino acid position in the compared sequence that is equivalent to the 500th position of SEQ ID NO: 1 when performing optimal alignment of a sequence with SEQ ID NO: 1, i.e., when a sequence is aligned with SEQ ID NO: 1 to obtain the highest percentage identity.

[0188] As used herein, the term "identity" refers to the sequence matching between two polypeptides or proteins, or between two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomeric subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum possible identity. Such comparisons can be performed using, for example, the method readily available through computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. Alternatively, the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0) can be used to determine the percentage identity between two amino acid sequences using a PAM120 weighted residue table, a 12-bit nick length penalty, and a 4-bit nick penalty. In addition, the Needleman and Wunsch (J MoI Biol.48:444-453(1970)) algorithm, which is integrated into the GAP program (available at www.gcg.com), can be used to determine the percentage identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6.

[0189] As used in this article, the term "cysteine" can also be abbreviated as "Cys," and it is a common amino acid found in living organisms. Cysteine ​​is the only amino acid among the more than 20 amino acids that make up proteins that has a reducing group, the thiol group (-SH).

[0190] As used in this article, the term "thiol group," also known as thiosulfate group or thiol group, is a negatively charged functional group consisting of a sulfur atom and a hydrogen atom bonded together, with the chemical formula -SH.

[0191] As used in this article, the term "protein tertiary structure" refers to the further coiling or folding of the polypeptide chain of a protein into a three-dimensional spatial structure with certain regularity based on various secondary structures.

[0192] As used in this article, the term "protein quaternary structure" refers to the spatial structure of a protein composed of two or more independent tertiary structures, which are linked together by secondary bonds.

[0193] As used in this article, the term "subunit" refers to each polypeptide chain in the quaternary structure of a protein, which has its own independent tertiary structure.

[0194] As used herein, the term "domain" refers to a basic unit that constitutes the tertiary structure of a protein and has a unique spatial conformation. Typically, different domains of a protein are spatially distinguishable. In some embodiments, when the protein consists of multiple polypeptide chains, the domains of the protein contain multiple subunits.

[0195] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and viral vectors. Viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0196] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0197] As used herein, the term "immunogen" refers to a factor (e.g., a microorganism (e.g., bacteria, virus, or fungus)) and / or a portion or component thereof (e.g., protein, nucleic acid)) capable of eliciting an immune response in a subject. In some embodiments, an immunogen elicits an immune response in a subject against the immunogen (e.g., a microorganism (e.g., a pathogen or pathogen product)).

[0198] As used herein, the term "FcRn" refers to a heterodimeric protein composed of a heavy chain and a light chain. In humans, the FcRn heavy chain is encoded by the Fcγ receptor and transporter (FCGRT) gene, located on chromosome 19q13.35, and is approximately 45 kDa in size. This gene contains seven exons and six introns, with exons 2-5 encoding the signal sequence and the extracellular α1-α2-α3 domain, exon 6 encoding the transmembrane region, and exon 7 encoding the intracellular tail. The FCGRT homolog is present in most mammals, and the encoded protein exhibits high amino acid conservation with the human receptor. The light chain, approximately 12 kDa, is encoded by the β2-microglobulin (β2m) gene. The α1-α2-α3 region of FcRn shows high structural homology to MHC class I molecules and non-covalently binds to β2-microglobulin (β2m) to form a heterodimer. FcRn is widely distributed in human tissues, including parenchymal tissues (epithelial cells, endothelial cells, hepatocytes, etc.) and hematopoietic cells. The main functions of FcRn in the human body are active and passive, namely active immunity and passive transport. pH-dependent ligand binding is crucial for both active and passive immune functions of FcRn. Its active functions include the transport of IgG-immune complexes (IgG-IC) and assisting in the presentation of IgG-IC, as well as the enhancement of innate and adaptive immunity. Passive functions include transport and endocytosis, preventing intracellular degradation of monomeric IgG and albumin and facilitating transcellular transport. This explains why both have significantly longer biological half-lives in the human body compared to most other proteins.

[0199] The relatively long biological half-life of HSA is mainly due to its recycling mediated by the neonatal Fc receptor (FcRn). FcRn is expressed in endothelial cells and other cell types, and its tight binding to HSA mediates its translocation across polar cell barriers. Furthermore, IgG, the most abundant antibody subtype in the human body, is similar to HSA, accounting for 10-20% of total serum protein. Through high affinity binding to FcRn, its circulating half-life is approximately 23 days. Compared to IgG and its Fc fragment conjugate complex, studies have shown that HSA exhibits better FcRn-mediated transmucosal transport efficiency.

[0200] HSA and IgG exhibit a strict pH dependence on FcRn. At physiological pH levels (e.g., in the extracellular environment of pH 7.4), they do not bind to FcRn or bind very little. However, under acidic pH conditions (e.g., in intracellular endosomes below pH 6.5), they show a very strong affinity for FcRn. Therefore, after HSA and IgG are taken up by cells, they bind to FcRn in the acidic intracellular endosomes and are transported to the extracellular environment via FcRn-mediated transport. They are then released at the physiological extracellular pH, thus achieving the function of circulating HSA and IgG and preventing their degradation within the cell like other serum proteins. This is the physiological basis for the long circulation of HSA and IgG. Furthermore, the binding sites of HSA, IgG, and FcRn do not overlap and do not interfere with each other. The binding sites of HSA and FcRn are mainly the interaction between the DIII domain of HSA and the α1 domain of FcRn, while the binding sites of IgG and FcRn are mainly located in the Fc segment (CH2,CH3) of IgG and the α1 and β2M domains of FcRn. Therefore, the transport and circulation of the two do not affect each other.

[0201] Beneficial effects of the invention

[0202] Compared to existing technologies, the mutant prepared in this application exhibits high FcRn affinity, showing stronger pH-dependent FcRn binding affinity compared to wild-type albumin, which is widely used as a drug carrier. Furthermore, it enhances the transmucosal delivery of small molecule drugs and functional protein drugs chemically conjugated or fused with it, while also increasing their biological half-life and prolonging serum drug stability. This results in a more uniform drug concentration in serum, leading to less frequent medication use, reduced potential drug toxicity, and improved patient compliance.

[0203] Furthermore, compared to full-length albumin, the mutant in this application has a smaller molecular weight, thus reducing the likelihood of steric hindrance when fused with functional protein drugs and expanding the molecular weight range of functional proteins that can be fused with albumin, thereby possessing better application potential. For example, as a therapeutic vaccine carrier, albumin and its variants do not compete with endogenously generated immune effect IgG antibodies for FcRn binding sites, theoretically resulting in better efficacy.

[0204] Furthermore, compared to traditional full-length IgG or IgG Fc conjugates, albumin and its variants have better transport and penetration efficiency in specific tissues (such as mucosal epithelial tissue), thereby achieving better drug target cell delivery efficiency and improving efficacy.

[0205] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0206] Figure 1 shows an example of the DIIIv Saccharomyces cerevisiae plasmid vector pYD1 constructed in Example 1.

[0207] Figures 2A to 2C show the fluorescence signals and their ratio changes of expression and binding of the initial library and different rounds of screening on the yeast surface.

[0208] Figure 3 shows an example of the DIII mutant Pichia pastoris plasmid vector pPIC9K constructed in Example 2.

[0209] Figure 4 shows the isolation and purification results of the Pichia pastoris expressing the DIII mutant.

[0210] Figures 5A and 5B show the results of intracellular colocalization of DIIIv and FcRn using fluorescence confocal microscopy. For ease of labeling, the sample names in the figures are abbreviated. V1 corresponds to D3V27, V2 to D3V28, V3 to D3V29, V4 to D3V30, and V5 to D3V31.

[0211] Figures 6A and 6B show the fluorescence intensity results of samples with different mutants and different cells detected by a near-infrared imager. For ease of labeling, the sample names in the figures are abbreviated. V1 corresponds to D3V27, V2 corresponds to D3V28, V3 corresponds to D3V29, V4 corresponds to D3V30, and V5 corresponds to D3V31.

[0212] Figure 7 shows the detection signal of DIIIv in different parts of mouse duodenum tissue.

[0213] Figure 8 shows the efficient transmembrane entry of DIIIv into the mouse circulation after intranasal administration.

[0214] Figure 9 shows the pharmacokinetic assay of DIIIv protein delivered via the nose in mice.

[0215] Sequence information

[0216] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0217] Table 1: Sequence Description Detailed Implementation

[0218] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0219] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CULTURE (edited by R.R. Freshney (1987)).

[0220] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0221] Example 1. Screening for FcRn high-affinity DIIIv mutants

[0222] First, a DIII mutant sequence library (hereinafter referred to as DIIIv) was constructed. By analyzing the crystal structure data of the HSA-FcRn complex, the binding interface between HSA and FcRn was obtained. Then, the amino acid residues that might bind HSA to FcRn were identified, and the mutation regions were determined. Site-directed mutagenesis was performed using the MPNN neural network deep learning method to obtain an initial mutant library. Next, Alphafold 2 was used to predict the structure of all mutants in the library. Further structural analysis and virtual screening were performed using RMSD, Tm-score, free energy DDG, competitive binding index PAE parameters, etc., to obtain the final mutant library. The mutant library was fabricated into a gene chip. First, the chip was centrifuged at high speed (12000 rpm, 1 min), and then dissolved in TE buffer (Tris-EDTA buffer). The mutant sequences were ligated to the vector pYD1 using a conventional seamless cloning method. An example of DIIIv and the vector plasmid is shown in Figure 1. The sequences were stored at -20℃ for later use.

[0223] Streaking EBY100 glycerol yeast onto YPD plates and incubate at 30°C for approximately 36 hours. Pick single colonies and transfer them to 3 mL of YPD medium, then incubate overnight at 30°C (to activate OD). 600 (Value 2-5 is recommended). Transfer an appropriate amount of yeast to 3 mL of fresh YPD medium to allow OD250 to rise. 600 The value was 0.1, and the OD was increased by incubation in a shaker at 30°C. 600The concentration should be between 0.4 and 1. Centrifuge the yeast at 4200 rpm for 2 min, resuspend in 700 μL of 0.1 M TE / LiAC, transfer to a 1.5 mL centrifuge tube, centrifuge at 4200 rpm for 2 min, discard the supernatant, and add 20 μL of 0.1 M TE / LiAC to prepare competent yeast cells for later use. Transformation solution preparation: Take 2 mg / mL salmon sperm DNA (ssDNA), denature at 100℃ for 5 min, vortex to mix, and immediately place on ice for 5 min. For each yeast transformation tube, add the following reagents to prepare the transformation solution: 62.4 μL 50% PEG3350, 8.22 μL 1 M TE / LiAC, 5 μL ssDNA, and mix thoroughly. For each yeast transformation tube, add 75.62 μL of transformation buffer, 20 μL of competent cells, and 100 ng of plasmid DNA. Mix thoroughly and incubate at 30°C on a shaker for 35 min. After incubation, heat shock at 42°C for 15 min. Centrifuge at 4200 rpm for 2 min to remove the supernatant. For each transformed yeast tube, resuspend in 100 μL of sterile ultrapure water, plate on an MD plate, and incubate at 30°C for 48-72 h until clear yeast colonies appear on the MD plate. Resuspend in 2-3 mL of 2*SC-URA-TRP selection medium and incubate on a shaker (30°C, 200-250 rpm) until OD500. 600 2-5, take 300 μL of transfer activation medium (2% glucose-YNB-C) and culture until OD. 600 2-5, dilute the yeast to 3-5 mL OD 600 Yeast culture was induced for 48-72 h in 0.5-1% induction medium (2% galactose-YNB-C) using a shaker (20℃, 200-250 rpm). It was then transferred to activation medium for simultaneous culture as a non-induced negative control. After induction, appropriate amounts of both induced and non-induced yeast cultures were diluted with PBS at pH 6.0 to an OD value. 600 Prepare 1.1 mL volume for later use.

[0224] The yeast was screened according to the following groups:

[0225] (1) Negative control: a. Uninduced group: Take 200 μL of uninduced yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 100 μL of PBS pH 6.0 to resuspend the culture, add 2 μL of (50:1) HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody, incubate at room temperature for 1 h, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS pH 6.0 to resuspend the culture for later use; b. Induced blank group: Take 200 μL of induced yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend the culture. Resuspend the bacterial culture at pH 6.0, wash the bacterial culture, centrifuge the bacterial culture at 4200 rpm for 2 min, discard the supernatant, add 600 μL PBS to pH 6.0 and resuspend the bacterial culture for later use;

[0226] (2) Sample groups: a. Single staining group: Take 200 μL of yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 100 μL of PBS pH 6.0 to resuspend the culture, add 2 μL of HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody, incubate at room temperature for 1 h, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS pH 6.0 to resuspend the culture for later use; b. Double staining group: Take 200 μL of yeast culture diluted with PBS pH 6.0, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend the culture. Resuspend the bacterial culture in PBS at pH 6.0, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 90 μL of PBS to resuspend the culture, add 10 μL of FcRn-β2M protein (Biotinylated, His-Avi, 100 μg / mL), incubate at room temperature for 1 h, add 2 μL of HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody and 0.3 μL of Streptavidin APC, incubate at room temperature for 1 h, add 1 mL of PBS at pH 6.0 to resuspend the culture, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS at pH 6.0 to resuspend the culture for later use.

[0227] After all samples were prepared, they were sequentially tested using flow cytometry and FACS sorting. The sorting results are shown in Figures 2A to 2C. After four to five rounds of sorting, it can be seen that both the fluorescence signals of the mutants and the fluorescence signals of the mutants binding to FcRn are significantly enhanced. This indicates that as the number of screening rounds increases, low-affinity mutants in the mutant library are gradually eliminated, while high-affinity mutants are continuously enriched (R.1 to R.5 in Figures 2B and 2C represent the first to fifth rounds of sorting, respectively). Finally, a high-affinity mutant library is obtained, which proceeds to the next stage of validation.

[0228] Example 2. Pichia pastoris expression of high affinity DIIIv

[0229] The mutants that passed the final screening by FACS were subjected to high-throughput sequencing. After comparison with the original mutant sequences, some example mutant sequences were selected, as shown in Tables 1 and 2. These mutants correspond to the third domain (SEQ ID NO:2) of wild-type human serum albumin.

[0230] The mutant was synthesized into the pPIC9K vector plasmid (the example plasmid map is shown in Figure 3). First, GS115 Pichia pastoris competent cells were prepared. GS115 cells were streaked on YPD plates and incubated at 30℃ for 2-3 days until single colonies grew. Single colonies were picked and transferred to 50mL centrifuge tubes containing 5ml of YPD medium and cultured overnight at 30℃ with shaking until OD. 600 With a value of 1, the overnight culture was transferred at a ratio of 1:100 to a 250 mL Erlenmeyer flask containing 50 mL of YPD, and cultured overnight at 30 °C with shaking until the Pichia pastoris OD value was reached. 600The concentration was 0.8-1.0. Yeast pellet was collected by centrifugation at 1500g for 10 min at room temperature. The pellet was washed twice with 25 mL of sterile water, centrifuged again at 1500g for 10 min at room temperature, and the supernatant was discarded. The cells were resuspended in 0.1M LiCl solution, centrifuged at 10000g for 15 s, the supernatant was discarded, and the yeast was resuspended in 0.1M LiCl solution. The mixture was transferred to 1.5 mL centrifuge tubes, aliquoted, and collected as competent yeast cells by centrifugation at 1500g for 5 min at room temperature. Plasmid linearization: Plasmid DNA was linearized by single enzyme digestion using standard methods. Salmon sperm DNA (ssDNA preparation): An appropriate amount of 2 mg / mL salmon sperm DNA was placed in a metal bath (100℃, 5 min) and immediately placed on ice (ice-water mixture) to prepare single-stranded DNA. Preparation of transformation solution: For each yeast transformation tube, add the following reagents to prepare the transformation solution: 240 μL 50% PEG3350, 36 μL 1M LiCl, 25 μL 2 mg / ml single-stranded salmon sperm DNA. For each yeast transformation tube, add the following reagents in the following volumes: 301 μL transformation buffer, 5-10 μg plasmid DNA (dissolved in 50 μL ddH2O); vortex vigorously for 1 min until the yeast precipitate is completely and evenly distributed; incubate at 30°C for 30 min (do not shake during incubation); then heat shock at 42°C for 20-25 min (do not shake during this process); centrifuge at 6000g for 1 min at room temperature, removing as much supernatant as possible, and collect the yeast precipitate to avoid the continued toxic effects of high-concentration LiCl solution on the cells; add 1 mL of YPD liquid medium and pipette until the yeast precipitate is dispersed and mixed (1 mL of sterile water can also be added); centrifuge at 6000g for 1 min at room temperature, removing as much supernatant as possible, and add 1 mL of... YPD liquid medium was used to disperse and mix the yeast precipitate by pipetting until well dispersed. Centrifuge tubes were sealed tightly with sealing film and incubated on a horizontal shaker at 30°C for 1-4 hours (centrifuge tubes should be placed horizontally, not vertically). 25-100 μL of the bacterial culture was spread onto MD plates and incubated at 30°C for 2-3 days (to avoid difficulty in adsorbing suspended cells, plates can be prepared 1-2 weeks in advance and stored at 4°C). After single colonies grew on MD plates after 2-3 days of incubation, 1 mL of sterile water was added to each plate. The single colonies were mixed and transferred to 1.5 mL centrifuge tubes for later use. 200 μL of each single colony was added to prepared YPD plates containing different gradients of G418 antibiotics for a second high-copy transformant selection. After incubation at 30°C for 3-4 days, single colonies were picked and added to 250 mL Erlenmeyer flasks containing 25 mL of BMGY medium. The flasks were incubated on a shaker at 30°C and 250 rpm until OD (out of 1). 600 Collect yeast by centrifugation at 3000g for 5 minutes at room temperature, then add it to a 1L shake flask containing 100mL BMMY medium until OD reaches 2-6. 600The culture medium was sealed with 6 layers of gauze and cultured on a shaker at 28℃ and 235rpm for 1-5 days. 1% methanol was added every 12-24 hours to induce expression. After induction, the culture medium supernatant was collected by centrifugation at 9000g for 20min. The supernatant was then filtered twice with a 0.8μm vacuum pump and once with a 0.22μm vacuum pump, and the filtrate was collected for later use.

[0231] Protein purification was performed using Albu purification matrix, following the manufacturer's recommended method. First, the purification column was equilibrated to 5 column volumes (5CV) using equilibration buffer. The filtered protein was then loaded, and the column was equilibrated again to 5CV using equilibration buffer. Buffer 1 was used to wash away impurities, followed by equilibration again to 5CV using equilibration buffer or UV back to baseline. Buffer 2 was used to wash away impurities, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. Buffer 3 was used to wash away impurities, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. Buffer 4 was used to elute the target protein, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. The column was then washed with 0.5M NaOH. Finally, the column was stored at 4°C after loading with 20% ethanol. During purification, the column buffer was collected as needed for subsequent purification effect evaluation. Washing buffers could be prepared selectively; the more cycles of impurities and column use, the more washing steps were required. All buffers were prepared according to the manufacturer's recommended method.

[0232] The purification results are shown in Figure 4, where the target protein band after purification is clearly visible at the black dotted line.

[0233] Table 2. Mutation sites of exemplary mutants

[0234] Example 3. Determination of the affinity between DIIIv and FcRn

[0235] First, the purified DIIIv protein was quantified using the BCA protein quantification method. FcRn-β2M ligand protein was prepared using a serial dilution method, resulting in antibody dilutions of 20,000 ng / mL, 4,000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, and 0.256 ng / mL.

[0236] The quantified protein was added at a concentration of 2 μg / mL (100 μL per well) to a polystyrene 96-well plate for ELISA. The plate was sealed with sealing film and incubated overnight at 4°C. 300 μL of PBS (pH 7.4) was added to each well for the first wash, followed by blocking buffer for 2 hours at room temperature. 300 μL of washing buffer was added to each well for the third wash. Serially diluted FcRn-β2M (His-tag) protein was added to each well and incubated at 37°C for 2 hours. 300 μL of washing buffer was added to each well for the third wash, followed by 100 μL of Anti-His-HRP mAb enzyme-labeled antibody. The plate was incubated at room temperature for 2 hours, followed by 300 μL of washing buffer for the third wash, and then 100 μL of TMB chromogenic solution was added. The plate was incubated at 37°C for 10 minutes. The reaction was terminated with sulfuric acid stop solution. The absorbance of the sample at 450 nm was immediately measured, and the affinity of all DIIIv samples for FcRn in Example 2 was calculated.

[0237] The example results for DIIIv are shown in Table 3. All detected DIIIv showed very strong binding affinity to FcRn under acidic pH conditions (e.g., pH 6.0). However, these DIIIv showed no directly measurable affinity for FcRn at physiological pH conditions (e.g., pH 7.4) (*: no signal detected; +: no obvious binding signal detected).

[0238] Table 3. Affinity results of representative mutants

[0239] Example 4. Determination of the affinity between the DIIIb mutant and FcRn

[0240] Based on the affinity determination of DIIIv for FcRn in Example 3, we further determined the affinity of mutants containing only the DIIIb portion (i.e., only containing SEQ ID NO:4 in Table 1) and lacking the DIIIa portion (i.e., SEQ ID NO:3 in Table 1) for FcRn. These mutants were named D3V1b, D3V2b, D3V4b, D3V8b, and D3V22b, respectively, and their specific sequences are shown in SEQ ID NO:49 to SEQ ID NO:53 in Table 1. This sequence corresponds to the fragment from amino acid 467 to amino acid 585 of SEQ ID NO:1.

[0241] Similar to the methods described above, the purified DIIIb mutant protein was first quantified using the BCA protein quantification method. FcRn-β2M ligand protein was prepared using a serial dilution method, resulting in antibody dilutions of 50,000 ng / mL, 10,000 ng / mL, 200 ng / mL, 40 ng / mL, 8 ng / mL, 1.6 ng / mL, 0.32 ng / mL, and 0 ng / mL.

[0242] The quantified protein was added at a concentration of 2 μg / mL (100 μL per well) to a polystyrene 96-well plate for ELISA. The plate was sealed with sealing film and incubated overnight at 4°C. 300 μL of PBS (pH 7.4) was added to each well for the first wash, followed by blocking buffer and blocking at room temperature for 2 hours. 300 μL of washing buffer was added to each well for the third wash. Serially diluted FcRn-β2M (His-tag) protein was added to each well and incubated at 37°C for 2 hours. 300 μL of washing buffer was added to each well for the third wash, followed by 100 μL of Ni-His-HRP mAb enzyme-labeled antibody and incubation at room temperature for 2 hours. 300 μL of washing buffer was added to each well for the third wash, followed by 100 μL of TMB chromogenic solution and incubation at 37°C for 10 minutes. The reaction was terminated with sulfuric acid stop solution. The absorbance of the samples at 450 nm was immediately measured, and the affinity of all DIIIv samples for FcRn was calculated.

[0243] The example results for the DIIIb mutants are shown in Table 4. All the DIIIb mutants tested showed a very strong binding affinity to FcRn under acidic pH conditions (e.g., pH 6.0). However, these DIIIb mutants showed no directly measurable affinity for FcRn at physiological pH conditions (e.g., pH 7.4) (*: no signal detected; +: no obvious binding signal detected).

[0244] Table 4. Affinity results of representative mutants

[0245] Example 5. DIIIv receptor-mediated transport and circulation

[0246] Based on previous research by our group, near-infrared fluorescent dyes containing cyclochloroethyl groups (such as IR-780) can undergo nucleophilic substitution reactions with albumin or DIII in a 1:1 molar ratio under certain conditions. The binding site is cysteine ​​residue 476 (Cys476) of albumin. Therefore, IR-780 is used as a labeling dye for albumin or DIII (for ease of labeling, the samples in the results are abbreviated. V1 corresponds to D3V27, V2 corresponds to D3V28, V3 corresponds to D3V29, V4 corresponds to D3V30, and V5 corresponds to D3V31, and so on).

[0247] IR-780 dye with a final concentration of 1 μM and albumin or DIII and DIIIv proteins were respectively adjusted to 500 μL of PBS buffer, vortexed for 15 s, incubated in a water bath at 60 °C for 10 min, and stored in the dark for later use. Prepare fresh each time.

[0248] The co-localization of DIIIv (DIII variants, DIII Vs) and FcRn in intracellular endosomes was verified using fluorescence confocal microscopy, confirming the efficient binding of high-affinity DIIIv to FcRn in intracellular endosomes. First, 300,000 293T cells per well were seeded in 35mm (15mm diameter) glass-bottomed culture dishes and cultured at 37°C with 5% CO2 for 24 hours. Protein and cyanine dye complexes were prepared using a similar method, and the protein and dye were prepared in serum-free medium (wild-type HSA, DIII, DIIIv, and cyanine dye concentrations were all 1μM) and stored in the dark. The cell culture medium was aspirated and the cells were washed three times with 500 μL PBS pH 7.4. 250 μL of protein and cyanine dye complex was added to each group and the cells were incubated at 37°C for 1 h. In addition to V4-IR-780, the negative control groups D3V30-Ab and D3V30-Ba A1 were also given DIII-specific polyclonal antibody (0.5 μM) to block their binding to the FcRn site and the binding condition (pH) disruptor Ba A1. After incubation, the sample was washed three times with 500 μL PBS buffer, then 250 μL of FcRn tracer (50 nm concentration DND-99, LysoTracker Red DND-99) was added and incubated at room temperature for 10 min. The sample was then washed three times with 500 μL PBS buffer, then 250 μL of Hoechst 33342 was added and incubated at room temperature for 10 min. The sample was then washed three times with 500 μL PBS buffer and the signal was detected using a near-infrared imager.

[0249] Experimental results showed that, compared with wild-type HSA and unmutated DIII, the high-affinity mutants D3V28 and D3V30 exhibited significantly stronger signals in cells, and their co-localization signals with FcRn were also significantly better than those of the control group (see lower right corner of Figure 5A). The Pearson product-moment correlation coefficient (PCCs) and overlap coefficient (Ol R) of the mutant co-localization signal distribution with FcRn were both significantly better than those of the control group. Furthermore, competitive blocking with antibodies and disruption of the acidic endosome environment resulted in a sharp decrease in the signal of the high-affinity mutant D3V30, and poorer co-localization data (Figure 5B).

[0250] Therefore, this experiment used fluorescence confocal microscopy to perform intracellular colocalization of DIII Vs and FcRn, verifying the efficient binding of high-affinity DIIIv to FcRn intracellularly, and providing cellular-level validation for its further transport and circulation via ligand receptor-mediated transport.

[0251] To further verify the transport efficiency of high-affinity DIIIv via FcRn, we performed a mutant transcytosis assay (VRA) in 293T and T84 cells that both expressed FcRn. First, 150,000 cells / well and 125,000 cells / well of 293T and T84 cells / well were seeded in 24-well plates and cultured at 37°C with 5% CO2 for 48 h, with medium changes every 24 h. Protein and cyanine dye complexes were prepared using a similar method. The protein and dye were prepared in serum-free medium (wild-type HSA, DIII, DIIIv, and cyanine dye concentrations were all 1 μM). The fluorescence intensity of the protein and cyanine dye complexes was recorded under near-infrared imaging to correct for the influence of inconsistent sample fluorescence intensity on the results. A D3V30-Ab control group was prepared using the same method and kept in the dark for later use. Aspirate the cell culture medium and wash three times with 500 μL PBS pH 7.4 buffer. Add 250 μL of protein and cyanine dye complex to each group and incubate at 37°C for 1 h. Aspirate the supernatant and wash five times with 1 mL PBS buffer. Add 250 μL of serum-free medium to each well to collect the protein transported back via FcRn. Incubate at 37°C for 30 min and collect the culture medium sample. Wash three times with 500 μL PBS buffer, add 250 μL of serum-free medium to each well again, and incubate at 37°C for 30 min. Collect the culture medium sample. This will give you the protein samples transported via FcRn at 30 min and 1 h time points. Collect protein samples at 2 h and 4 h in the same way. Centrifuge 10000 g of the collected samples at room temperature for 2 min and collect the supernatant (to remove possible influence of cells and debris on the results). Finally, detect the fluorescence intensity of the samples under near-infrared imaging.

[0252] As can be seen from Figure 6A (293T cells) and Figure 6B (T84 cells), regardless of the time points of 30 min, 1 h, 2 h, or 4 h, the high-affinity mutants D3V28 and D3V30 showed significantly higher amounts of protein transported back to the extracellular space via the receptor compared to the wild-type HSA, DIII, and D3V30-Ab control groups. This further suggests that the high-affinity DIIIv can be efficiently transported and circulated in cells via the receptor, avoiding degradation by lysosomes within the cell.

[0253] Example 6. Absorption of DIIIv in the digestive system mucosa

[0254] To further explore the absorption of DIIIv in the digestive mucosa, the mutant D3V1 was labeled using the same method with IR-780, and mice (C57BL / 6) were gavaged with 80 μM, 500 μL. Two hours later, the duodenum was harvested and frozen sectioned. At the same time, FcRn antibody was used to trace the expression of FcRn in the mouse duodenum.

[0255] As shown in Figure 7, after gavage, the D3V1 group showed widespread sample signals in different parts of the mouse duodenum, and these signals were highly colocalized with FcRn signals, indicating that D3V1 binds to FcRn expressed in the mouse duodenum villi epithelial cells. Conversely, the IR-780 control group did not show widespread signal detection in the duodenum, indicating that IR-780 alone is rarely absorbed by the mouse duodenum. Therefore, this experiment verifies that the D3V1 sample, after gavage, can be efficiently transported across the intestinal mucosa via mouse duodenal villi cells for absorption.

[0256] Example 7. DIIIv efficiently transmembrane enters the systemic circulation after intranasal administration.

[0257] Following the same method described above, IR-780 was used as the labeling dye for DIIIv. IR-780 was mixed with DIII and D3V1 at a molar ratio of 1:1 (50 μM) and incubated in a 60°C water bath for 10 min, protected from light throughout. Mice (C57BL / 6) were anesthetized, and 50 μL of DIII and D3V1 were administered intranasally to each group of mice (n=4). Fluorescence was detected using a near-infrared II imaging system.

[0258] By monitoring the time points after intranasal administration, the fluorescence signal of DIIIv protein could be clearly detected in organs such as the lungs of mice, and the D3V1 sample group could still be detected in the blood circulation after 13 days (Figure 8), which shows the sustained-release characteristics of D3V1 administered intranasally. At the same time, the fluorescence signal intensity in the blood circulation of mice showed that after intranasal administration, the signal of D3V1 in the blood circulation was significantly higher than that of DIII, indicating that the transmucosal efficiency of D3V1 was significantly higher than that of DIII.

[0259] Example 8. Pharmacokinetic determination of mutant protein administered intranasally in mice

[0260] Based on previous research, near-infrared fluorescent dyes containing cyclochloroethyl groups (such as IR-780) can covalently bind to albumin or DIII in a 1:1 molar ratio under certain conditions through a nucleophilic substitution reaction. Therefore, IR-780 was used as the labeling dye for DIIIv. First, IR-780 was mixed with DIII, D3V1, and D3V2 in a 1:1 molar ratio (20 μM) and incubated in a 60°C water bath for 10 min, protected from light throughout. Mice (Balb / c-nu) were anesthetized, and 100 μL of DIII, D3V1, and D3V2 were administered intranasally to each group of mice (n=3). Fluorescence detection was performed using a near-infrared II imaging system. Through intranasal administration, DIIIv protein fluorescence signals were clearly detected in organs such as the lungs of mice (as shown in Figure 9a; due to display requirements, the minimum / maximum display values ​​of the fluorescence signal at each time point were adjusted according to...). Adjustments were made within the same time period, and the absolute values ​​are shown in the quantitative data, i.e., b and c in Figure 9). Compared with the original DIII, the high-affinity mutant protein has a significantly higher transmembrane efficiency. After intranasal administration, the mutant protein reached its peak concentration in 8-12 hours (as shown in b in Figure 9). It can be seen that the DIIIv protein exhibits the characteristic of rapidly reaching peak plasma concentration. At the same time, after a detection period of up to 14 days, the fluorescence signal of DIIIv protein can still be detected in the blood circulation of mice (as shown in a and c in Figure 9), indicating that the DIIIv protein retains and is released slowly in the lungs of mice after intranasal administration.

[0261] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

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 acids in native albumin 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 corresponding to positions 497 to 585 of SEQ ID NO:1 have amino acid insertions, substitutions, deletions and / or mutations; 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, having 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 according to 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 natural albumin corresponding to SEQ ID NO:1 in the mutant 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 L 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 natural albumin corresponding to SEQ ID NO:1 in the mutant is H or Y; (7) The amino acid at position 531 of natural albumin corresponding to SEQ ID NO:1 in the mutant is L; (8) The amino acid at position 547 of 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 in natural albumin corresponding to SEQ ID NO:1 is R or N; (11) The amino acid at position 498 of natural albumin corresponding to SEQ ID NO:1 in the mutant is E; (12) The amino acid at position 512 of natural albumin corresponding to SEQ ID NO:1 in the mutant 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 the 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 sequences. composition: (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, for example, (GGGGS). m (RGD) n The sequence shown, where, 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 (GGGGS) m (ZF9) n The sequence shown is where m and n are each an independent positive integer (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 according to claim 7 or 8, wherein, The fusion protein comprises one or more (e.g., two, three, four, five) 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., two, three, four, five).

10. The fusion protein of claim 9, wherein, The fusion protein has one or more of the following characteristics: (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 of any one of claims 1-5, or albumin of claim 6, or a portion thereof or a derivative thereof, or a fusion protein of 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: Candidate mutants, albumin or a portion thereof or its derivatives, or fusion proteins were contacted with FcRn and their affinity for FcRn was detected. 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 said dye molecule being 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 injection, transmucosal delivery, 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 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 use in 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 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, in the preparation of a pharmaceutical composition; 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.

21. A method for increasing the affinity of a molecule for FcRn, or for prolonging the half-life of a molecule, the method comprising: 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, is fused, conjugated or covalently bound to the molecule. Preferably, the molecule is selected from small molecule drugs, macromolecule drugs (e.g., nanoparticles, peptides, proteins), or any combination thereof.

22. A method for preparing the complex of claim 15, the method comprising: The mutant, the albumin, a portion thereof, a derivative thereof, or the fusion protein are reacted with the cyanine dye molecule at room temperature or under heating conditions.

23. A method for targeting cells, the method comprising contacting the cells with the complex of claim 15; optionally, after contact, irradiating the cells with a laser to obtain an image of the cells; Preferably, the complex targets the cell by binding to cell surface molecules, cell surface proteins, or cell surface receptors expressed on the cell; Preferably, the cells are selected from stem cells, proliferating cells, cells in the process of proliferation, inflammatory cells, negatively regulating immune cells, cells infected by pathogens, neurons, adipocytes, or lipid cells; Preferably, the cells are 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. Preferably, the cells are present in tissues or living organisms.

24. 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, in the preparation of a kit for diagnosing whether a subject is infected with a pathogen or has a disease caused by a pathogen infection (e.g., a respiratory disease); Preferably, the administration method of the kit is selected from oral, sublingual, nasal, intrathecal, bronchial, rectal, transdermal, inhalation, parenteral, or any combination thereof; Preferably, the complex targets the cell by binding to cell surface molecules, cell surface proteins, or cell surface receptors expressed on the cell; Preferably, the cells are selected from stem cells, proliferating cells, cells in the process of proliferation, inflammatory cells, negatively regulating immune cells, cells infected by pathogens, neurons, adipocytes, or lipid cells; Preferably, the cells are 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. Preferably, the cells are present in tissues or living organisms.

25. An imaging method, the method comprising using the complex of claim 15 as an imaging developer; Preferably, the imaging is near-infrared II fluorescence imaging; Preferably, the imaging method is fluorescence imaging of cells, tissues, or living organisms.

Images