Drug for treating urinary system diseases

Abstract

Provided is a drug for treating urinary system diseases. Specifically provided is a complex, comprising a carrier protein, and a functional molecule used for the diagnosis, prevention and / or treatment of urinary system diseases. The carrier protein is selected from a mutant DIIIbV of a subdomain structure (for example, DIIIb) of albumin domain III, an albumin domain III DIIIV containing the mutant, and multimers of the DIIIbV or DIIIV. The carrier protein exhibits an excellent transmucosal efficiency, can achieve transmucosal administration by means of pulmonary administration and gastrointestinal administration, and exhibits long-acting and sustained-release characteristics in the body. By means of the modes such as fusion expression and chemical grafting, etc., the functional molecule used for the diagnosis, prevention and / or treatment of urinary system diseases is carried, so as to achieve transmucosal drug delivery, thereby achieving targeted diagnosis and treatment of urinary system diseases.

Description

Medications used to treat urinary system diseases

[0001] This application is based on and claims priority to Chinese patent application No. 202411547095.9, filed on October 31, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This application relates to the field of biological agents, specifically to drugs for the treatment of urinary system diseases. Background Technology

[0003] Mucosa, as the first line of defense in the body's immune system, is widely distributed in the oral cavity, nasal cavity, eyes, gastrointestinal tract, vagina, and respiratory tract. It is a membranous structure composed of epithelial and connective tissues; the epithelial portion is called the epithelium, which is richly supplied with blood vessels and nerves and covered by mucus. Mucosal drug delivery systems are a novel drug delivery method that delivers drugs, along with suitable carrier materials, to the mucosal site for local action or absorption into the systemic circulation to induce systemic therapeutic effects. Mucosal drug delivery offers several advantages: First, compared to oral administration, drugs are absorbed into the systemic circulation through the mucosa, bypassing the liver and effectively avoiding the first-pass effect. Second, the limited types and quantities of enzymes in the mucosa result in relatively low enzymatic degradation activity of drugs, increasing drug stability. Furthermore, local administration increases drug concentration at the site of action, thereby improving bioavailability. Finally, local application makes the drug delivery process more convenient, allowing patients to self-administer the procedure and improving patient compliance. These advantages make mucosal drug delivery a promising area for drug delivery.

[0004] The kidneys are crucial organs for maintaining homeostasis, performing vital physiological functions such as filtering blood, eliminating metabolic waste, regulating water and electrolyte balance, and secreting hormones. The complexity of their structure and function determines the challenges and unique characteristics of targeted drug delivery. The inherent physiological characteristics of the kidneys present technical obstacles to drug delivery. For a long time, targeted drug delivery to the kidneys has faced two major challenges: First, as a vital metabolic organ, drugs entering the bloodstream are rapidly excreted by the kidneys, resulting in the elimination of drugs before reaching effective concentrations. This necessitates frequent dosing to maintain local effective drug concentrations, increasing the burden on patients and potentially raising the risk of adverse drug reactions. Second, the complex hierarchical structure and physiological barriers of the kidneys (such as the glomerular filtration barrier and the renal tubular epithelial cell barrier) make it difficult for exogenous drugs to accurately penetrate and accumulate in key diseased areas such as the renal tubules, leading to low drug utilization and limited therapeutic efficacy. Therefore, a targeted drug delivery system capable of effective kidney-targeting is needed to address the current challenges in kidney disease treatment. Summary of the Invention

[0005] The research unexpectedly discovered a protein with affinity for the neonatal Fc receptor (FcRn), which can efficiently deliver various payloads (small molecule drugs, proteins, nucleic acids, etc.). Administered via the mucosal system or blood, it can be reabsorbed into the bloodstream through the proximal renal tubules, thus achieving long-term kidney targeting. Depending on different needs, administration can be done via nasal spray, bronchial administration, oral mucosa, gastrointestinal mucosa, or blood, providing a new tool for the precise diagnosis, long-term monitoring, and efficient treatment of kidney diseases.

[0006] The neonatal Fc receptor (FcRn) is the receptor for human serum albumin (HSA), mediating lysosomal escape of HSA to enable long-term circulation of albumin in vivo. The FcRn protein structure consists of a heterodimer composed of an α-chain and a β2-microglobulin bonded non-covalently. It is widely expressed in various epithelial cells, endothelial cells, and immune cells in vivo, and is highly expressed on the surface of mucosal epithelial cells (such as the nasal airway and olfactory epithelium).

[0007] By constructing FcRn mutants with different affinities (high or low affinity) and employing high-throughput screening methods, a high-affinity albumin DIII mutant of FcRn was identified as a renal targeting warhead. This high-affinity mutant exhibits excellent transmucosal efficiency and demonstrates long-acting, sustained-release characteristics. Through fusion expression and chemical grafting, the identified high-affinity mutant and functional molecules, such as small molecule compounds, peptides or proteins, and nucleic acid molecules, that can be used for the diagnosis, prevention, and / or treatment of kidney diseases were further developed.

[0008] Specifically, in one aspect, this application provides a complex comprising a carrier protein and a functional molecule for the diagnosis, prevention and / or treatment of urinary system diseases;

[0009] The carrier protein is selected from the DIIIbV mutant of the albumin third domain substructure (e.g., DIIIb), the albumin third domain DIIIV containing the mutant, and the DIIIbV or DIIIV multimer.

[0010] In some embodiments, compared to the wild type, the mutant comprises one or more (e.g., 40-50, 30-40, 20-30, 15-20, 10-15, 5-10, 1-5) amino acid insertions, substitutions, deletions, and / or mutations. In some embodiments, the mutant comprises DIII and its subunit structures containing one or more amino acid insertions, substitutions, deletions, and / or mutations, such as smaller fragments of the DIIIb mutant and its subunits containing one or more amino acid insertions, substitutions, deletions, and / or mutations.

[0011] In some embodiments, the inserted, substituted, deleted, and / or mutated amino acids correspond to amino acids at positions 467 to 585 of SEQ ID NO:1.

[0012] In some embodiments, the inserted, substituted, deleted, and / or mutated amino acids correspond to the amino acids at positions 497 to 585 of SEQ ID NO:1;

[0013] In some embodiments, the inserted, substituted, deleted, and / or mutated amino acids correspond to the amino acids at positions 500 to 573 of SEQ ID NO:1.

[0014] In some embodiments, the DIIIbV or DIIIV exhibits a higher FcRn affinity under acidic or weakly acidic conditions compared to the wild type. Those skilled in the art understand suitable methods for determining whether the affinity of the DIIIbV or DIIIV for FcRn is higher or lower than that of natural albumin for FcRn. An exemplary approach is to determine and compare the binding constant Kd. Therefore, according to the invention, mutants with Kd lower than that of natural albumin or its DIII are considered to have a higher plasma half-life than natural albumin or its DIII, and mutants with Kd higher than that of natural albumin or its DIII are considered to have a lower plasma half-life than natural albumin or its DIII. In some embodiments, the DIIIV's FcRn affinity Kd value is in the range of 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 - 10 Or 10 -11 M level; preferably, the DIIIbV affinity for FcRn, Kd value, is 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Or 10 -11M level.

[0015] In some implementations, the DIIIbV or DIIIV contains mutations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids compared to the wild type.

[0016] In some embodiments, the DIIIbV or DIIIV comprises a mutation at one or more amino acid sites selected from those corresponding to amino acids 500, 505, 523, 524, 527, 528, 531, 547, 509, 510, 498, 512 and 573 of natural albumin having the amino acid sequence shown in SEQ ID NO:1.

[0017] In some implementations, the mutation is a conserved mutation. A conserved mutation means that amino acid residues within a specific group are interchangeable.

[0018] 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).

[0019] In some embodiments, the DIII portion comprises a fragment of natural albumin corresponding to amino acids 467 through 585 of SEQ ID NO:1.

[0020] In some embodiments, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 523 and 573.

[0021] In some embodiments, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 505, 523, 547, and 573.

[0022] In some embodiments, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 500, 505, 523, 524, 527, 528, 531, 547, and 573.

[0023] In some implementations, the albumin DIII mutant:

[0024] (1) The 500th amino acid in the natural albumin having the amino acid sequence shown in SEQ ID NO:1 is L or D;

[0025] (2) The amino acid at position 505 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is Q, N, or T;

[0026] (3) The amino acid at position 523 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L or M;

[0027] (4) The amino acid at position 524 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L;

[0028] (5) The amino acid at position 527 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is K;

[0029] (6) The amino acid at position 528 of natural albumin having the amino acid sequence shown in SEQ ID NO:1 is H or Y;

[0030] (7) The amino acid at position 531 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L;

[0031] (8) The amino acid at position 547 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is A or C;

[0032] (9) The amino acid at position 509 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L;

[0033] (10) The amino acid at position 510 of natural albumin having the amino acid sequence shown in SEQ ID NO:1 is R or N;

[0034] (11) The amino acid at position 498 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is E;

[0035] (12) The amino acid at position 512 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is G;

[0036] (13) The amino acid at position 573 of the natural albumin having the amino acid sequence shown in SEQ ID NO:1 is any amino acid other than K (e.g., P); or,

[0037] (14) The amino acid at position 508 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L;

[0038] (15) Any combination of the above.

[0039] In some implementations, the DIIIbV or DIIIV:

[0040] (1) The amino acid at the 500th position is mutated from K to L or D;

[0041] (2) The amino acid at position 505 is mutated from E to Q, N or T;

[0042] (3) The amino acid at position 523 is mutated from I to L or M;

[0043] (4) The amino acid at position 524 is mutated from K to L;

[0044] (5) The amino acid at position 527 is mutated from T to K;

[0045] (6) The amino acid at the 528th position is mutated from A to H or Y;

[0046] (7) The amino acid at position 531 is mutated from E to L;

[0047] (8) The amino acid at position 547 is mutated from V to A or C;

[0048] (9) The amino acid at position 509 is mutated from F to L;

[0049] (10) The amino acid at the 510th position is mutated from H to R or N;

[0050] (11) The amino acid at position 498 is mutated from V to E;

[0051] (12) The amino acid at the 512th position is mutated from D to G;

[0052] (13) The amino acid at the 573rd position is mutated from K to any amino acid other than K (e.g., P);

[0053] (14) The amino acid at position 508 is mutated from T to L; or,

[0054] (15) Any combination of the above.

[0055] In some implementations, the DIIIbV or DIIIV contains the mutations I523G and K573P.

[0056] In some implementations, the DIIIbV or DIIIV contains mutations such as E505Q, I523G, V547A, and K573P.

[0057] In some implementations, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P.

[0058] In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, V547C, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505N, T508L, F509L, H510R, I523L, K524L, T527K, A528H, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528H, E531L, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528H, E531L, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505Q, I523L, K524L, T527K, A528Y, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505Q, I523L, T527K, A528H, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505Q, I523L, T527K, A528H, E531L, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505T, I523L, T527K, A528Y, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505Q, I523L, K524L, T527K, A528H, E531L, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528Y, E531L, K573P.In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, T508L, F509L, H510R, D512G, I523L, K524L, T527K, A528H, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500D, E505T, I523L, T527K, A528H, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528Y, E531L, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528Y, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528Y, K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505N, I523L, K524L, T527K, A528H, E531L, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E,K500L,E505N,I523L,T527K,A528H,E531L,K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E,K500L,E505T,A511T,I523L,T527K,A528H,E531L,K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505N,I523L,K524L,T527K,A528H,V547A,K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505Q, I523M, K524L, T527K, A528H, E531L, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505T, I523L, T527K, A528H, K573P.In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505Q, F507V, T508K, F509L, H510N, D512G, I523L, K524L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutation K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505Q, T527M, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505Q, I523G, T527M, V547A, and K573P.

[0059] In some embodiments, the wild type of DIII or DIIIb is derived from natural serum albumin of mammals; preferably, the mammals are selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs, and pigs. In some embodiments, the wild type of DIII or DIIIb is derived from natural human serum albumin.

[0060] In some embodiments, the natural human serum albumin comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:1.

[0061] In some embodiments, the natural human serum albumin DIII comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:2.

[0062] In some embodiments, the natural human serum albumin DIIIb comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:4.

[0063] In some embodiments, the DIIIbV is selected from the amino acid sequences shown in SEQ ID NO:36-40.

[0064] In some embodiments, the DIIIV is selected from the amino acid sequences shown in SEQ ID NO:5-35.

[0065] In some embodiments, the DIIIV has the amino acid sequence shown in SEQ ID NO:5.

[0066] In some embodiments, the carrier protein further comprises an albumin first domain DI, a portion thereof, a derivative thereof, or a mutant thereof, and / or an albumin second domain DII, a portion thereof, a derivative thereof, or a mutant thereof.

[0067] In some embodiments, the DI and DII are each independently derived from the natural serum albumin of mammals; preferably, the mammals are selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs and pigs; preferably, the mammals are humans.

[0068] In some embodiments, the polymer is a homo- or hetero-dimer, trimer, tetramer, or any polymer that can exist stably in physiological solutions of DIIIbV or DIIIV.

[0069] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0070] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0071] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0072] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0073] In some implementations, the urinary system disease is selected from kidney disease, ureteral disease, bladder disease, and urethral disease.

[0074] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0075] In some embodiments, the functional molecule is selected from diagnostic reagents, imaging agents, therapeutic radionuclides, or macromolecular or small molecule drugs, such as peptides, proteins, antibodies, nanobodies, nucleic acid drugs, or chemotherapeutic drugs. In some embodiments, the peptide or protein is a peptide chain or a cyclic peptide. In some embodiments, the antibody is a monoclonal antibody or its antigen-binding fragment; the nucleic acid drug is mRNA or a protein-nucleic acid complex.

[0076] In some embodiments, the drug is an anti-inflammatory, anti-apoptotic, or anti-oxidative stress drug targeting the urinary system disease. In some embodiments, the drug is selected from amifostine, cimetidine, glutathione, RNLS agonists (e.g., RP81), cortisone, curcumin, dexamethasone, and MMAE.

[0077] In some embodiments, the diagnostic reagent is selected from substances that are highly expressed or secreted by the renal system (such as interleukin, intrinsic factor-vitamin B12 receptor (cubilin), low-density lipoprotein receptor-associated protein 2 (megalin), kidney injury-associated molecule (KIM-1), neutrophil gelatinase-associated lipotransferase (NGAL), and other proteins).

[0078] In some embodiments, the imaging agent is selected from cyanine dyes such as IR-780, IR-783, and other photosensitizers.

[0079] In some embodiments, the therapeutic radionuclide is selected from... 64 Cu、 18 F, 68 Ga、 177 Lu、 125 I, 90 Y、 89 Sr、 32 P, 233 Ra.

[0080] In some implementations, the carrier protein and the functional molecule are directly linked or linked via a connector.

[0081] In some embodiments, the linker may be cleavable or non-cleavable; preferably, the cleavable linker is of the acid-cleavable, disulfide-cleavable, protease-cleavable, glycosidase-cleavable, or phosphatase-cleavable type. In some embodiments, the linker is selected from DBCO-NHS ester, Sulfo-SMCC sodium, CL2 linker, DSP Cross linker, Mc-Val-Cit-PABC-PNP, Val-Cit-PAB, MC-Val-Cit-PAB, MAC glucuronide linker-2, Fmoc-PEA, Mal-PEG4-OH, 3-Mercaptopropionic acid NHS ester, and tBoc-NH-PEG-NH2.

[0082] In some embodiments, the carrier protein and the functional molecule are coupled together by gene fusion or chemical methods to form the complex.

[0083] In some embodiments, the complex has the following structure: C-(LD) n

[0084] Wherein, C represents the carrier protein;

[0085] D represents the functional molecule;

[0086] L represents a bond or a linker connecting the carrier protein and the functional molecule, the linker preferably being one of the linkers described above;

[0087] n is an integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0088] In some implementations, C is selected from DIIIV1 to DIIIV31, DIIIbV1, DIIIbV2, DIIIbV4, DIIIbV8 and DIIIbV22.

[0089] In some implementations, D is selected from RP81, cortisone, curcumin, and MMAE.

[0090] In some implementation schemes, L is selected from

[0091] Nucleic acid molecules, vectors, and host cells

[0092] In another aspect, this application provides a nucleic acid molecule that encodes the complex described in any of the preceding claims.

[0093] In some embodiments, the complex is a fusion protein.

[0094] In another aspect, this application provides a vector comprising the nucleic acid molecules described above. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a vector of eukaryotic bacteria (e.g., Pichia pastoris, and further, pPIC9K, pCDNA3.4).

[0095] 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.

[0096] In another aspect, this application provides a host cell comprising the nucleic acid molecules or vectors described above. In some embodiments, the cell is a eukaryotic or prokaryotic cell. In some embodiments, the eukaryotic cell is a yeast cell (e.g., *Saccharomyces cerevisiae*, *Pichia pastoris*), or a 293T or 293F cell. In some embodiments, the prokaryotic cell is an *Escherichia coli* cell, a *Bacillus subtilis* cell, or any combination thereof.

[0097] Delivery of combination or pharmaceutical composition

[0098] In another aspect, this application provides a delivery combination or pharmaceutical composition comprising any of the complexes described above.

[0099] In some embodiments, the delivery combination or pharmaceutical composition is delivered intravenously.

[0100] In some embodiments, the delivery combination or pharmaceutical composition is delivered via nasal or oral inhalation, preferably via nasal delivery, such as nasal drops, nasal sprays, or combinations thereof.

[0101] In some implementations, the complex is delivered to the mucosal surface of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa, vaginal mucosa).

[0102] In some embodiments, the delivery combination or pharmaceutical composition further comprises one or more mucosal adhesives to enhance the residence time of the effector molecules on the mucosal surface of the subject.

[0103] In some embodiments, the delivery combination or pharmaceutical composition is an aerosol, powder inhaler, spray, or other dosage form suitable for inhalation administration.

[0104] In some embodiments, the pharmaceutical composition contains one or more pharmaceutically acceptable excipients.

[0105] Medical uses and methods

[0106] In another aspect, this application provides the use of any of the foregoing complexes, delivery combinations, or pharmaceutical compositions in the preparation of a medicament for the treatment of urinary system diseases.

[0107] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0108] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0109] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0110] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0111] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0112] In another aspect, this application provides the complex, delivery combination, or pharmaceutical composition described in any of the foregoing claims for the treatment of urinary system diseases.

[0113] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0114] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0115] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0116] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0117] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0118] In another aspect, this application provides a method for treating urinary system diseases, comprising the steps of administering an effective amount of any of the preceding compound, delivery combination, or pharmaceutical composition to a subject in need of such treatment.

[0119] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0120] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0121] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0122] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0123] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0124] In another aspect, this application provides the use of the carrier protein as defined in any of the preceding claims in the preparation of medicaments for treating diseases of the urinary system.

[0125] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0126] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0127] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0128] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0129] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0130] In another aspect, this application provides a carrier protein as defined in any of the preceding claims for the treatment of urinary system diseases.

[0131] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0132] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0133] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0134] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0135] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0136] In another aspect, this application provides a method for treating urinary system diseases, comprising administering a carrier protein as defined in any of the preceding claims to a subject in need of such treatment.

[0137] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0138] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0139] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0140] In some implementations, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy).

[0141] In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0142] In another aspect, this application provides a medicament for treating diseases of the urinary system, which contains a carrier protein as defined in any of the preceding claims.

[0143] In some implementations, the urinary tract disease is kidney damage, urinary tract inflammation, or urinary tract cancer.

[0144] In some implementations, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; and urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis.

[0145] In some implementations, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureteral cancer, bladder cancer, and urethral cancer.

[0146] In some embodiments, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy). In some embodiments, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritic syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

[0147] Terminology Definition

[0148] 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 those related to molecular genetics, chemistry, molecular biology, biochemistry, 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.

[0149] As used in this article, the term "carrier" or "carrier protein" refers to a protein that can carry different biomolecules and circulate throughout the body to different parts of the body.

[0150] As used in this article, the term "small molecule drug" mainly refers to chemically synthesized drugs, which are usually organic compounds with a molecular weight of less than 1,000. They can enter cells and act on intracellular targets. Small molecule drugs are usually signal transduction inhibitors that can specifically block the signal transduction pathways necessary for tumor growth and proliferation, thereby achieving the purpose of treatment.

[0151] As used in this article, the term "monoclonal antibody drug" refers to highly homogeneous antibodies produced by B cells that target only a specific antigenic epitope. These antibodies can exert their therapeutic effects in cancer treatment by mediating ADCC pathways, targeting cancer cells to induce apoptosis, targeting the tumor microenvironment, and targeting immune checkpoints. Monoclonal antibodies can also be modified, such as by conjugating them with radiopharmaceuticals, to achieve therapeutic goals for cancer treatment.

[0152] As used in this article, the term "targeting molecule" refers to a targeting group that can specifically recognize and bind tightly to an antigen protein, mainly including antibodies, antibody fragments, scaffold proteins, peptides and other small molecules.

[0153] 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.

[0154] 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).

[0155] As used herein, “variant” or “mutant” is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains its essential characteristics. A typical variant of a polynucleotide differs from another reference polynucleotide in its nucleotide sequence. Changes in the nucleotide sequence of a variant may alter or not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the differences are limited, and thus the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. The amino acid sequences of the variant and the reference polypeptide can differ in any combination by one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variants, or may be unknown variants. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis or direct synthesis. Variants may also include, but are not limited to, polypeptides or fragments thereof with chemical modifications to one or more amino acid side groups. Chemical modification includes, but is not limited to, the addition of chemical groups, the formation of new bonds, and the removal of chemical groups. Modification of amino acid side groups includes, but is not limited to, acylation of the ε-amino group of lysine, N-alkylation of arginine, histidine, or lysine, alkylation of the carboxylic acid group of glutamic acid or aspartic acid, and deamidation of glutamine or asparagine. Modification of terminal amino groups includes, but is not limited to, deamination, N-lower alkyl, N-dilower alkyl, and N-acyl modifications. Modification of terminal carboxyl groups includes, but is not limited to, modifications of amides, lower alkylamides, dialkylamides, and lower alkyl esters. Furthermore, one or more side groups or terminal groups may be protected by protecting groups known to those skilled in the art.

[0156] As used herein, a "polypeptide" refers to any peptide or protein containing two or more amino acids linked together by peptide bonds or modified peptide bonds, i.e., peptide isoelectronic arrangement. "Polypeptide" refers both to short-chain peptides, commonly called peptides, oligopeptides, or oligomers, and long-chain peptides, commonly called proteins. Polypeptides can contain all amino acids except the 20 amino acids encoded by genes. "Polypeptides" include amino acid sequences modified by natural processes (such as post-translational processing) or by chemical modification techniques well known in the art. These modifications are described in detail in basic materials, monographs, and a large body of research literature in the field. Modifications can occur anywhere on a polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl terminals. It should be understood that the same type of modification can be present at several sites on a given polypeptide in the same or different amounts. Furthermore, a given polypeptide can contain many types of modifications. Polypeptides can branch due to ubiquitination, and they can be cyclic, with or without branching. Cyclic, branched, and branched-cyclic polypeptides can be produced by natural post-translational processes or prepared by synthetic methods. Modifications include acetylation, acylation, ADP ribosylation, amidation, covalent linkage of flavin, partial covalent linkage of heme, covalent linkage of nucleotides or nucleotide derivatives, covalent linkage of lipids or lipid derivatives, covalent linkage of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, proteolytic treatment, phosphorylation, propionylation, racemization, selenylation, sulfation, and tRNA-mediated addition of amino acids to proteins, such as arginylation and ubiquitination. Reference "PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES", 2nd Ed., TECreighton et al, 1993; Seifter, et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. (1990) 182:626-646; Rattan et al. Protein Synthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci(1992)663:48-62. et al.

[0157] As used herein, the terms “wild” or “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, peptides, or proteins, they indicate that the nucleic acid molecule, peptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing.

[0158] 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.

[0159] 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.

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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. In this document, the first domain of albumin may be abbreviated as DI, the second domain as DII, the third domain as DIII, the sub-domain a of the third domain as DIIIa, and the sub-domain b of the third domain as DIIIb. DIIIV can be used to describe the DIII mutant of the present invention. DIIIbV or DIIIVb can be used to describe the DIIIb mutant of the present invention.

[0166] 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.

[0167] 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.

[0168] 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)).

[0169] 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.

[0170] 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.

[0171] 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.

[0172] The term "treatment" refers to the successful treatment or improvement of any symptom of an injury, lesion, or condition, including any objective or subjective parameter, such as the elimination, relief, or reduction of symptoms, or making the patient more tolerant of the injury, lesion, or condition. Treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of physical examination, neuropsychiatric examination, and / or psychiatric evaluation.

[0173] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or underlying causes, prevent the occurrence of symptoms and / or underlying causes, and / or improve or cure damage caused by or associated with a disease state. In some embodiments, an effective amount is a therapeutic effective amount or a preventive effective amount. A "therapeutic effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with a disease state, or otherwise prevent, block, delay, or reverse the progression of a disease state or any other undesirable symptom in any way associated with the disease.

[0174] "Prophylactic effective dose" is the amount of a pharmaceutical composition that, when administered to a subject, will have the expected preventive effect, such as preventing or delaying the onset (or recurrence) of a disease state, or reducing the likelihood of the onset (or recurrence) of a disease state or related symptoms. Complete therapeutic or preventive effect may not occur with a single dose and may only occur after a series of doses. Therefore, therapeutic or preventive effective doses may be administered once or multiple times.

[0175] As used herein, the terms “therapeutic effective dose” and “therapeutic effective amount” refer to the amount of a fusion protein in an tissue system, animal, or human that elicits a biological or medical response sought by a researcher, physician, or other clinician (including the reduction or improvement of symptoms of the treated disease or disorder), i.e., the amount that supports one or more desired biological or medical responses at observable levels.

[0176] As used herein, the term "diagnostic reagent" refers to a diagnostic reagent prepared using principles or methods of immunology, microbiology, molecular biology, etc., and used in vitro or in vivo for the diagnosis, detection, and epidemiological investigation of human diseases.

[0177] In this article, acute kidney injury (AKI) refers to a rapid decline in kidney function over hours to days, characterized by elevated serum creatinine levels or decreased urine output. It is often caused by dehydration, infection, medications, or reduced renal blood flow and is an emergency requiring rapid treatment.

[0178] In this article, acute kidney disease generally refers to acute kidney injury (AKI), which is a situation in which kidney function declines rapidly in a short period of time, accompanied by elevated creatinine and decreased urine output.

[0179] In this article, chronic kidney disease (CKD) refers to the gradual loss of kidney function over a period of three months or longer, manifested as a decreased glomerular filtration rate (GFR) or persistent structural damage (such as proteinuria). It is divided into five stages and is a major cause of kidney failure.

[0180] In this article, diabetic nephropathy is a common complication in diabetic patients, characterized by proteinuria, decreased glomerular filtration rate (GFR), and hypertension, which can lead to end-stage renal disease. Microalbuminuria is an early sign.

[0181] In this article, systemic lupus erythematosus (SLE) refers to a multisystem autoimmune disease characterized by various autoantibodies that can affect the skin, joints, kidneys, and other organs. Lupus nephritis is a common renal manifestation of SLE.

[0182] In this article, IgA nephropathy is a common primary glomerular disease caused by IgA deposition in the glomeruli. It manifests as hematuria, possibly accompanied by proteinuria, hypertension, and progressively deteriorating renal function.

[0183] Beneficial effects of the invention

[0184] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0185] 1. Excellent transmucosal efficiency and enhanced affinity of FcRn enable optimized kidney targeting. High bioavailability and delivery system that carries drugs or imaging reagents can be recycled through mucosal epithelial cells, thus achieving long-lasting effects.

[0186] 2. The complex of the present invention can be delivered to the kidneys via nasal delivery, gastrointestinal mucosal epithelial delivery, or blood injection, depending on the sustained-release properties and acid-base tolerance of the drug.

[0187] 3. This invention enables mucosal delivery, making the drug administration process more convenient, allowing patients to operate independently, and significantly improving patient compliance.

[0188] 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

[0189] Figure 1a shows the *Saccharomyces cerevisiae* plasmid vector pYD1, constructed in Example 1, and Figures 1b, 1c, and 1d show the fluorescence signals and their ratio changes of the initial library and different rounds of screening for expression and binding on the yeast surface as described in Example 1. Figure 1e shows the *Pichia pastoris* plasmid vector pPIC9K, constructed in Example 1, and Figure 1f shows the isolation and purification results of the DIII mutant. In the plasmid schematic diagrams of Figures 1a and 1e, DIII refers to the DIII mutant.

[0190] Figure 2-1 shows the ELISA results of DIII mutants DIIIV1, DIIIV2, DIIIV3, wild-type DIII, and HAS in Example 2-1.

[0191] Figure 2-2 shows the results of intracellular colocalization of DIII mutant and FcRn using fluorescence confocal microscopy in Examples 2-3. For ease of labeling, the sample names in the figure are abbreviated. V1 corresponds to DIIIV27, V2 corresponds to DIIIV28, V3 corresponds to DIIIV29, V4 corresponds to DIIIV30, and V5 corresponds to DIIIV31.

[0192] Figures 2-3 show the Pearson product-moment correlation coefficients (PCCs) and overlap coefficients (O1R) of the DIII mutant and FcRn colocalization signal distribution in Example 2-3 using fluorescence confocal microscopy for intracellular colocalization analysis. For ease of labeling, the sample names in the figures are abbreviated. V1 corresponds to DIIIV27, V2 to DIIIV28, V3 to DIIIV29, V4 to DIIIV30, and V5 to DIIIV31.

[0193] Figure 2-4a shows the fluorescence intensity results of DIII mutants and 293T cells detected by a near-infrared imager in Examples 2-3. For ease of labeling, the sample names in the figure are abbreviated. V1 corresponds to DIIIV27, V2 corresponds to DIIIV28, V3 corresponds to DIIIV29, V4 corresponds to DIIIV30, and V5 corresponds to DIIIV31.

[0194] Figure 2-4b shows the fluorescence intensity results of DIII mutants and T84 cells detected by a near-infrared imager in Examples 2-3. For ease of labeling, the sample names in the figure are abbreviated. V1 corresponds to DIIIV27, V2 corresponds to DIIIV28, V3 corresponds to DIIIV29, V4 corresponds to DIIIV30, and V5 corresponds to DIIIV31.

[0195] Figure 3 shows the in vivo metabolism of the IR783 and IR783@DIII mutants described in Example 3 after tail vein injection, as shown in near-infrared II imaging.

[0196] Figure 4 shows the metabolism of Cy5-DIIIV as described in Example 4 under a two-photon laser confocal microscope.

[0197] Figure 5 shows the expression of FcRn in the kidney under a confocal microscope as described in Example 5.

[0198] Figure 6a shows the pharmacokinetic monitoring of DIII, DIIIV1, and DIIIV2 after intranasal delivery and renal metabolism in mice as described in Example 6.

[0199] Figure 7 shows the PK metabolism via the kidneys of mice after oral administration of IR783@DIIIV1 described in Example 7.

[0200] Figure 8a shows the yeast expression system plasmid designed by DIIIV1-Spytag as described in Example 8, Figure 8b shows the protein gel image of yeast expression purification, and Figure 8c shows its renal metabolism in vivo.

[0201] Figure 9a shows the prokaryotic expression plasmid designed by Spycatcher-eGFP as described in Example 9, Figure 9b shows the prokaryotic expression purification gel image of Spycatcher-eGFP, Figure 9c shows the protein gel image of DIIIV1-Spytag-Spycatchere-GFP, and Figure 9d shows the renal metabolism of DIIIV1-eGFP in vivo.

[0202] Figure 10 is a design diagram of the DIIIV1-ZF9 plasmid for delivering nucleic acid systems as described in Example 10.

[0203] Figure 11a is a WB verification diagram of the small amount of DIIIbV1 expression described in Example 11, and Figure 11b is the result of in vivo two-photon imaging performed in C57BL / 6J mice.

[0204] Figure 12a shows the plasmid designed by DIIIV1-RP81 as described in Example 12. Figures 12b and 12c are protein gel electrophoresis images and Western blot verification images of the fusion protein DIIIV1-RP81. Figure 12d shows the renal metabolism of DIIIV1-RP81 in vivo. Figure 12e is the renal HE image of DIIIV1-RP81 administered via tail vein for the treatment of AKI. Figure 12f shows the pathological score data corresponding to the HE image.

[0205] Figure 13a shows the renal urea nitrogen (UREA / BUN) results after nasal administration of DIIIV1-RP81 for the treatment of AKI as described in Example 13. Figure 13a shows the serum urea (UREA) results. Figure 13b shows the serum creatinine (CREA / Scr) results. Figure 13c shows the renal HE and TUNLE results. Figure 13d shows the pathological score results corresponding to HE.

[0206] Figure 14 shows the results of blood urea nitrogen (UREA / BUN) in the kidneys after oral administration of DIIIV1-RP81 for the treatment of AKI as described in Example 14. Figure 14a shows the serum urea (UREA) results, Figure 14b shows the serum creatinine (CREA / Scr) results, Figure 14c shows the neutrophil gelatinase-associated lipocalin (NGAL) results, and Figure 14d shows the kidney injury molecule (KIM-1) results.

[0207] Figure 15a shows the synthetic pathway of DIIIV1-Cortisone described in Example 15, Figure 15b is a protein gel electrophoresis image, and Figure 15c shows the serum creatinine (CREA / Scr) data of mice after treatment.

[0208] Figure 16a shows the synthetic pathway of DIIIV1-Curcumin as described in Example 16, Figure 16b is a protein gel electrophoresis image, and Figure 16c shows the serum creatinine (CREA / Scr) data of mice after treatment.

[0209] Figure 17a shows the synthesis pathway of DIIIV1-MMAE described in Example 17, Figure 17b shows a protein gel electrophoresis image, Figure 17c shows the mass spectrometry verification data of DIIIV-MMAE, and Figure 17d shows the tumor cell killing effect of DIIIV-MMAE verified at the cellular level.

[0210] Figure 18a shows the synthetic route of DIIIV1-Dexamethasone described in Example 18, Figure 18b shows the protein gel electrophoresis verification image, and Figure 18c shows the kidney HE results of DIIIV1-Dexamethasone used to treat glomerular basement membrane nephritis.

[0211] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0212] Table 1: Sequence Information Detailed Implementation

[0213] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0214] 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)).

[0215] 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.

[0216] Example 1. Screening, preparation and purification of FcRn high-affinity DIII mutants

[0217] First, a DIII mutant sequence library 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 MPNN deep learning to obtain an initial mutant library. Next, Alphafold2 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, and competitive binding index PAE parameters to obtain the final mutant library. The mutant library was fabricated into a gene chip. The chip was first centrifuged at high speed (12000 rpm, 1 min), and then dissolved in TE buffer (Tris-EDTA buffer). The mutant sequences were ligated to the pYD1 vector using a conventional seamless cloning method. An example DIII mutant and vector plasmid are shown in Figure 1a. The sequences were stored at -20℃ for later use.

[0218] Streaking *Saccharomyces cerevisiae* EBY100 glycerol culture onto YPD plates and incubate at 30℃ for approximately 36 hours. Pick single colonies and transfer them to 3 mL of YPD medium, incubating overnight at 30℃ (activation OD600 value should be 2-5). Transfer an appropriate amount of yeast to 3 mL of fresh YPD medium, adjusting the OD600 value to 0.1, and incubate at 30℃ with a shaker until the OD600 value is 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 later use. For each yeast transformation tube, prepare the transformation solution by adding the following reagents in the following volumes: 62.4 μL 50% PEG3350, 8.22 μL 1MTE / LiAC, and 5 μL ssDNA. Mix the transformation solution thoroughly. For each yeast transformation tube, add 75.62 μL of transformation solution, 20 μL of competent cells, and 100 ng of plasmid DNA. Mix thoroughly and incubate at 30°C in an incubator / shaker for 35 min. After incubation, heat shock at 42°C for 15 min. Centrifuge at 4200 rpm for 2 min, discard the supernatant. For each tube of transformed yeast, resuspend in 100 μL of sterile ultrapure water, plate onto MD plates, and incubate at 30℃ for 48-72 h until obvious yeast colonies appear on the MD plates. Resuspend in 2-3 mL of 2*SC-URATRP selective medium and incubate on a shaker (30℃, 200-250 rpm) until OD600 2⁻⁵. Transfer 300 μL to activation medium (2% glucose-YNB-C) and incubate until OD600 2⁻⁵. Dilute the yeast to 3-5 mL of induction medium (2% galactose-YNB-C) with an OD600 of 0.5-1 and incubate on a shaker for 48-72 h (20℃, 200-250 rpm). Transfer to activation medium again using the same method for simultaneous incubation as a non-induced negative control. After induction, take appropriate amounts of induced and non-induced yeast culture and dilute with PBS pH 6.0 to an OD600 of 1, 1 mL each, for later use.

[0219] The yeast was screened according to the following groups:

[0220] (1) Negative control: a. Uninduced group: Take 200 μL of uninduced yeast culture diluted in 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 in PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS Resuspend the bacterial culture at pH 6.0, wash the culture, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 600 μL PBS at pH 6.0 to resuspend the bacterial culture for later use;

[0221] (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-TagMouse 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 Resuspend the bacterial culture at pH 6.0, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 90 μL PBS, resuspend the culture at pH 6.0, add 10 μL FcRn-β2M protein (Biotinylated, His-Avi, 100 μg / mL), incubate at room temperature for 1 h, add 2 μL HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody and 0.3 μL Streptavidin APC, incubate at room temperature for 1 h, add 1 mL PBS, resuspend the culture at pH 6.0, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL PBS, resuspend the culture at pH 6.0 for later use.

[0222] After all samples were prepared, they were sequentially tested using flow cytometry and FACS sorting. The sorting results are shown in Figures 1b-1d. 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 were 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 1c and 1d 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.

[0223] The mutants selected through FACS final screening were subjected to high-throughput sequencing. After comparison with the original mutant sequences, the preferred mutation sites (combinations) are shown in Table 2, and the mutant sequences are shown in Table 1. These sequences correspond to amino acids 497 to 585 of wild-type human serum albumin SEQ ID NO:1 (unless otherwise specified, the amino acid positions involved in this invention are numbered from the N-terminus to the C-terminus). The experiments conducted in this application used the third domain (DIII) containing these sequences; that is, the sequences of the mutants shown in Table 1 were substituted at the corresponding positions of the third domain.

[0224] The mutant was synthesized into the pPIC9K vector plasmid (the plasmid map of the example is shown in Figure 1e). 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 YPD medium and cultured overnight at 30℃ with shaking until the OD600 value reached 1. The overnight culture was transferred at a ratio of 1:100 to 250mL Erlenmeyer flasks containing 50mL YPD medium and cultured overnight at 30℃ with shaking until the Pichia pastoris OD600 reached 0.8-1.0. The yeast pellet was collected by centrifugation at 1500g for 10min at room temperature, washed twice with 25mL sterile water, centrifuged at 1500g for 10min at room temperature, and the supernatant was discarded. The cells were resuspended in 0.1M LiCl solution, centrifuged at 10000g for 15s, the supernatant was discarded, and 0.1M LiCl solution was added. Resuspend the yeast in LiCl solution, transfer to a 1.5 mL centrifuge tube, mix well, aliquot, and centrifuge at 1500g for 5 min at room temperature to collect competent yeast cells for later use. Plasmid linearization: Linearize the plasmid DNA by single-enzyme digestion using standard methods; Salmon sperm DNA (ssDNA preparation): Add an appropriate amount of 2 mg / mL salmon sperm DNA to a metal bath (100℃, 5 min) and immediately place on ice (ice-water mixture) to prepare single-stranded DNA. Transformation solution preparation: For each yeast transformation tube, add the following reagents in the following volumes 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 culture medium was agitated until the yeast precipitate was dispersed and mixed. The centrifuge tubes were sealed with sealing film and shaken on a horizontal shaker at 30°C for 1-4 hours (the 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 for 2-3 days, 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 plate was added to prepared YPD plates containing different gradients of G418 antibiotics for a second high-copy transformant screening. Culture at 30℃ for 3-4 days. Once single colonies have grown, pick a single colony and add it to a 250mL Erlenmeyer flask containing 25mL of BMGY medium. Incubate on a shaker at 30℃ and 250rpm until the OD600 reaches 2-6. Centrifuge at 3000g for 5min at room temperature to collect the yeast. Add the yeast to a 1L shaker flask containing 100mL of BMMY medium until the OD600 reaches 1.0. Seal the flask with 6-8 layers of gauze and incubate on a shaker at 28℃ and 235rpm for 1-5 days. Add 1% methanol every 12-24h to induce expression. After induction, centrifuge at 9000g for 20min to collect the supernatant. Filter twice with a 0.8μm vacuum pump and once with a 0.22μm vacuum pump. Collect the filtrate for later use.

[0225] 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 to 5CV using equilibration buffer. The column was washed with Buffer 1 (50mM pH 6.0 sodium phosphate buffer), and the column was equilibrated to 5CV or UV back to baseline using equilibration buffer. The column was then washed with Buffer 2 (50mM pH 6.0 sodium phosphate buffer), and the column was equilibrated to 5CV or UV back to baseline using equilibration buffer. The column was then washed with Buffer 3 (50mM pH 8.0 ammonium acetate buffer), and the column was equilibrated to 5CV or UV back to baseline using equilibration buffer. The target protein was eluted with Buffer 4 (pH 8.0 50mM ammonium acetate 10mM sodium caprylate buffer), and the column was equilibrated to 5CV or UV back to baseline using equilibration buffer. The column was washed with 0.5M NaOH, and 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 and used selectively; the more contaminating proteins and column cycles, the more washing steps were required. All buffers were prepared according to the manufacturer's recommended methods. The purified mutant was verified by protein gel electrophoresis, and the results are shown in Figure 1f, yielding a purified DIIIV protein solution.

[0226] Table 2. Mutation sites of exemplary mutants

[0227] Example 2-1. Determination of the affinity between the DIII mutant and FcRn

[0228] First, the purified DIII mutant 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.

[0229] 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 Anti-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 DIII mutants in Example 2-1 to FcRn was calculated.

[0230] The dissociation constants (Kd) of the DIII mutants with FcRn are summarized in Table 3. The ELISA results of representative DIII mutants DIIIV1, DIIIV2, and DIIIV3 with wild-type DIII and HSA are shown in Figure 2-1. All detected DIIIV mutants exhibited very strong binding affinity to FcRn under acidic pH conditions (e.g., pH 6.0). However, these DIIIV 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). Furthermore, these DIII mutants generally exhibited higher affinity for FcRn under acidic pH conditions compared to wild-type DIII and HSA.

[0231] Table 3. Affinity results of representative mutants

[0232] Example 2-2 Determination of the affinity between the DIIIb mutant and FcRn

[0233] Based on the affinity determination of DIIIV with FcRn in Example 2-1, we further determined the affinity of the mutant containing only the DIIIb portion (i.e., only SEQ ID NO:4) and lacking the DIIIa portion (i.e., SEQ ID NO:3) with FcRn. The specific mutant sequences are shown in Table 1. This sequence corresponds to the fragment from amino acid 497 to amino acid 585 of SEQ ID NO:1.

[0234] 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.

[0235] 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 Anti-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 sample at 450 nm was immediately measured, and the affinity between DIIIbV and FcRn was calculated.

[0236] The 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).

[0237] Table 4. Affinity results of representative mutants

[0238] Examples 2-3. Transport and circulation of DIII mutants in cells

[0239] IR-780 dye with a final concentration of 1 μM was mixed with albumin or DIII and DIII mutant protein at a molar ratio of 1:1 in 500 μL of PBS buffer. The mixture was vortexed for 15 s, incubated in a water bath at 60 °C for 10 min, and stored in the dark for later use. The mixture was prepared fresh each time it was used. (For ease of labeling, the samples in the results are abbreviated. V1 corresponds to DIIIV27, V2 corresponds to DIIIV28, V3 corresponds to DIIIV29, V4 corresponds to DIIIV30, and V5 corresponds to DIIIV31. The same applies below.)

[0240] The co-localization of DIIIV (DIII variants, DIII Vs) and FcRn in intracellular endosomes was verified using fluorescence confocal microscopy, demonstrating the efficient co-localization of high-affinity DIIIV with 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 DIIIV30-Ab and DIIIV30-Ba A1 were also given DIII-specific polyclonal antibody (0.5 μM) to block its binding to the FcRn site and bind 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.

[0241] Experimental results showed that, compared with wild-type HSA and unmutated DIII, the high-affinity mutants DIIIV28 and DIIIV30 exhibited significantly stronger signals in cells, and their co-localization signals with FcRn were also significantly better than the control group (see lower right corner of Figure 2-2). The Pearson product-moment correlation coefficient (PCCs) and overlap coefficient (Ol R) of the mutant co-localization signal distribution with FcRn were significantly better than those of the control group. Furthermore, competitive blocking with antibodies and disruption of the endosome acidic environment resulted in a sharp decrease in the signal of the high-affinity mutant DIIIV30, and poorer co-localization data (Figure 2-3).

[0242] Therefore, this experiment used fluorescence confocal microscopy to perform intracellular colocalization of DIIIV and FcRn, verifying the efficient intracellular colocalization of high-affinity DIIIV with FcRn, and providing cellular-level evidence for its further transport and circulation mediated by ligand FcRn.

[0243] 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 DIIIV30-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.

[0244] As can be seen from Figures 2-4a (293T cells) and 2-4b (T84 cells), regardless of the time points of 30 min, 1 h, 2 h, or 4 h, the high-affinity mutants DIIIV28 and DIIIV30 showed significantly higher amounts of protein transported back into the extracellular space via FcRn compared to the wild-type HSA, DIII, and DIIIV30-Ab control groups. This further suggests that high-affinity DIIIV can undergo efficient transport and circulation in cells.

[0245] Example 3: Pharmacokinetic characteristics of the DIII mutant after tail vein injection in vivo

[0246] IR-783 was dissolved in DMSO to obtain a stock solution with a concentration of 2 mmol / L. The concentration of DIIIV1 protein was determined to be 2 mg / mL using the BCA method, which is equivalent to a molar concentration of 85.5 μmol / L. IR-783 and DIIIV1 were mixed at a molar ratio of 1:1, vortexed for 30 s, and then heated in a water bath at 60 °C for 10 min to form a stable covalent cyanine dye complex IR-783@DIIIV1.

[0247] 100 μL of 20 μM IR783 and IR783@DIIIV1 complex were injected via tail vein into different Balb / c mice. After administration, brightness was characterized in the near-infrared window. Imaging was performed using an indium gallium arsenide (InGaAs) camera under 808 nm laser excitation and a 1000 nm long-pass filter to verify the kidney-targeting ability of DIII and its ability to carry small molecule compounds. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence data in prone, supine, and lateral positions at 5 min, 10 min, 30 min, 1 h, 2 h, and 4 h post-administration.

[0248] As shown in Figure 3, after tail vein injection of DIIIV1, DIIIV1 showed specific enrichment in the kidney region. Replacing DIIIV1 with other DIII mutants of the present invention yielded similar results for the resulting complex.

[0249] Example 4: Metabolism of the DIII mutant in the kidney

[0250] Cy5-NHS was dissolved in DMSO to obtain a stock solution with a concentration of 10 mM. The concentration of DIIIV1 was determined by the BCA method to be 2 mg / mL, which is equivalent to a molar concentration of 85.5 μmol / L. Cy5-NHS and DIIIV1 were mixed at a molar ratio of 5:1 and reacted at room temperature for 4 h on a shaking reactor. After the reaction was completed, the mixture was filtered once through a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 10 min to remove free Cy5-NHS.

[0251] Mice were anesthetized with isoflurane and disinfected with povidone-iodine. The kidneys of Balb / c mice were exposed using surgical instruments, and a negative pressure adsorbent was used to adsorb the kidneys, placing them under a two-photon fluorescence microscope. First, the confocal plane was located at 900 nm. Then, 100 μL of 20 μM Cy5-DIIIV1 and 100 μL of FITC-Dex were injected via the tail vein. Fluorescence signals in the 495-540 nm (FITC) and 575-645 nm (Cy5) channels were monitored, and the tubular distribution of Cy5-DIIIV1 was observed at 0 min, 2 min, 15 min, and 30 min.

[0252] As shown in Figure 4, after Cy5-DIIIV1 injection, DIIIV1 filled the renal tubules. Over time, DIIIV1 migrated towards the tubular wall, where the signal intensity increased and exhibited punctate patterns. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complex. These results confirm the reabsorption of DIIIV1 within the renal tubular wall. The DIII mutants are reabsorbed into the bloodstream via FcRn in the proximal tubules, leading to long-term circulating accumulation in the kidneys.

[0253] Example 5: FcRn expression in the kidney region

[0254] Live mouse kidneys were dissected, surface blood was aspirated, and the sections were placed in embedding cassettes and embedded using a cryostat. The sections were then fixed overnight at -80°C. The sections were cut into 5-10 μm thicknesses using a cryostat. They were washed clean with 1X PBS, allowed to dry, and fixed with 4% paraformaldehyde for 10 min, followed by repeated PBS washing. The sections were then blocked with goat serum for 30 min without washing, and incubated overnight with FcRn primary antibody (1:100) and LTL (FITC-lotus root agglutinin, specifically labeled for renal tubules, 1:200). After washing with PBS for 1 h, the sections were incubated with secondary antibody (1:500) at room temperature for 1 h, followed by washing with PBS for 1 h. Finally, the sections were mounted with DAPI-containing mounting medium.

[0255] The results were observed under a confocal microscope. As shown in Figure 5, FcRn is mainly located in the tubular wall and glomerulus, which corresponds to Figure 4. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complex. These results demonstrate that DIIIV achieves effective enrichment in the kidneys via FcRn, and mutants with high FcRn affinity will have even higher kidney enrichment capacity, providing a highly efficient targeting vector for kidney diseases.

[0256] Example 6: Pharmacokinetics of the DIII mutant after nasal drop administration

[0257] As in Example 3, the same covalent cyanine dye complexes IR-783@DIII, IR-783@DIIIV1, and IR-783@DIIIV2 were prepared. Balb / c mice were administered 100 μL of 20 μM IR783@DIII and IR-783@DIIIV1 complexes via intranasal instillation. After administration, brightness was characterized in the near-infrared window using an indium gallium arsenide (InGaAs) camera with 808 nm laser excitation and a 1000 nm long-pass filter. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence in the lateral decubitus position at 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 48 h, 72 h, 4 d, 6 d, 8 d, 10 d, 12 d, and 14 d after administration. The results are shown in Figure 6, clearly detecting DIII and the DIII mutant DIII V protein fluorescence signals in the mouse kidneys. Compared to the original DIII, the FcRn affinity of the DIII mutant was increased by 100-1000 times. Quantitative data on the relative average fluorescence intensity were obtained by comparing the mean fluorescence intensity of the kidney region with the mean fluorescence intensity of the adjacent muscle region in mouse imaging data at different time points (as shown in Figure 6d). These results show that the high-affinity DIII mutant has a higher signal-to-noise ratio in the kidney region and a significantly longer retention time during 14 days of monitoring. Replacing DIIIV1 or DIIIV2 with other DIII mutants of this invention yielded similar results for the resulting complexes. These results indicate that both DIII and its mutants can be delivered across the nasal mucosa, but the high-affinity DIII mutant exhibits higher transnasal mucosal efficiency and a longer retention time in the kidney, enabling more efficient transnasal drug delivery and kidney targeting.

[0258] Example 7: Pharmacokinetics of the DIII mutant after oral administration

[0259] As in Example 3, the same covalent cyanine dye complex IR-783@DIIIV1 was prepared. Balb / c mice were orally administered 200 μL of the 20 μM IR783@DIIIV1 complex, and brightness characterization was performed in the near-infrared window. Imaging was performed using an indium gallium arsenide (InGaAs) camera under 808 nm laser excitation and a 1000 nm long-pass filter. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence data in the lateral decubitus position at 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, 4 d, 6 d, 8 d, 10 d, 12 d, and 14 d after administration.

[0260] As shown in Figure 7, after oral administration, IR-783@DIIIV1 can remain in the stomach for a prolonged period and be slowly absorbed through the mucosa, thereby entering the bloodstream and achieving renal targeting. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complexes. These results demonstrate that DIII mutants can achieve renal targeting across the gastrointestinal mucosa and can be used for drug delivery across the gastric mucosa.

[0261] Example 8: Pharmacokinetics of the DIII mutant loaded with Spytag protein in vivo

[0262] First, a vector plasmid was synthesized from the DIIIV1 and Spytag fusion protein, as shown in Figure 8a. The protein was then expressed using both prokaryotic and eukaryotic systems (GS115 Pichia pastoris and HEK293T cells). Subsequently, the protein expressed in Pichia pastoris was purified using an affinity column to obtain the purified DIIIV1-Spytag fusion protein. The protein expressed in HEK293T cells was purified using a nickel column to obtain the purified DIIIV1-Spytag fusion protein, as shown in Figure 8b.

[0263] Then, as in Example 3, IR-783 and DIII were mixed at a 1:1 molar ratio, vortexed for 30 seconds, and then heated in a 60°C water bath for 10 minutes to form a stable covalent cyanine dye complex IR-783@DIIIV1-Spytag. 100 μL of the 20 μM complex was injected into Balb / c mice via the tail vein. After administration, brightness was characterized in the near-infrared window using an indium gallium arsenide (InGaAs) camera with 808 nm laser excitation and a 1000 nm long-pass filter. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence data in the lateral decubitus position at 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, 4 d, 6 d, 8 d, 10 d, 12 d, and 14 d after administration.

[0264] As shown in Figure 8c, DIIIV1-Spytag can be effectively enriched in the kidney region. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complexes. These results indicate that the DIII mutants possess good protein loading capacity while retaining their original pharmacokinetic characteristics and kidney-targeting properties, making them suitable for loading kidney diagnostic or therapeutic proteins and peptides.

[0265] Example 9: Pharmacokinetics of the DIII mutant loaded with eGFP protein in vivo

[0266] First, a vector plasmid was synthesized from the Spycatcher and eGFP fusion protein, as shown in Figure 9a. The protein was then expressed using a prokaryotic system (E. coli). The expressed protein was subsequently purified using a nickel column to obtain the purified Spycatcher-eGFP fusion protein, as shown in Figure 9b. Then, DIIIV1-Spytag and Spycatcher-eGFP were incubated together to form the DIIIV1-Spytag-Spycatcher-eGFP fusion protein, as shown in Figure 9c.

[0267] Then, as in Example 3, IR-783 and DIIIV1-Spytag-Spycatcher-eGFP were mixed at a 1:1 molar ratio, vortexed for 30 s, and then heated in a 60°C water bath for 10 min to form a stable covalent cyanine dye complex IR-783@DIIIV1-Spytag-Spycatcher-eGFP. 50 μL of the 20 μM complex was injected intrabronchially into Balb / c mice. After administration, brightness was characterized in the near-infrared window using an indium gallium arsenide (InGaAs) camera under 808 nm laser excitation and a 1000 nm long-pass filter. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence data from the lateral decubitus position, back, and abdomen of the mice after administration.

[0268] As shown in Figure 9d, DIIIV1-Spytag-Spycatcher-eGFP can be effectively enriched in the kidney region. Replacing DIIIV1 with other DIII mutants of the present invention yields complexes with similar results. Example 9 further demonstrates that the DIII mutant has good protein loading capacity and retains the kidney-targeting characteristics, making it suitable for loading kidney diagnostic or therapeutic proteins and peptides, providing an efficient tool for the diagnosis and treatment of kidney diseases.

[0269] Example 10: Pharmacokinetics of the DIII mutant after loading ZF9 protein and delivering nucleic acid in vivo.

[0270] A plasmid for synthesizing the DIIIV1 and ZF9 fusion protein was synthesized (as shown in Figure 10).

[0271] Example 11: Pharmacokinetics of the DIIIb mutant in vivo

[0272] First, a plasmid for the DIIIb mutant was designed and expressed using a eukaryotic expression system. Then, the DIIIb mutant was purified using an affinity column to obtain a high-purity DIIIb mutant. Finally, Western blot verification was performed using a polyclonal antibody against albumin, as shown in Figure 11a.

[0273] Then, as in Example 3, Cy5-NHS was dissolved in DMSO to obtain a mother liquor with a concentration of 10 mM. The concentration of DIIIbV1 was determined to be 1 mg / mL using the BCA method. Cy5-NHS and DIIIbV1 were mixed at a molar ratio of 5:1 and reacted at room temperature for 4 h on a shaking reactor. After the reaction was completed, the mixture was filtered once through a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 10 min to remove free Cy5-NHS.

[0274] Mice were anesthetized with isoflurane and disinfected with povidone-iodine. The kidneys of C57BL / 6J mice were exposed using surgical instruments, and the kidneys were adsorbed using a negative pressure adsorbent and placed under a two-photon fluorescence microscope. First, the confocal plane was located at 900 nm. Then, 100 μL of 20 μM Cy5-DIIIVb and 100 μL of Hochest were injected via the tail vein. The fluorescence signals in both channels were monitored, and the tubular distribution of Cy5-DIIIbV1 was observed at 0 min, 1 min, 5 min, 10 min, 15 min, and 20 min. The results are shown in Figure 11b. It can be seen that DIIIbV1 appeared in the proximal tubules over time, while no obvious signal appeared in the distal tubules. This indicates that DIIIbV1 remained in the proximal tubules of the kidney, and the signal gradually weakened, which is similar to the results of the DIII mutant in Example 3. Replacing DIIIbV1 with other DIIIb mutants of the present invention yields a complex with similar results, achieving reabsorption via FcRn in the proximal tubules, thereby enabling long-term enrichment of the DIIIVb mutant in the kidneys.

[0275] Example 12: Treatment of AKI by tail vein administration of DIII mutant-RP81

[0276] The DIIIV1-RP81 fusion protein was synthesized into a vector plasmid, as shown in Figure 12a. The protein was then expressed using a eukaryotic system (yeast). The expressed protein was subsequently purified using an albumin affinity column to obtain the purified DIIIV1-RP81 fusion protein. The protein gel electrophoresis pattern and Western blotting results are shown in Figures 12b and 12c.

[0277] Then, as in Example 3, IR-783 and DIIIV1-RP81 were mixed at a 1:1 molar ratio, vortexed for 30 seconds, and then heated in a 60°C water bath for 10 minutes to form a stable covalent cyanine dye complex IR-783@DIIIV1-RP81. 100 μL of the 20 μM complex was injected into Balb / c mice via tail vein. After administration, brightness was characterized in the near-infrared window using an indium gallium arsenide (InGaAs) camera with 808 nm laser excitation and a 1000 nm long-pass filter. Long-term fluorescence detection was performed in the near-infrared II imaging system, acquiring fluorescence data from the lateral decubitus position, back, and abdomen of the mice after administration. The imaging results are shown in Figure 12d, indicating that DIIIV1-RP81 was effectively enriched in the kidney region. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complex.

[0278] A mouse model of renal ischemia / reperfusion AKI was established: C57BL / 6J mice were anesthetized, and the abdominal cavity was exposed through 0.5cm incisions below the costal margins on both sides of the back. The renal pedicles were separated, and each pedicle was clamped with an arterial clamp. After 30 minutes, the clamps were released, and the restoration of renal blood supply was assessed based on the color change of the kidneys. Finally, the abdomen was sutured closed. The control group mice underwent the same procedure, except for the separation of the renal pedicles. 100μL of DIII-RP81 protein solution was administered via the tail vein. Blood was collected 24 hours later and incubated at room temperature for 1 hour. The blood was then centrifuged at 3500rpm for 15 minutes at 4°C to obtain the supernatant serum. The levels of BUN, Scr, KIM-1, and NGAL were detected using an ELISA kit. HE staining was used to assess the treatment effect on renal damage. HE results and pathological scoring data are shown in Figures 12e and 12f, indicating that DIIIV1-RP81 can alleviate renal damage. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complex.

[0279] Example 13: Treatment of AKI by intranasal administration of DIII mutant-RP81

[0280] As in Example 12, a mouse model of renal ischemia / reperfusion AKI was constructed: C57BL / 6J mice were anesthetized, and the abdominal cavity was exposed through 0.5cm incisions below the costal margins on both sides of the back. The renal pedicles were separated, and each pedicle was clamped with an arterial clamp. After 30 minutes, the clamps were released, and the restoration of renal blood supply was assessed based on the color change of the kidneys. Finally, the abdomen was sutured closed. The control group mice underwent the same procedure, except that the renal pedicles were separated. 50μL of 20μM MDIIIV1, RP81, and DIIIV1-RP81 protein solutions were administered via nasal drops. After 24 hours, mouse blood was collected and incubated at room temperature for 1 hour. The serum was then separated by centrifugation at 3500rpm for 15 minutes at 4°C. The levels of BUN, Scr, KIM-1, and NGAL were detected using an ELISA kit, and HE staining was used to assess the treatment effect on renal damage. The results showed that, compared with the model group, DIIIV1-RP81 reduced serum levels of UREA and CREA, as shown in Figures 13a and 13b. Kidney pathology also showed that DIIIV1-RP81 protected the kidneys by reducing renal tubular cell apoptosis, as shown in Figures 13c and 13d. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the resulting complex.

[0281] Example 14: Treatment of AKI with oral administration of DIII mutant-RP81

[0282] As in Example 12, a mouse model of renal ischemia / reperfusion AKI was constructed: C57BL / 6J mice were anesthetized, and the abdominal cavity was exposed through 0.5cm incisions below the costal margins on both sides of the back. The renal pedicles were separated, and each pedicle was clamped with an arterial clamp. After 30 minutes, the arterial clamps were released, and the restoration of renal blood supply was assessed based on the color change of the kidneys. Finally, the abdomen was sutured closed. The control group mice underwent the same procedure, except that the renal pedicles were separated. 200μL of 20μM DIIIV1, RP81, and DIIIV1-RP81 protein solutions were administered orally. After 24 hours, mouse blood was collected, and after being left at room temperature for 1 hour, the serum was separated by centrifugation at 3500rpm for 15 minutes at 4°C. The levels of BUN, Scr, KIM-1, and NGAL were detected using an ELISA kit, and HE staining was used to assess the treatment effect on renal damage. The results showed that, compared with the model group, DIIIV1-RP81 reduced the levels of UREA, CREA, KIM-1, and NGAL in serum (as shown in Figures 14a, 14b, 14c, and 14d). DIIIV-RP81 protected the kidney by reducing renal tubular cell apoptosis. Replacing DIIIV1 with other DIII mutants of the present invention yielded similar results for the DIII mutant-RP81 complex.

[0283] Example 15: Treatment of IgA Nephropathy with the DIII Mutant-Cortisone

[0284] DIIIV1-Cortisone was synthesized according to the synthetic route shown in Figure 15a: Equimolar amounts of Cortisone and MAL-PEG4-OH were dissolved in DMF and magnetically stirred at 80°C. Then, an equal volume of DMAP and three times the volume of DCC in DMF solution were added. After reacting overnight, urea generated from DCC was removed through cellulose / cotton filter paper, and the product was purified by preparative high-performance liquid chromatography to obtain Cortisone-MAL. A DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide (10 eq.) and 0.2% DIPEA were added to a PBS solution (40 mM, 1.0 eq.) of DIIIV1 protein, and the mixture was stirred at 25°C for approximately 3 h. The filtrate was collected after desalting column chromatography. Add 5 eq. of TCEP in PBS to the filtrate and reduce at 25°C for about 3 h. Then add 10 eq. of Cortisone-MAL in DMSO and stir at 25°C for about 18 h. Then add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench at 25°C for about 3 h. After centrifugation on a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Cortisone. The protein gel electrophoresis results are shown in Figure 15b.

[0285] Establishing an IgA nephropathy model: Balb / c mice were acclimatized for one week. The model group was administered bovine serum albumin (BSA) acidified water (800 mg / kg) by gavage every other day; castor oil and carbon tetrachloride (CCl4) were mixed in a 5:1 ratio and injected subcutaneously (0.1 mL) once a week, and simultaneously injected intraperitoneally (0.06 mL) once every two weeks; lipopolysaccharide (LPS) (50 μg) was injected via tail vein at weeks 6 and 8. The control group was given the corresponding dose of physiological saline. At the end of week 8, urine was collected from each group of mice, and the total urine volume was recorded. The collected urine was centrifuged at 3500 r / min for 5 min, and the supernatant was aliquoted into multiple centrifuge tubes and stored at -20℃ for later use. A portion of the aliquoted fresh urine was used for testing urinary biochemical indicators. Blood was collected from each group of mice and, after being left at room temperature for 1 hour, centrifuged at 4°C and 3500 rpm for 15 minutes to separate the supernatant serum. The serum supernatant was stored at -20°C for the determination of blood biochemical indicators. Mice were euthanized by cervical dislocation, and both kidneys were removed. After rinsing with physiological saline and drying with filter paper, the kidneys were photographed and weighed. The right kidney was flash-frozen in liquid nitrogen and then stored in aluminum foil at -80°C for proteomics analysis. The left kidney was fixed in 10% formalin for histopathological (H&E staining, PAS staining), immunofluorescence, and immunohistochemical analysis. Serum creatinine (CREA / Scr) data are shown in Figure 15c. It can be seen that DIIIV1-Cortisone has a certain protective effect on the kidneys, reducing serum creatinine and indicating a reduction in the deterioration of kidney function. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the DIII mutant-Cortisone complex.

[0286] Example 16: Treatment of Diabetic Nephropathy with the DIII Mutant-Curcumin

[0287] DIIIV1-Curcumin was synthesized according to the reaction route shown in Figure 16a: Curcumin was reacted with glutaric anhydride in anhydrous tetrahydrofuran using 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) as catalysts, under reflux at 90 °C and stirred for 24 hours under a nitrogen atmosphere. After the reaction, compound 1 was purified by solvent evaporation, washing with hydrochloric acid solution, and silica gel column chromatography (eluent: dichloromethane / methanol, 96:4, v / v). Its structure was confirmed by 1H NMR. Compound 1 was activated with EDC·HCl and NHS in anhydrous methanol for 30 min, followed by the addition of tBoc-NH-PEG-amine, and stirred at 25 °C for 12 hours. The reaction solution was purified by dialysis (molecular weight cutoff 1 kDa) and freeze-drying to obtain compound 2. Compound 2 was reacted with trifluoroacetic acid (TFA) in anhydrous methanol for 30 minutes to remove the Boc protecting group, followed by coupling with 6-maleimide hexanoic acid in the presence of EDC·HCl and NHS for 12 hours. The reaction solution was purified by dialyzing (molecular weight cutoff 1 kDa) and freeze-drying to obtain Curcumin-MAL. A DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide ester (10 eq.) and 0.2% DIPEA were added to a PBS solution (40 mM, 1.0 eq.) of DIIIV1 protein, and the mixture was stirred at 25 °C for approximately 3 hours. The filtrate was collected after desalting column chromatography. Add 5 eq. of TCEP in PBS to the filtrate and reduce at 25°C for about 3 h. Then add 10 eq. of Curcumin-MAL in DMSO and stir at 25°C for about 18 h. Then add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench at 25°C for about 3 h. After centrifugation on a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Curcumin. The protein gel electrophoresis data are shown in Figure 16b.

[0288] Establishment of a diabetic nephropathy model: Six-week-old C57BL / 6J mice were used to establish a diabetic nephropathy model. They were fed a high-fat diet (HFD) and intraperitoneally injected with STZ 50 mg / kg for 5 consecutive days until week 8. Treatment began in week 4; the experimental group received 100 μL of 10 μg DIIIV1-Curcumin via tail vein injection, while the control group received the same volume of physiological saline. After the experiment, urine was collected from each group, and the total urine volume was recorded. The collected urine was centrifuged at 3500 rpm for 5 min, and the supernatant was aliquoted into multiple centrifuge tubes and stored at -20℃ for later use. A portion of the aliquoted fresh urine was used for urinary biochemical assays; blood was collected from each group of mice, and after being left at room temperature for 1 hour, it was centrifuged at 3500 rpm for 15 min at 4℃ to separate the serum. The serum supernatant was stored at -20℃ for the determination of blood biochemical parameters. Mice were euthanized by cervical dislocation, and both kidneys were harvested. The kidneys were rinsed with physiological saline, dried with filter paper, photographed, and weighed. The right kidney was flash-frozen in liquid nitrogen and then stored in aluminum foil at -80°C for proteomics analysis. The left kidney was fixed in 10% formalin for histopathological examination (H&E staining, PAS staining), immunofluorescence, and immunohistochemical analysis. Serum creatinine (CREA / Scr) data are shown in Figure 16c. It can be observed that DIIIV1-Cortisone has a certain protective effect on the kidneys, reducing blood creatinine and decreasing the deterioration of renal function. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the DIII mutant-Curcumin complex.

[0289] Example 17: Treatment of Renal Cell Carcinoma with DIII Mutant-MMAE

[0290] The DIIIV1-MMAE was synthesized according to the synthetic pathway shown in Figure 17a: DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide (10 eq.) and 0.2% DIPEA were added to a PBS solution (40 mM, 1.0 eq.) of DIIIV1 protein. The mixture was stirred at 25°C for approximately 3 h, and the filtrate was collected after desalting. TCEP solution in PBS (5 eq.) was added to the filtrate, and the mixture was reduced at 25°C for approximately 3 h. Then, DMSO solution of VcMMAE (10 eq.) was added, and the mixture was stirred at 25°C for approximately 18 h. Next, PBS solution of N-acetyl-L-cysteine ​​(10 eq.) was added, and the mixture was quenched at 25°C for approximately 3 h. After centrifugation using a desalting column, the filtrate was collected and concentrated by ultrafiltration to obtain DIIIV1-MMAE. The synthesis of the complex was verified by protein gel electrophoresis, and the results are shown in Figure 17b, confirming the successful synthesis of DIIIV1-MMAE. The molecular weight of DIIIV1-MMAE was then determined using MALDI TOF. DIIIV1-MMAE and SA (sinapic acid) were mixed in a 1:1 ratio (2 μL DIIIV1-MMAE and 2 μL SA). After mixing the sample matrix, it was loaded onto a MALDI target plate using the direct drop method. After the sample dried, its molecular weight was determined using an Autoflex maX (Bruker) spectrometer. The results, shown in Figure 17c, indicate that the measured complex is the molecule we desired.

[0291] The therapeutic results of DIIIV1-MMAE were validated at the renal cell carcinoma level using CCK-8 assays. Human renal cell adenocarcinoma cells in the logarithmic growth phase were injected at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and pre-cultured at 37°C in a 5% CO2 incubator for 24 hours. Afterward, the old culture medium was discarded, and 100 μL of serum-free medium containing a series of concentrations (0.001 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM) of DIIIV1-MMAE was added to each experimental group, while the control group received an equal volume of serum-free medium. Cell-free blank wells were also included, with 5 replicates per group. The cells were cultured for another 48 hours. After culture, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 2 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the cell viability (%) was calculated using the formula: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% (where As, Ac, and Ab are the average absorbance values ​​of the experimental group, control group, and blank group, respectively). The results, shown in Figure 17d, demonstrate that DIIIV1-MMAE exhibits concentration-dependent toxicity and can effectively kill renal cell carcinoma cells. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the DIII mutant-MMAE complex.

[0292] Construction of a renal cell carcinoma model: Tumor cells labeled with luciferase were cultured to the logarithmic growth phase, digested with trypsin, and resuspended in PBS + Matrigel (1:1) (concentration: 5 × 10⁻⁶). 6 Cells / mL were collected and stored on ice to prevent Matrigel from solidifying. Balb / c mice were anesthetized by inhalation of isoflurane (3% for induction, 1.5% for maintenance). The kidneys were exposed using surgical instruments: the kidneys were gently pulled out with curved forceps (avoiding compression). 50 μL of cell suspension (approximately 2.5 × 10⁻⁶ cells / mL) was slowly injected subcapsularly using a blunt needle. 5 After injection, the needle is slowly withdrawn, and a local bulge can be seen on the surface of the kidney (success is indicated by no fluid leakage). The kidney is placed back into the abdominal cavity, the muscle layer is sutured (using 5-0 absorbable sutures), and the skin is clamped (or sutured). Postoperatively, a heating pad is used to maintain body temperature until awakening. D-fluorescein (150 mg / kg) is administered intraperitoneally daily, and tumor monitoring is performed using a small animal in vivo imaging system.

[0293] After the experiment, urine was collected from each group of mice, and the total urine volume was recorded. The collected urine was centrifuged at 3500 rpm for 5 min, and the supernatant was aliquoted into multiple centrifuge tubes and stored at -20℃ for later use. A portion of the aliquoted fresh urine was used to detect urinary biochemical indicators. Blood was collected from each group of mice and, after being left at room temperature for 1 hour, was centrifuged at 3500 rpm for 15 min at 4℃ to separate the upper serum layer. The supernatant serum was stored at -20℃ for the determination of blood biochemical indicators. Mice were euthanized by cervical dislocation, and both kidneys were removed. After rinsing with physiological saline and drying with filter paper, the kidneys were photographed and weighed. The right kidney was flash-frozen in liquid nitrogen and then wrapped in aluminum foil and stored at -80℃ for proteomics detection. The left kidney was fixed in 10% formalin for histopathological (H&E staining, PAS staining), immunofluorescence, and immunohistochemical analysis.

[0294] Example 18: Treatment of Glomerular Basement Membrane Nephritis with the DIII Mutant-Dexamethasone

[0295] DIIIV1-Dexamethasone was prepared according to the procedure shown in Figure 18a: Equimolar amounts of Dexamethasone and MAL-PEG4-OH were dissolved in DMF and magnetically stirred at 80°C. Then, an equal amount of DMAP and three times the amount of DCC in DMF solution were added. After reacting overnight, urea generated from DCC was removed through cellulose / cotton filter paper. The product was purified by preparative high-performance liquid chromatography to obtain Dexamethasone-MAL. A DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide (10 eq.) and 0.2% DIPEA were added to a PBS solution (40 mM, 1.0 eq.) of DIIIV1 protein, and the mixture was stirred at 25°C for approximately 3 h. The filtrate was collected after desalting column chromatography. Add 5 eq. of TCEP in PBS to the filtrate and reduce at 25°C for about 3 h. Then add 10 eq. of Dexamethasone-MAL in DMSO and stir at 25°C for about 18 h. Next, add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench at 25°C for about 3 h. After centrifugation using a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Dexamethasone. The gel protein of the prepared DIIIV1-Dexamethasone is shown in Figure 18b, which is the result we wanted.

[0296] Thirty 6-8 week old BALB / c mice were acclimatized for one week, and an anti-basal membrane (GBM) nephritis model was established. First, 6 mg of goat IgG antibody was dissolved in 0.5 mL of 0.9% NaCl solution, followed by the addition of 0.5 mL of Freund's complete adjuvant. After complete emulsification, the solution was injected into the right paw of the mice for pre-immunization. One week later, formal immunization was performed by injecting 40 μg of anti-GBM antibody via the tail vein. After modeling, the 24-hour urinary protein content was measured; a level exceeding 20 mg was considered a successful model. Mice were randomly assigned to Nephritis, Dexamethasone, or DIIIV1-Dexamethasone groups, with each group receiving the corresponding drug via tail vein injection at a dose of 4 μg. Several mice served as control mice, receiving an equal volume of physiological saline in the same manner. Immunization was administered once a week for four consecutive weeks.

[0297] After the experiment, urine was collected from each group of mice, and the total urine volume was recorded. The collected urine was centrifuged at 3500 rpm for 5 min, and the supernatant was aliquoted into multiple centrifuge tubes and stored at -20℃ for later use. A portion of the aliquoted fresh urine was used to detect urinary biochemical indicators. Blood was collected from each group of mice and, after being left at room temperature for 1 hour, was centrifuged at 3500 rpm for 15 min at 4℃ to separate the upper serum layer. The supernatant serum was stored at -20℃ for the determination of blood biochemical indicators. Mice were euthanized by cervical dislocation, and both kidneys were removed. After rinsing with physiological saline and drying with filter paper, the kidneys were photographed and weighed. The right kidney was flash-frozen in liquid nitrogen and then wrapped in aluminum foil and stored at -80℃ for proteomics detection. The left kidney was fixed in 10% formalin for histopathological (H&E staining, PAS staining), immunofluorescence, and immunohistochemical analysis.

[0298] The HE results, as shown in Figure 18c, initially demonstrated a relatively good therapeutic effect, and the degree of renal tubular damage was effectively alleviated. Replacing DIIIV1 with other DIII mutants of this invention yielded similar results for the DIII mutant-Dexamethasone complex.

[0299] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A complex comprising a carrier protein and a functional molecule for the diagnosis, prevention, and / or treatment of urinary system diseases; in, The carrier protein is selected from DIIIbV, a mutant of the albumin third domain substructure (e.g., DIIIb), albumin third domain DIIIV containing the mutant, and a multimer of DIIIbV or DIIIV.

2. The complex of claim 1, wherein, compared with the wild type, the mutant comprises one or more (e.g., 40-50, 30-40, 20-30, 15-20, 10-15, 5-10, 1-5) amino acid insertions, substitutions, deletions and / or mutations; Preferably, the inserted, substituted, deleted, and / or mutated amino acids correspond to the amino acids at positions 467 to 585 of SEQ ID NO:1; More preferably, the inserted, substituted, deleted and / or mutated amino acids correspond to the amino acids at positions 497 to 585 of SEQ ID NO:1; More preferably, the inserted, substituted, deleted and / or mutated amino acids correspond to the amino acids at positions 500 to 573 of SEQ ID NO:

1.

3. The complex of claim 1 or 2, wherein the DIIIbV or DIIIV: (1) Compared with the wild type, it has a higher FcRn affinity under acidic or weakly acidic conditions; preferably, the FcRn affinity Kd value of the DIIIV is 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Or 10 -11 M level; preferably, the DIIIbV affinity for FcRn, Kd value, is 10. -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Or 10 -11 M level; (2) Mutations containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids compared to the wild type; (3) Mutations containing one or more amino acid sites selected from the following: corresponding to amino acids 500, 505, 523, 524, 527, 528, 531, 547, 509, 510, 498, 512 and 573 of natural albumin having the amino acid sequence shown in SEQ ID NO:

1. (4) The 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 amino acid mutations contained in DIIIbV or DIIIV are located at positions 523 and 573; Preferably, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 505, 523, 547, and 573; Preferably, the amino acid mutations in DIIIbV or DIIIV are located at positions 500, 505, 523, 524, 527, 528, 531, 547, and 573; or, (6) Any combination of the above.

4. The complex according to any one of claims 1-3, wherein the DIIIbV or DIIIV: (1) The 500th amino acid in natural albumin having the amino acid sequence shown in SEQ ID NO:1 is L or D; (2) The amino acid at position 505 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is Q, N, or T; (3) The amino acid at position 523 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L or M; (4) The amino acid at position 524 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L; (5) The amino acid at position 527 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is K; (6) The amino acid at position 528 of natural albumin having the amino acid sequence shown in SEQ ID NO:1 is H or Y; (7) The amino acid at position 531 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L; (8) The amino acid at position 547 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is A or C; (9) The amino acid at position 509 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L; (10) The amino acid at position 510 of natural albumin having the amino acid sequence shown in SEQ ID NO:1 is R or N; (11) The amino acid at position 498 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is E; (12) The amino acid at position 512 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is G; (13) The amino acid at position 573 of the natural albumin having the amino acid sequence shown in SEQ ID NO:1 is any amino acid other than K (e.g., P); or, (14) The amino acid at position 508 of natural albumin, which corresponds to the amino acid sequence shown in SEQ ID NO:1, is L; (15) Any combination of the above.

5. The complex of claim 4, wherein the DIIIbV or DIIIV: (1) The amino acid at the 500th position 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 the 528th position 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 the 510th position 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 the 512th position is mutated from D to G; (13) The amino acid at the 573rd position 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; or, (15) Any combination of the above; Preferably, the DIIIbV or DIIIV contains mutations of I523G and K573P; Preferably, the DIIIbV or DIIIV contains mutations of E505Q, I523G, V547A and K573P; Preferably, the DIIIbV or DIIIV contains mutations of K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A and K573P.

6. The complex according to any one of claims 1-5, wherein, The wild type of DIII or DIIIb is derived from natural serum albumin of mammals; preferably, the mammals are selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs and pigs; preferably, the wild type of DIII or DIIIb is derived from natural human serum albumin. Preferably, the natural human serum albumin contains, or is composed of, the amino acid sequence shown in SEQ ID NO:1; Preferably, the natural human serum albumin DIII comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:2; Preferably, the natural human serum albumin DIIIb contains, or is composed of, the amino acid sequence shown in SEQ ID NO:

4.

7. The complex according to any one of claims 1-6, wherein the DIIIbV is selected from the amino acid sequences shown in SEQ ID NO: 36-40; Preferably, the DIIIV is selected from the amino acid sequences shown in SEQ ID NO:5-35; Preferably, the DIIIV has the amino acid sequence shown in SEQ ID NO:

5.

8. The complex according to any one of claims 1-7, wherein the carrier protein further comprises an albumin first domain DI, a portion thereof, a derivative thereof, or a mutant thereof, and / or an albumin second domain DII, a portion thereof, a derivative thereof, or a mutant thereof; Preferably, the DI and DII are each independently derived from the natural serum albumin of mammals; preferably, the mammals are selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs and pigs; preferably, the mammals are humans.

9. The complex according to any one of claims 1-8, wherein the polymer is a homo- or hetero-dimer, trimer, tetramer, or any polymer that can exist stably in physiological solutions of DIIIbV or DIIIV.

10. The complex according to any one of claims 1-9, wherein the urinary tract disease is kidney injury, urinary tract inflammation, or urinary tract cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the urinary system disease is selected from kidney disease, ureteral disease, bladder disease, and urethral disease; Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer; preferably, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerular lesions (e.g., diabetic nephropathy); preferably, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

11. The complex according to any one of claims 1-10, wherein the functional molecule is selected from diagnostic reagents, imaging agents, therapeutic radionuclides, or macromolecular or small molecule drugs, such as polypeptides, proteins, antibodies, nanobodies, nucleic acid drugs, or chemotherapeutic drugs; preferably, the polypeptide or protein is a peptide chain or a cyclic peptide; preferably, the antibody is a monoclonal antibody or its antigen-binding fragment; and the nucleic acid drug is mRNA or a protein-nucleic acid complex. Preferably, the drug is an anti-inflammatory, anti-apoptotic, or anti-oxidative stress drug for the urinary system disease; preferably, the drug is selected from amifostine, cimetidine, glutathione, RNLS agonists (e.g., RP81), cortisone, curcumin, dexamethasone, and MMAE. Preferably, the diagnostic reagent is selected from substances highly expressed or secreted by the renal system (such as interleukin, intrinsic factor-vitamin B12 receptor (cubilin), low-density lipoprotein receptor-associated protein 2 (megalin), kidney injury-associated molecule (KIM-1), neutrophil gelatinase-associated lipotransferase (NGAL), and other proteins). Preferably, the imaging agent is selected from cyanine dyes such as IR-780 and IR-783, and other photosensitizers; Preferably, the therapeutic radionuclide is selected from... 64 Cu、 18 F, 68 Ga、 177 Lu、 125 I, 90 Y、 89 Sr、 32 P, 233 Ra.

12. The complex according to any one of claims 1-11, wherein the carrier protein and the functional molecule are directly linked or linked through a linker; Preferably, the connector is a cuttable or non-cuttable connector; preferably, the cuttable connector is of the acid-cleaving type, disulfide bond-cleaving type, protease-cleaving type, glycosidase-cleaving type, or phosphatase-cleaving type; preferably, the connector is selected from DBCO-NHS ester, Sulfo-SMCC sodium, CL2 linker, DSP Cross linker, Mc-Val-Cit-PABC-PNP, Val-Cit-PAB, MC-Val-Cit-PAB, MAC glucuronide linker-2, Fmoc-PEA, Mal-PEG4-OH, 3-Mercaptopropionic acid NHS ester, and tBoc-NH-PEG-NH2.

13. The complex according to any one of claims 1-12, wherein the carrier protein and the functional molecule are coupled by gene fusion or chemical methods to form the complex.

14. A nucleic acid molecule encoding the complex according to any one of claims 1-13; Preferably, the complex is a fusion protein.

15. A vector comprising the nucleic acid molecule of claim 14; preferably, the vector is an expression vector; Preferably, the vector is a vector of eukaryotic bacteria (e.g., pPIC9K, pCDNA3.4).

16. A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15; Preferably, the cells are eukaryotic cells or prokaryotic cells; Preferably, the eukaryotic cells are yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris) and 293T cells; Preferably, the prokaryotic cells are Escherichia coli cells, Bacillus subtilis cells, or any combination thereof.

17. A delivery combination or pharmaceutical composition comprising the complex according to any one of claims 1-13; Preferably, the delivery combination or pharmaceutical composition is delivered intravenously; Preferably, the delivery combination or pharmaceutical composition is delivered via nasal or oral inhalation, preferably via nasal delivery, such as nasal drops, nasal spray, or a combination thereof; Preferably, the complex is delivered to the mucosal surface of the subject (e.g., oral mucosa, nasal mucosa, tracheal mucosa, eyelid mucosa, vaginal mucosa); Preferably, the delivery combination or pharmaceutical composition further comprises one or more mucosal adhesives to enhance the residence time of the effector molecules on the mucosal surface of the subject; Preferably, the delivery combination or pharmaceutical composition is an aerosol, powder inhaler, spray, or other dosage form suitable for inhalation administration; Preferably, the pharmaceutical composition contains one or more pharmaceutically acceptable excipients.

18. Use of the complex of any one of claims 1-13, the delivery combination of claim 17, or the pharmaceutical composition in the preparation of a medicament for treating urinary system diseases; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

19. The complex according to any one of claims 1-13, the delivery combination or pharmaceutical composition according to claim 17, for treating urinary system diseases; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

20. A method of treating a urinary tract disease, comprising administering to a subject in need an effective amount of the complex of any one of claims 1-13, the delivery combination of claim 17, or the pharmaceutical composition; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

21. Use of the carrier protein as defined in any one of claims 1-13 in the preparation of a medicament for treating urinary system diseases; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

22. The carrier protein as defined in any one of claims 1-13, for the treatment of urinary system diseases; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

23. A method of treating a urinary tract disease, comprising administering to a subject in need a carrier protein as defined in any one of claims 1-13; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

24. A medicament for treating diseases of the urinary system, comprising a carrier protein as defined in any one of claims 1-13; Preferably, the urinary system disease is kidney damage, urinary system inflammation, or urinary system cancer; Preferably, the urinary tract inflammation is selected from allergic inflammation such as glomerulonephritis; urinary tract infections such as renal tuberculosis, pyelonephritis, cystitis, and urethritis; Preferably, the urinary system tumor is selected from renal cancer, urothelial carcinoma such as renal pelvis cancer, ureter cancer, bladder cancer, and urethral cancer. Preferably, the kidney injury is selected from acute kidney injury, acute kidney disease, chronic kidney disease, or complications of other chronic diseases and autoimmune diseases (e.g., diabetic nephropathy, systemic lupus erythematosus nephropathy, IgA nephropathy); Preferably, the kidney disease is selected from kidney injury (e.g., acute kidney injury), glomerular disease, tubulointerstitial disease, and renal cancer. In some embodiments, the glomerular disease is selected from nephritis syndrome, nephrotic syndrome, and diabetic glomerulonephropathy (e.g., diabetic nephropathy). In some embodiments, the tubulointerstitial disease is selected from tubulointerstitial nephritis (e.g., acute or chronic tubulointerstitial nephritis), acute pyelonephritis, acute tubular necrosis, acute renal papillary necrosis, obstructive or reflux nephropathy, nephrocalcinosis, pyonephrosis, and renal or perirenal abscess.

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