Diagnostic and therapeutic drug for kidney injury

Abstract

Provided is a diagnostic and therapeutic drug for kidney injury. Specifically provided is a complex, containing a carrier protein and a functional molecule for the diagnosis, prevention and / or treatment of kidney injury. The carrier protein is selected from a mutant DIIIbV of a subdomain (e.g., DIIIb) of albumin domain III, an albumin domain III (DIIIV) containing the mutant, and a polymer of the DIIIbV or DIIIV. The carrier protein has improved affinity for FcRn and excellent transmucosal efficiency, and exhibits long-acting and sustained-release characteristics. Loading, by means of fusion expression, chemical grafting, etc., the carrier protein with the diagnostic and therapeutic drug for kidney injury can be used for the diagnosis, prevention and / or treatment of kidney injury.

Description

Diagnosis and treatment drug for kidney damage

[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 hereby incorporated by reference in its entirety into the present application. TECHNICAL FIELD

[0002] The present application relates to the field of biological agents, in particular to a diagnosis and treatment drug for kidney damage. BACKGROUND

[0003] Kidney damage refers to a clinical syndrome of abnormal structure or reduced function of the kidney caused by various causes, with the core features of decreased kidney metabolic waste excretion and water-electrolyte / acidity balance disorder, and accompanied by health damage risk. According to the progression speed and persistence, it is mainly divided into two subtypes of acute kidney injury (AKI) and chronic kidney injury in clinical, which together constitute an important inducement of global end-stage renal disease (ESRD) and cardiovascular disease. As a core metabolic organ for maintaining homeostasis, the rapid impairment of kidney function can trigger systemic chain reactions, leading to a significantly increased mortality rate in patients with kidney damage. This brings heavy pressure and burden of clinical diagnosis and treatment, so kidney damage prevention and treatment needs further research and technical optimization.

[0004] The current treatment of kidney damage follows the core logic of "cause control-function maintenance-replacement support". The key principle of AKI treatment is to identify and control the cause in time, maintain hemodynamic stability, and avoid exposure to nephrotoxic drugs. If the kidney function is severely impaired (such as severe uremia symptoms, difficult-to-correct electrolyte disorders, etc.), kidney replacement therapy is needed to maintain life. The core of chronic kidney damage treatment is "long-term cause control-delay progression-complication prevention and control", to avoid progression to ESRD. However, there is currently no specific treatment drug for kidney damage pathological process (such as kidney tubular injury repair and kidney fibrosis reversal) in clinical. The existing scheme is mostly "symptomatic supportive treatment", which can only relieve symptoms and maintain basic body functions, and cannot block the progression of kidney damage or promote kidney function repair from the pathological mechanism level. Some patients still face the risk of worsening kidney function and developing into chronic kidney disease or even end-stage renal disease.

[0005] Therefore, the development of specific treatment drugs that can target kidney damage pathological features and directly act on pathological targets to block the pathological process has become the primary requirement to solve the clinical treatment dilemma of kidney damage.

[0006] In summary, it is of great significance to screen a carrier that can precisely, durably and efficiently deliver drugs to the kidney SUMMARY

[0007] This study provides a drug for the detection, monitoring, and / or treatment of kidney injury via mucosal administration or efficient blood delivery. This drug can be administered via nasal spray, bronchial mucosa, oral mucosa, gastrointestinal mucosa, or blood injection, depending on different needs, for the diagnosis, imaging, and treatment of kidney diseases.

[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 kidney injury;

[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 -11 M 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:5.

[0063] In some embodiments, the DIIIbV is selected from the amino acid sequences shown in SEQ ID NO:39-43.

[0064] In some embodiments, the DIIIV is selected from the amino acid sequences shown in SEQ ID NO:8 to 38.

[0065] In some embodiments, the DIIIV has the amino acid sequence shown in SEQ ID NO:8.

[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 kidney injury is selected from acute kidney injury and chronic kidney injury.

[0070] 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.

[0071] In some embodiments, the drug is an anti-inflammatory, anti-apoptotic, or anti-oxidative stress drug targeting the kidney injury. In some embodiments, the drug is selected from amifostine, cimetidine, glutathione, and RNLS agonists (e.g., RP81, RP220).

[0072] 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).

[0073] In some embodiments, the imaging agent is selected from cyanine dyes such as IR-780, IR-783, and other photosensitizers.

[0074] 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.

[0075] In some implementations, the carrier protein and the functional molecule are directly linked or linked via a connector.

[0076] 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.

[0077] In some embodiments, the carrier protein and the functional molecule are coupled together by gene fusion or chemical methods to form the complex.

[0078] In some embodiments, the complex has the following structure: C-(LD) n

[0079] Wherein, C represents the carrier protein;

[0080] D represents the functional molecule;

[0081] 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;

[0082] 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.

[0083] In some implementations, C is selected from DIIIV1 to DIIIV31, DIIIbV1, DIIIbV2, DIIIbV4, DIIIbV8 and DIIIbV22.

[0084] In some implementations, D is selected from amifostine, cimetidine, glutathione, and RNLS agonists (e.g., RP81, RP220).

[0085] In some implementation schemes, L is selected from

[0086] Nucleic acid molecules, vectors, and host cells

[0087] In another aspect, this application provides a nucleic acid molecule that encodes the complex described in any of the preceding claims.

[0088] In some embodiments, the complex is a fusion protein.

[0089] 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).

[0090] 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.

[0091] 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.

[0092] Delivery of combination or pharmaceutical composition

[0093] In another aspect, this application provides a delivery combination or pharmaceutical composition comprising any of the complexes described above.

[0094] In some embodiments, the delivery combination or pharmaceutical composition is delivered intravenously.

[0095] 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.

[0096] 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).

[0097] 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.

[0098] In some embodiments, the delivery combination or pharmaceutical composition is an aerosol, powder inhaler, spray, or other dosage form suitable for inhalation administration.

[0099] In some embodiments, the pharmaceutical composition contains one or more pharmaceutically acceptable excipients.

[0100] Medical uses and methods

[0101] 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 kidney injury.

[0102] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0103] In another aspect, this application provides the complex, delivery combination, or pharmaceutical composition described in any of the preceding claims for the treatment of kidney injury.

[0104] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0105] In another aspect, this application provides a method for treating kidney injury, 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.

[0106] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0107] In another aspect, this application provides the use of the carrier protein as defined in any of the preceding claims in the preparation of a medicament for treating kidney injury.

[0108] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0109] In another aspect, this application provides a carrier protein as defined in any of the preceding claims for the treatment of kidney injury.

[0110] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0111] In another aspect, this application provides a method for treating kidney injury, comprising administering a carrier protein as defined in any of the preceding claims to a subject in need of such treatment.

[0112] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0113] In another aspect, this application provides a medicament for treating kidney injury, which contains a carrier protein as defined in any of the preceding claims.

[0114] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0115] Terminology Definition

[0116] 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.

[0117] As used in this article, the term "carrier" refers to a protein that can carry different biomolecules and circulate throughout the body to different parts of the body.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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).

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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)).

[0137] 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.

[0138] 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.

[0139] "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.

[0140] 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.

[0141] 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.

[0142] In this article, kidney injury refers to damage to the structure or function of the kidneys caused by various factors. This damage interferes with the normal physiological functions of the kidneys, including the excretion of metabolic waste, regulation of water and electrolyte balance, maintenance of acid-base balance, and hormone secretion. Factors that can cause kidney injury include ischemic factors, nephrotoxic substances, infection, immune responses, and trauma. In this article, acute kidney injury (AKI) refers to a rapid decline in kidney function within hours to days, characterized by elevated serum creatinine levels or decreased urine output. It is often caused by dehydration, infection, drugs, or reduced renal blood flow and is an emergency requiring rapid treatment. Chronic kidney injury (CKD) refers to the gradual loss of kidney function over 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 the main cause of kidney failure.

[0143] Beneficial effects of the invention

[0144] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0145] 1. Excellent transmucosal efficiency, which can improve the affinity level of FcRn and thus control its kidney or bladder targeting. High bioavailability. The delivery system carries drugs or imaging or diagnostic reagents and can be recycled through mucosal epithelial cells to achieve long-term efficacy.

[0146] 2. The complex of the present invention can be delivered to the urinary system via nasal delivery, gastrointestinal mucosal epithelium delivery, or blood injection, depending on the sustained-release properties and acid-base tolerance of the drug.

[0147] 3. This invention can enable the diagnosis, imaging, and / or treatment of kidney injury.

[0148] 4. This invention enables mucosal delivery, making the drug administration process more convenient, allowing patients to operate independently, and significantly improving patient compliance.

[0149] 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

[0150] Figure 1 shows the DIII mutant Saccharomyces cerevisiae plasmid vector pYD1 constructed in Example 1, where DIII specifically refers to the DIII mutant.

[0151] Figures 2A to 2C show the fluorescence signals and their ratio changes of the initial library and different rounds of screening expression and binding on the yeast surface as described in Example 1.

[0152] Figure 3 shows the DIIIV1 Pichia pastoris plasmid vector pPIC9K constructed in Example 2, where DIII specifically refers to the DIII mutant.

[0153] Figure 4 shows the isolation and purification results of the Pichia pastoris expressing the DIII mutant described in Example 2.

[0154] Figures 5A and 5B show the results of intracellular colocalization of DIIIV and FcRn using fluorescence confocal microscopy in Example 5. 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.

[0155] [Revised according to Rule 26, 12.01.2026] Figure 6 shows the fluorescence intensity results of samples of different DIII mutants and different cells as described in Example 6 under near-infrared imaging; wherein, 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.

[0156] Figure 7 shows the H&E staining results of mouse kidneys of different grades as described in Example 7.

[0157] Figure 8 shows the urinary protein detection results among the AKI biochemical indicators described in Example 7.

[0158] Figure 9 shows the blood urea nitrogen detection results among the AKI biochemical indicators described in Example 7.

[0159] Figure 10 shows the serum creatinine detection results among the AKI biochemical indicators described in Example 7.

[0160] Figure 11 shows dynamic NIR-II imaging of mouse in vivo kidneys at different grades as described in Example 7.

[0161] Figure 12 shows the results of the correlation analysis between the inflection point of renal fluorescence intensity change and GFR in mice with different grades of kidney injury as described in Example 7.

[0162] Figure 13 is a flowchart of the NIR-II imaging omics data combined with deep learning to predict GFR as described in Example 7.

[0163] Figure 14 shows the fusion expression plasmid map of DIIIV1 and RP81 described in Example 8, where DIII in the schematic diagram of each plasmid refers to DIIIV1.

[0164] Figure 15 shows the SDS-PAGE and WB results of DIIIV1-RP81 expressed using Pichia pastoris GS115 as described in Example 8.

[0165] Figure 16 shows the SDS-PAGE and WB results of DIIIV1-RP81 expressed in HEK293F cells as described in Example 8.

[0166] Figure 17 is a map of the fusion expression plasmid of DIIIV1 and RP220 described in Example 8, where DIII refers to DIIIV1.

[0167] Figure 18 shows the SDS-PAGE and WB results of DIIIV1-RP220 expressed in HEK293F cells as described in Example 8.

[0168] Figure 19 shows the SDS-PAGE results of DIIIbV1 expressed in HEK293F cells as described in Example 8.

[0169] Figure 20 shows the fusion expression plasmid of DIIIbV1 and RP81 described in Example 8.

[0170] Figure 21 shows the fusion expression plasmid of DIIIbV1 and RP220 described in Example 8.

[0171] Figure 22 shows the synthesis of DIIIV1-amifostine as described in Example 9.

[0172] Figure 23 shows the SDS-PAGE results of the synthesized DIIIV1-amifostine described in Example 9.

[0173] Figure 24 shows the synthesis of DIIIV1-cimetidine as described in Example 9.

[0174] Figure 25 is a schematic diagram of real-time imaging 0-30 min after DIIIbV1@ICG is injected via the tail vein as described in Example 10.

[0175] Figure 26 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after tail vein injection of DIIIbV1@ICG as described in Example 10.

[0176] Figure 27 is a schematic diagram of real-time imaging 0-30 min after RP81@ICG is injected via the tail vein as described in Example 11.

[0177] Figure 28 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after administration of RP81@ICG via tail vein injection as described in Example 11.

[0178] Figure 29 is a schematic diagram of real-time imaging 0-30 min after administration of DIIIV1-RP81@IR780 via tail vein injection as described in Example 11.

[0179] Figure 30 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after administration of DIIIV1-RP81@IR780 via tail vein injection as described in Example 11.

[0180] Figure 31 shows a two-photon in vivo imaging of the kidney region in a small animal after injection of Cy5-labeled DIIIV1 via the tail vein, as described in Example 12.

[0181] Figure 32 shows a two-photon in vivo image of the kidney region in a small animal after injection of Cy5-labeled RP81 via the tail vein, as described in Example 12.

[0182] Figure 33 shows a two-photon in vivo imaging of the kidney region in a small animal after injection of Cy5-labeled DIIIV1-RP81 via the tail vein, as described in Example 12.

[0183] Figure 34 shows the immunofluorescence results of FcRn colocalization in the kidney region after tail vein injection of DIIIV1-RP81@IR780 as described in Example 13.

[0184] Figure 35 shows the blood biochemistry results of the AKI mouse model constructed by clamping both renal arteries for 30 minutes as described in Example 14.

[0185] Figure 36 shows the blood biochemistry results of treating AKI by administering 2 μg DIIIV1-RP81 via the tail vein in the IRI-induced AKI model described in Example 15.

[0186] Figure 37 shows the ELISA results of AKI treatment via tail vein administration of 2 μg DIIIV1-RP81 in the IRI-induced AKI model described in Example 15.

[0187] Figure 38 shows the H&E and TUNEL results of the kidneys in an IRI-induced AKI model as described in Example 15, where 2 μg of DIIIV1-RP81 was administered via the tail vein to treat AKI.

[0188] Figure 39 shows the scoring results of kidney damage in the IRI-induced AKI model described in Example 15, after treatment with 2 μg DIIIV1-RP81 via tail vein administration for AKI.

[0189] Figure 40 shows the blood biochemistry results of AKI treatment via tail vein administration of DIIIV1, RP81, and DIIIV1-RP81 in an IRI-induced AKI model as described in Example 16.

[0190] Figure 41 shows the ELISA results of AKI treatment via tail vein administration of DIIIV1, RP81, and DIIIV1-RP81 in the IRI-induced AKI model described in Example 16.

[0191] Figure 42 shows the H&E and TUNEL results of the kidneys in an IRI-induced AKI model as described in Example 16, when DIIIV1, RP81, and DIIIV1-RP81 were administered via the tail vein.

[0192] Figure 43 shows the scoring results of kidney damage in the IRI-induced AKI model described in Example 16, after treatment with DIIIV1, RP81, and DIIIV1-RP81 via tail vein administration for AKI.

[0193] Figure 44 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after administration of RP81@ICG via nasal drops as described in Example 17.

[0194] Figure 45 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after administration of DIIIV1-RP81@IR780 via nasal drops as described in Example 17.

[0195] Figure 46 shows the immunofluorescence results of the kidney site after intranasal administration of DIIIV1-RP81@IR780 as described in Example 18.

[0196] Figure 47 shows the blood biochemistry results of treating AKI in an IRI-induced AKI model as described in Example 19 by administering 20 μg DIIIV1-RP81 via nasal drops.

[0197] Figure 48 shows the ELISA results of treating AKI with 20 μg DIIIV1-RP81 via nasal drops in an IRI-induced AKI model as described in Example 19.

[0198] Figure 49 shows the H&E and TUNEL results of the kidneys in an IRI-induced AKI model as described in Example 19, where 20 μg of DIIIV1-RP81 was administered via nasal drops.

[0199] Figure 50 shows the scoring results of kidney damage in the IRI-induced AKI model described in Example 19, after treatment with 20 μg DIIIV1-RP81 via nasal drops.

[0200] Figure 51 shows the blood biochemistry results of treating AKI with intranasal administration of DIIIV1, RP81, and DIIIV1-RP81 in an IRI-induced AKI model as described in Example 20.

[0201] Figure 52 shows the H&E and TUNEL results of the kidneys in an IRI-induced AKI model as described in Example 20, when DIIIV1, RP81, and DIIIV1-RP81 were administered via nasal drops.

[0202] Figure 53 shows the scoring results of kidney damage in the IRI-induced AKI model described in Example 20, after treatment with nasal drops of DIIIV1, RP81, and DIIIV1-RP81 for AKI.

[0203] Figure 54 shows the blood biochemistry results of AKI treatment by oral administration of 65 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 21.

[0204] Figure 55 shows the ELISA results of AKI treatment by oral administration of 65 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 21.

[0205] Figure 56 shows the blood biochemistry results of AKI treatment by oral administration of DIIIV1, RP81, and DIIIV1-RP81 in the IRI-induced AKI model described in Example 22.

[0206] Figure 57 shows the ELISA results of AKI treatment by oral administration of DIIIV1, RP81, and DIIIV1-RP81 in the IRI-induced AKI model described in Example 22.

[0207] Figure 58 shows the blood biochemistry results of treating AKI in an IRI-induced AKI model as described in Example 23 by administering 1 μg of DIIIV1-RP220 via the tail vein.

[0208] Figure 59 shows the blood biochemistry results of the cisplatin-induced AKI mouse model described in Example 24.

[0209] Figure 60 shows the blood biochemical results of the mouse model of chronic kidney injury induced by UUO as described in Example 25.

[0210] Figure 61 is a schematic diagram of real-time imaging from 0 to 30 minutes after administration of DIIIV1-RP81 to mice with UUO-induced chronic kidney injury via tail vein as described in Example 26.

[0211] Figure 62 shows the NIR II fluorescence results of mice in prone, supine, and lateral positions after being injected with DIIIV1-RP81 via the tail vein in mice with UUO-induced chronic kidney injury as described in Example 26. Detailed Implementation

[0212] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0213] 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)).

[0214] 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.

[0215] Example 1. Screening for FcRn high-affinity DIII mutants

[0216] First, a DIIIV 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, Alphafold 2 was used to predict the structure of all mutants in the library. Further structural analysis and virtual screening were performed using RMSD, Tm-score, free energy DDG, 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 of DIIIV and the vector plasmid is shown in Figure 1. The chips were stored at -20℃ for later use.

[0217] Saccharomyces cerevisiae EBY100 glycerol culture was streaked onto YPD plates and incubated at 30℃ for approximately 36 hours. Single colonies were picked and transferred to 3 mL of YPD medium and incubated overnight at 30℃ (activation OD600 value of 2-5 is recommended). An appropriate amount of yeast was transferred to 3 mL of fresh YPD medium to achieve an OD600 value of 0.1, and incubated at 30℃ with a shaker until the OD600 value was between 0.4 and 1. The yeast was centrifuged at 4200 rpm for 2 min, resuspended in 700 μL of 0.1 M TE / LiAC, transferred to a 1.5 mL centrifuge tube, centrifuged at 4200 rpm for 2 min, the supernatant was discarded, and 20 μL of 0.1 M TE / LiAC was added to prepare competent yeast cells for later use. Transformation solution preparation: 2 mg / mL salmon sperm DNA (ssDNA) was heat-denatured at 100℃ for 5 min, vortexed to mix, and immediately placed 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 1M TE / 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 to remove supernatant. For each tube of transformed yeast, resuspend in 100 μL of sterile ultrapure water, plate on 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-URA-TRP 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 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 and incubate simultaneously as a non-induced negative control. After induction, take appropriate amounts of induced and uninduced yeast culture solutions and dilute them with PBS at pH 6.0 to an OD600 of 1, then keep 1 mL for later use.

[0218] The yeast was screened according to the following groups:

[0219] (1) Negative control: a. Uninduced group: Take 200 μL of uninduced yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 100 μL of PBS pH 6.0 to resuspend the culture, add 2 μL of (50:1) HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody, incubate at room temperature for 1 h, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS pH 6.0 to resuspend the culture for later use; b. Induced blank group: Take 200 μL of induced yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend the culture. Resuspend the bacterial culture at pH 6.0, wash the bacterial culture, centrifuge the bacterial culture at 4200 rpm for 2 min, discard the supernatant, add 600 μL PBS to pH 6.0 and resuspend the bacterial culture for later use;

[0220] (2) Sample groups: a. Single staining group: Take 200 μL of yeast culture diluted with PBS pH 6.0, centrifuge the culture at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 100 μL of PBS pH 6.0 to resuspend the culture, add 2 μL of HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody, incubate at room temperature for 1 h, add 1 mL of PBS pH 6.0 to resuspend, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS pH 6.0 to resuspend the culture for later use; b. Double staining group: Take 200 μL of yeast culture diluted with PBS pH 6.0, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 1 mL of PBS pH 6.0 to resuspend the culture. Resuspend the bacterial culture in PBS at pH 6.0, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 90 μL of PBS to resuspend the culture, add 10 μL of FcRn-β2M protein (Biotinylated, His-Avi, 100 μg / mL), incubate at room temperature for 1 h, add 2 μL of HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody and 0.3 μL of Streptavidin APC, incubate at room temperature for 1 h, add 1 mL of PBS at pH 6.0 to resuspend the culture, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, repeat twice, and finally add 600 μL of PBS at pH 6.0 to resuspend the culture for later use.

[0221] After all samples were prepared, they were sequentially tested using flow cytometry and FACS sorting. The sorting results are shown in Figures 2A to 2C. After four to five rounds of sorting, it can be seen that both the fluorescence signals of the mutants and the fluorescence signals of the mutants binding to FcRn are significantly enhanced. This indicates that as the number of screening rounds increases, low-affinity mutants in the mutant library are gradually eliminated, while high-affinity mutants are continuously enriched (R.1 to R.5 in Figures 2B and 2C represent the first to fifth rounds of sorting, respectively). Finally, a high-affinity mutant library is obtained, which proceeds to the next stage of validation.

[0222] Example 2. Pichia pastoris expression of high-affinity DIII mutant

[0223] The mutants selected through FACS final screening were subjected to high-throughput sequencing. After comparison with the original mutant sequences, some example mutant sequences were selected, as shown in Table 1. These sequences correspond to amino acids 497 to 585 of wild-type human serum albumin SEQ ID NO:1. The experiments conducted in this application used the third domain (DIII) containing these exemplary sequences; that is, the sequences of the mutants shown in Table 2 were substituted at the corresponding positions of the third domain.

[0224] The mutant was synthesized into the pPIC9K vector plasmid (the example plasmid map is shown in Figure 3). First, GS115 Pichia pastoris competent cells were prepared. GS115 cells were streaked on YPD plates and incubated at 30℃ for 2-3 days until single colonies grew. Single colonies were picked and transferred to 50mL centrifuge tubes containing 5mL 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 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 again to 5CV using equilibration buffer. Buffer 1 was used to wash away impurities, followed by equilibration again to 5CV using equilibration buffer or UV back to baseline. Buffer 2 was used to wash away impurities, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. Buffer 3 was used to wash away impurities, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. Buffer 4 was used to elute the target protein, and the column was equilibrated again to 5CV using equilibration buffer or UV back to baseline. The column was then washed with 0.5M NaOH. Finally, the column was stored at 4°C after loading with 20% ethanol. During purification, the column buffer was collected as needed for subsequent purification effect evaluation. Washing buffers could be prepared selectively; the more cycles of impurities and column use, the more washing steps were required. All buffers were prepared according to the manufacturer's recommended method.

[0226] The purification results are shown in Figure 4. The purified target protein band is clearly visible at the black dotted line in the figure.

[0227] Table 1. Mutation sites of exemplary mutants

[0228] Example 3. Affinity detection of DIII mutant FcRn

[0229] First, the purified DIIIV protein was quantified using the BCA protein quantification method. FcRn-β2M ligand protein was prepared using a serial dilution method, resulting in antibody dilutions of 20,000 ng / mL, 4,000 ng / mL, 800 ng / mL, 160 ng / mL, 32 ng / mL, 6.4 ng / mL, 1.28 ng / mL, and 0.256 ng / mL.

[0230] 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 of all DIIIV samples for FcRn in Example 2 was calculated.

[0231] The example results for DIIIV are shown in Table 2. All detected DIIIVs exhibited very strong binding affinity to FcRn under acidic pH conditions (e.g., pH 6.0). However, these DIIIVs showed no directly measurable affinity for FcRn at physiological pH conditions (e.g., pH 7.4) (*: no signal detected; +: no obvious binding signal detected).

[0232] Table 2. Affinity results of representative mutants

[0233] Example 4. Determination of the affinity between the DIIIb mutant and FcRn

[0234] Based on the affinity determination of DIIIV with FcRn in Example 3, we further determined the affinity of the mutant containing only the DIIIb portion (i.e., only the portion of Table 3 SEQ ID NO:5) and excluding the DIIIa portion (i.e., Table 3 SEQ ID NO:4) with FcRn. This sequence corresponds to the fragment from amino acid 467 to amino acid 585 of SEQ ID NO:1.

[0235] 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.

[0236] The quantified protein was added at a concentration of 2 μg / mL (100 μL per well) to a polystyrene 96-well plate for ELISA. The plate was sealed with sealing film and incubated overnight at 4°C. 300 μL of PBS (pH 7.4) was added to each well for the first wash, followed by blocking buffer 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 of all DIIIV samples for FcRn in Example 2 was calculated.

[0237] The example results for the DIIIb mutants are shown in Table 3. 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).

[0238] Table 3. Affinity results of representative mutants

[0239] Example 5. DIIIV transport and circulation mediated by FcRn

[0240] IR-783 was dissolved in DMSO to obtain a stock solution with a concentration of 2 mmol / L. The concentration of DIII mutant 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 DIIIV 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@DIIIV.

[0241] First, 300,000 293T cells per well were seeded in 35mm glass-bottomed culture dishes (15mm diameter glass bottom) 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 kept in the dark. The cell culture medium was aspirated, and the cells were washed three times with 500µL PBS pH 7.4. 250µL of the protein and cyanine dye complex was added to each group, and the cells were incubated at 37°C for 1 hour. For the control groups V4-Ab and V4-Ba A1, in addition to V4-IR-780, a DIII-specific polyclonal antibody (0.5µM) was added to block its binding to the FcRn site. 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.

[0242] 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 5A). 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 5B).

[0243] 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.

[0244] Example 6. Pharmacokinetic determination of mutant protein administered intranasally in mice

[0245] DIIIV1 was labeled with IR783 to obtain the IR-783@DIIIV1 complex. 100 μL of 20 μM IR783 and the 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 with an 808 nm laser excitation and a 1000 nm long-pass filter, simultaneously verifying 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 after administration.

[0246] As shown in Figure 6, after tail vein injection of DIIIV1, DIIIV1 was specifically enriched in the kidney region. Replacing DIIIV1 with any other DIII mutant yielded similar results for the resulting complex.

[0247] Example 7: A complex of the DIII mutant and cyanine dye IR-783 was used to detect a cisplatin-induced AKI mouse model.

[0248] I. Construction of a cisplatin-induced AKI mouse model

[0249] Cisplatin was dissolved in a 0.9% sodium chloride solution, protected from light by aluminum foil, and shaken and mixed at 37°C for 2 hours to obtain a cisplatin solution. Six- to eight-week-old female BALB / c mice were randomly divided into six groups according to the cisplatin induction dose, from low to high: G1 = 2 mg / kg, G2 = 5 mg / kg, G3 = 7.5 mg / kg, G4 = 10 mg / kg, G5 = 15 mg / kg, and G6 = 20 mg / kg, with at least three mice in each group. The intraperitoneal injection dose for each mouse was calculated based on its body weight. Intraperitoneal injection of cisplatin was used to establish mouse models of different degrees of acute kidney injury. Healthy mice under the same conditions served as the normal control group and did not require any treatment. Kidney tissue was collected from all mice after 24 hours, fixed by immersion in 4% paraformaldehyde, and histological morphology was observed by hematoxylin and eosin (H&E) staining.

[0250] As shown in Figure 7, with increasing cisplatin dosage, more and more aggregated elongated vacuoles were observed in H&E staining, and the number of nephrons decreased significantly. These pathological findings indicate that the higher the cisplatin induction dose, the more severe the kidney damage, and also reflect the successful establishment of the kidney injury model.

[0251] II. Detection of AKI Biochemical Indicators

[0252] Urinary protein detection: Urine samples from each mouse before modeling were used as the baseline. Urine samples were then collected 24 hours after cisplatin-induced kidney injury. Following a urinary protein detection kit, the OD value was measured at 595 nm, and the urinary protein concentration was calculated. Changes in urinary protein before and after kidney injury induction were compared. As shown in Figure 8, with increasing kidney injury levels, there was no significant pattern or difference in the ratios among healthy mice, groups G1, G2, and G3. Urinary protein levels increased slightly in groups G4, G5, and G6, with only a significant increase in G6. Here, G represents the kidney injury grade, and the numbers 1, 2, 3, 4, 5, and 6 represent the degree of kidney injury. For example, G1 represents grade I kidney injury, i.e., mild kidney injury.

[0253] Blood urea nitrogen (UREA) determination: Serum samples were collected before and after modeling to measure blood urea nitrogen. After the reaction was completed, 0.2 mL of the reaction solution was added to a 96-well plate. The concentration was calculated by measuring the OD value at 640 nm according to the blood urea nitrogen detection kit. The changes in blood urea nitrogen before and after kidney injury induction were compared. As shown in Figure 9, there were no obvious patterns or differences between the groups.

[0254] Serum creatinine (CREA) measurement: Serum creatinine was measured before and after modeling. The concentration was calculated by measuring the OD value at a wavelength of 546 nm using a serum creatinine assay kit. Changes in serum creatinine before and after kidney injury induction were compared. As shown in Figure 10, the changes in serum creatinine values ​​in the six groups (healthy mice, G1, G2, G3, G4, and G5) were all less than 1.5, which did not meet the clinical criteria for AKI (a surge in serum creatinine value of more than 1.5 times). This demonstrates that this method can more sensitively reflect the degree of kidney injury.

[0255] III. Dynamic NIR-II Imaging of Mouse Kidneys with Different Grades of Injury

[0256] A 10 μM mixture of IR-783 and DIII mutant was prepared by mixing IR-783 and DIII at a molar ratio of 1:1, and then incubated in a 60°C water bath for 10 min to obtain the IR-783@DIII complex. 100 μL of 10 μM IR-783@DIII was injected intravenously via a medical catheter, and the entire dynamic process of the IR-783@DIII complex reaching the kidney was monitored in real time starting from time 0. During imaging, the mice were in a lateral recumbent position. After recording video for the first 30 minutes, images were taken at seven time points (40 min, 50 min, 60 min, 75 min, 90 min, 105 min, and 120 min) in lateral, supine, and prone positions, as shown in Figure 11A. The first 30 minutes of video were used as the NIR-II imaging omics database for deep learning diagnosis of AKI. Simultaneously, the fluorescence intensity of the kidney in the lateral recumbent position was analyzed to obtain characteristic curves for key time points of dynamic imaging of the mouse kidney at different grades. As shown in Figure 11B, the changes in renal fluorescence intensity in different groups were significantly different.

[0257] IV. Analysis of changes in renal fluorescence intensity in mice of different grades

[0258] Statistical analysis of the changes in renal fluorescence intensity in different mice over the first 30 minutes of video recording was performed, as shown in Figure 12a. With increasing cisplatin-induced dose, the inflection point of renal fluorescence intensity gradually decreased, clearly distinguishing the degree of kidney damage. Furthermore, the glomerular filtration rate (GFR) of mice with different cisplatin-induced doses was measured using FITC-inulin. As shown in Figure 12b, it was found that GFR gradually decreased with increasing cisplatin-induced dose. Correlation analysis revealed a strong correlation between the two, which can be used to evaluate or predict the degree of kidney damage.

[0259] V. Predicting GFR using NIR-II imaging omics data combined with deep learning

[0260] As shown in Figure 13a, we propose a spatiotemporal deep learning method for GFR prediction. A deep learning network is established by combining NIR II imaging of the IR-783@DIII complex in mouse kidneys with GFR to predict GFR, thereby achieving the goal of diagnosing kidney injury through the IR-783@DIII complex. First, we construct a variational autoencoder (VAE) framework to extract temporal features in the latent space. Based on this, we propose a spatiotemporal fusion network based on cross-attention mechanism to achieve GFR prediction. Furthermore, each mouse is hot-coded once according to the injection dose, induction time, and scan time, and this is used as a prior to improve prediction accuracy. As shown in Figure 13b, we performed 10-fold cross-validation, calculated the accuracy of each fold, and found the average value as the final accuracy of the model, which is 0.8627. We also plotted the 10-fold cross-validation ROC curves for each GFR group, obtaining an average AUC of 0.9377.

[0261] Example 8: Expression, isolation, and purification of the fusion protein

[0262] First, a vector plasmid was synthesized from the DIII mutant and RP81 fusion protein (the composite plasmid map is shown in Figure 14), and the protein was expressed in both prokaryotic and eukaryotic systems (GS115 Pichia pastoris and HEK293F cells). The protein expressed in Pichia pastoris was then purified using an affinity column to obtain the purified DIIIV1 / RP81 fusion protein (Figure 15). The protein expressed in HEK293F cells was purified using a nickel column to obtain the purified DIIIV1 / RP81 fusion protein (Figure 16).

[0263] The DIIIV1-RP220 fusion protein was synthesized into a vector plasmid (the composite plasmid map is shown in Figure 17), and the protein was expressed using a eukaryotic system (HEK293F cells). The protein expressed in HEK293F cells was purified and separated by nickel column chromatography to obtain the purified DIIIV1-RP220 fusion protein. Figure 18 shows the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) of the purified DIIIV1-RP220.

[0264] DIIIbV1 was expressed using the GS115 Pichia pastoris system and then purified using an albumin affinity column to obtain DIIIbV1 (as shown in Figure 19).

[0265] A vector plasmid was synthesized from the DIIIbV1 and RP81 fusion protein (the composite plasmid map is shown in Figure 20), and the protein was expressed in HEK293F cells. The protein expressed in HEK293F cells was purified and separated by nickel column chromatography to obtain the purified DIIIbV1 and RP81 fusion protein.

[0266] A vector plasmid was synthesized from the DIIIbV1 and RP220 fusion protein (the composite plasmid map is shown in Figure 21), and the protein was expressed in HEK293F cells. The protein expressed in HEK293F cells was purified and separated by nickel column chromatography to obtain the purified DIIIbV1 and RP220 fusion protein.

[0267] Example 9: Synthesis of DIIIV1-Amifostine and DIIIV1-Cimitidine

[0268] Amifostine was grafted onto DIIIV1 protein via chemical synthesis (as shown in Figure 22). Amifostine (1 eq.) was dissolved in DMF and cooled to 0°C. Then, a DMF solution of maleimide hexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol p-nitrophenyl carbonate (Mc-VC-PAB-PNP, 2 eq.) was added. The reaction mixture was slowly heated to room temperature and stirred overnight. Liquid chromatography monitoring continued until the starting material was completely consumed. The product, Mc-VC-PAB-Amifostine, was then purified by semi-preparative HPLC. A DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide (10 eq.) and 0.2% DIPEA were added to a PBS solution (1.0 eq.) of DIIIV1 protein. 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 it at 25 °C. Then add 10 eq. of Mc-VC-PAB-Amifostine in DMSO and stir overnight at 25 °C. Then add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench the reaction at 25 °C. After centrifugation by a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Amifostine (as shown in Figure 23).

[0269] Cimetidine was grafted onto DIIIV1 via chemical synthesis (as shown in Figure 24). Cimetidine (1 eq.) and DIPEA (3 eq.) were dissolved in DMF and cooled to 0°C. Then, a DMF solution of maleimide hexanoyl-L-valine-L-citrulline p-aminobenzyl alcohol p-nitrophenyl carbonate (Mc-VC-PAB-PNP, 2 eq.) was added. The reaction mixture was slowly heated to room temperature and stirred overnight. Liquid chromatography monitoring continued until the reactants were completely consumed. The product, Mc-VC-PAB-Cimetidine, was then purified by semi-preparative HPLC. A DMSO solution of 3-mercaptopropyl-N-hydroxysuccinimide ester (10 eq.) and 0.2% DIPEA were added to a PBS solution (1.0 eq.) of DIIIV1 protein. 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 it at 25 °C. Then add 10 eq. of Mc-VC-PAB-Cimetidine in DMSO and stir overnight at 25 °C. Then add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench the reaction at 25 °C. After centrifugation by desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Cimetidine.

[0270] Example 10: Pharmacokinetics of DIIIbV1 in mice

[0271] ICG-NHS was used as a labeling dye to observe the pharmacokinetics (PK) of DIIIbV1 in mice. First, ICG-NHS and DIIIbV1 were mixed at a concentration of 20 μM and a molar ratio of 5:1, and the mixture was reacted with shaking at room temperature for 2 h. Subsequently, the reaction mixture was centrifuged in a desalting column to remove unreacted ICG-NHS, finally obtaining ICG-labeled DIIIbV1. 100 μL of the DIIIbV1@ICG complex was injected into C57BL / 6J mice via tail vein, and fluorescence was detected using a near-infrared II imaging system. Real-time imaging was performed within 0–30 min after administration (as shown in Figure 25), and fluorescence was acquired at 30 min, 1 h, 2 h, 4 h, 12 h, and 24 h post-injection in prone, supine, and lateral positions. The imaging results in Figures 25 and 26 show that DIIIV1b accumulates efficiently and in large quantities in the kidneys.

[0272] Example 11 Pharmacokinetics of RP81 and DIIIV1-RP81 in mice

[0273] ICG-NHS was used as a labeling dye to observe the pharmacokinetics (PK) of RP81 in mice. First, ICG-NHS and RP81 were mixed at a concentration of 20 μM and a molar ratio of 5:1, and reacted with shaking at room temperature for 2 h. Then, the reaction mixture was placed in a 3 kDa ultrafiltration centrifuge tube and centrifuged at 13000 rpm for 10 min to remove unreacted ICG-NHS, finally obtaining ICG-labeled RP81. 100 μL of the RP81@ICG complex was injected into C57BL / 6J mice via tail vein, and fluorescence was detected using a near-infrared II imaging system. Real-time imaging was performed within 0-30 min after administration (as shown in Figure 27), and fluorescence was acquired at 24 h and 72 h post-injection in prone, supine, and lateral positions. Figure 28 shows that although RP81 is a small polypeptide, it does not accumulate in the kidneys, but rather in the liver.

[0274] The pharmacokinetics of DIIIV1-RP81 were observed using IR-780 as a labeling dye. C57BL / 6J mice were injected with 100 μL of the DIIIV1-RP81@IR-783 complex via tail vein. Fluorescence was detected using a near-infrared II imaging system. Real-time imaging was performed within 0-30 min after administration (Figure 29). Fluorescence was acquired at 24 h, 48 h, 72 h, 4 d, 5 d, 6 d, 7 d, and 10 d after administration in prone, supine, and lateral positions. NIR II imaging results showed that DIIIV1-RP81 was enriched in the kidneys, and the enrichment time reached 10 days (Figure 30).

[0275] Example 12: Distribution of DIII, RP81, and DIIIV1-RP81 in the kidneys

[0276] First, DIIIV1, RP81, and DIIIV1-RP81 were labeled with 20 μM Cy5-NHS at a molar ratio of 5:1 and reacted with shaking at room temperature for 2 h. Then, the reaction mixture was placed in a 3 kDa or 20 kDa ultrafiltration centrifuge tube and centrifuged at 13000 rpm for 10 min to remove unreacted Cy5-NHS, ultimately obtaining Cy5-labeled DIIIV1, RP81, and DIIIV1-RP81. Mice (C57BL / 6J) were anesthetized and placed laterally on the operating table. The kidneys were then removed under aseptic conditions and externally placed, using a negative pressure device to adhere them to a glass slide. An indwelling needle was inserted into the mouse's tail vein for fixation to facilitate dye infusion. Observation was performed under a multiphoton laser scanning microscope. First, the focal plane was located, and then 100 μL of FITC-dextran (average relative molecular mass of 500,000) and Cy5-labeled DIIIV1, RP81, and DIIIV1-RP81 were injected via the tail vein. The distribution of DIIIV1, RP81, and DIIIV1-RP81 in the kidney was recorded every 30 seconds for 30 minutes after injection. The fluorescence signals of DIIIV1 (as shown in Figure 31) or DIIIV1-RP81 (as shown in Figure 32) could be clearly seen in the lumen of the renal tubules. FITC-dextran was distributed in the capillaries around the renal tubules; while RP81 did not appear in the kidney (as shown in Figure 33).

[0277] Example 13: Co-location of DIIIV1-RP81 and FcRn

[0278] The DIIIV1-RP81@IR-780 complex was prepared according to the above method. Then, 100 μL of the DIIIV1-RP81@IR-780 complex was injected into mice (C57BL / 6J) via tail vein. Kidney tissue was obtained from the mice at 3 h and 12 h after administration, and frozen at -80℃ to prepare frozen sections. Subsequently, the kidney tissue was stained with lotus tetragonolobus lectin (LTL), mouse FcRn antibody, and DAPI for immunofluorescence staining. The co-localization of DIIIV1-RP81 and FcRn was observed using confocal microscopy. As shown in Figure 34, DIIIV1-RP81 was distributed in the proximal tubules labeled with LTL and co-localized with FcRn expressed in the proximal tubular cells.

[0279] Example 14: Establishment and Evaluation of AKI

[0280] Establishment of a mouse model of AKI induced by bilateral renal ischemia / reperfusion (IRI): After anesthesia, mice were exposed to the abdominal cavity through 0.5 cm incisions below the costal margins on both sides of the back. The renal pedicles were separated, and arterial clamps were used to close both renal pedicles. 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 sham-operated group followed the same procedure, except that the renal pedicles were separated. Blood was collected from the mice 24 hours later, and the serum levels of UREA and CREA were measured using a fully automated biochemical analyzer. The blood biochemistry results showed that the levels of UREA and CREA in the model group were more than 1.5 times higher than those in the sham-operated group, meeting the clinical criteria for the occurrence of AKI (as shown in Figure 35).

[0281] Example 15: Treatment of AKI mice by tail vein administration of DIIIV1-RP81

[0282] After establishing the IRI-induced AKI model, mice were injected with 2 μg of DIIIV1-RP81 via the tail vein. Subsequently, 24 h after treatment, blood was collected from each group of mice. After being left at room temperature for 1 h, the serum was separated by centrifugation at 4000 rpm for 10 min at 4°C. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. Figure 36 shows that 2 μg of DIIIV1-RP81 effectively reduced the levels of UREA and CREA in the serum of AKI mice. The levels of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) in the mouse serum were measured by ELISA. The results showed that 2 μg of DIIIV1-RP81 effectively reduced the levels of KIM-1 and NGAL in the serum of AKI mice (as shown in Figure 37). In addition, the collected kidney tissues were subjected to H&E staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining to observe the pathology and morphology of the kidney tissues. The results of H&E and kidney pathological scoring showed that 2 μg DIIIV1-RP81 could effectively slow down kidney damage (as shown in Figure 38); similarly, the results of TUNEL staining showed that 2 μg DIIIV1-RP81 could reduce kidney cell apoptosis and thus slow down AKI (as shown in Figure 39).

[0283] Example 16: Comparison of AKI relief in different treatment groups via tail vein administration.

[0284] After confirming that 2 μg DIIIV1-RP81 effectively alleviated AKI, its therapeutic effect was compared with all control groups in IRI-induced AKI. After establishing the AKI model, mice were injected via tail vein with DIIIV1 (1.56 μg), RP81 (0.27 μg), and DIIIV1-RP81 (2 μg), respectively. Subsequently, 24 h after treatment, blood was collected from each group of mice. After being incubated at room temperature for 1 h, the serum was separated by centrifugation at 4000 rpm for 10 min at 4°C. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. The levels of KIM-1 and NGAL in the mouse serum were measured by ELISA. Furthermore, the collected kidney tissue was stained with H&E and TUNEL to observe the pathology and morphology of the kidney tissue. The results showed that, compared with the model group, DIIIV1-RP81 could reduce the levels of UREA, CREA, KIM-1 and NGAL in serum (as shown in Figures 40 and 41). The pathological results of the kidneys also showed that DIIIV-RP81 protected the kidneys by reducing renal tubular cell apoptosis (as shown in Figures 42 and 43).

[0285] Example 17: PK of RP81 and DIIIV1-RP81 in mice via intranasal administration.

[0286] RP81@ICG complex was administered nasally to C57BL / 6J mice, and fluorescence was detected using a near-infrared II imaging system. Fluorescence was observed in prone, supine, and lateral positions at 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, and 7 d post-administration. Imaging results showed that RP81 was cleared by the lung mucosa, resulting in a short accumulation time in lung tissue and no accumulation in the kidneys (as shown in Figure 44).

[0287] Fluorescence was detected in C57BL / 6J mice by intranasal instillation of 30 μL of the DIIIV1-RP81@IR-780 complex in a near-infrared II imaging system. Fluorescence was measured in prone, supine, and lateral positions at 15 min, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, 4 d, 6 d, and 9 d post-administration. NIR II imaging results showed that DIIIV1-RP81 rapidly entered the bloodstream from the lung tissue and then accumulated in the kidneys, with an accumulation period of up to 9 days (as shown in Figure 45).

[0288] Example 18: Distribution of DIIIV1-RP81 in the kidney

[0289] Kidney tissues were collected from C57BL / 6J mice 24 and 48 hours after administration by intranasal instillation of 30 μL of the DIIIV1-RP81@IR-783 complex. The tissues were frozen at -80°C and prepared into frozen sections. Immunofluorescence staining with LTL and DAPI was performed on the kidney tissues, and the distribution of DIIIV1-RP81 in the kidneys was observed using confocal microscopy. The immunofluorescence results in Figure 46 show that after intranasal administration, DIIIV1-RP81 accumulated in the proximal renal tubules.

[0290] Example 19: Treatment of AKI mice with DIIIV1-RP81 via intranasal administration

[0291] After establishing the IRI-induced AKI model, mice were administered 20 μg of DIIIV1-RP81 via intranasal drip. Subsequently, 24 h after treatment, blood was collected from each group of mice. After being left at room temperature for 1 h, the blood was centrifuged at 4000 rpm for 10 min at 4°C to obtain the supernatant serum. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. The levels of KIM-1 and NGAL were measured using ELISA. Furthermore, the collected kidney tissue was stained with H&E and TUNEL to observe the pathology and morphology of the kidney tissue. The results showed that 20 μg of DIIIV1-RP81 could reduce the levels of UREA, CREA, KIM-1, and NGAL in the serum of AKI mice and reduce renal tubular cell apoptosis, effectively alleviating kidney damage (as shown in Figures 47-50).

[0292] Example 20: Comparison of AKI relief in different treatment groups via nasal drops.

[0293] After confirming that 20 μg of DIIIV1-RP81 could effectively alleviate AKI, its therapeutic effect was compared with that of all control groups in IRI-induced AKI. After establishing the AKI model, mice were injected via tail vein with DIIIV1 (15.63 μg), RP81 (2.73 μg), and DIIIV1-RP81 (20 μg), respectively. Subsequently, 24 h after treatment, blood was collected from each group of mice. After being left at room temperature for 1 h, the supernatant serum was separated by centrifugation at 4000 rpm for 10 min at 4°C. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. Furthermore, the collected kidney tissue was stained with H&E and TUNEL to observe the pathology and morphology of the kidney tissue. The results showed that, compared with the model group, DIIIV1-RP81 reduced the levels of UREA and CREA in the serum (as shown in Figure 51). The pathological results of the kidneys also showed that DIIIV1-RP81 protected the kidneys by reducing renal tubular cell apoptosis (as shown in Figures 52 and 53).

[0294] Example 21: Treatment of AKI mice with oral administration of DIIIV1-RP81

[0295] After establishing the IRI-induced AKI model, mice were orally administered 65 μg of DIIIV1-RP81. Subsequently, 24 h after treatment, blood was collected from each group of mice. After incubation at 37°C for 1 h, the supernatant serum was separated by centrifugation at 4000 rpm for 10 min at 4°C. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. Blood biochemistry results showed that, compared to the model group, the 65 μg treatment group effectively reduced the levels of UREA and CREA in the serum (Figure 54). ELISA results also showed that the 65 μg treatment group significantly reduced the level of NGAL in the serum, indicating a reduction in kidney damage (Figure 55).

[0296] Example 22 compares the relief of AKI in different treatment groups by oral administration.

[0297] After confirming that oral administration of 65 μg DIIIV1-RP81 could effectively alleviate AKI, the treatment effect was compared with all control groups in IRI-induced AKI. After the AKI model was established, mice were injected via tail vein with DIIIV1 (50.82 μg), RP81 (8.86 μg), and DIIIV1-RP81 (65 μg), respectively. Subsequently, after treatment (24 h after administration), blood was collected from each group of mice, and after being left at room temperature for 1 h, the supernatant serum was separated by centrifugation at 4000 rpm for 10 min at 4 °C. The levels of UREA and CREA in the serum were measured using a fully automated biochemical analyzer. The results in Figure 56 show that, compared with oral administration of DIIIV1 and RP81, oral administration of 65 μg DIIIV1-RP81 significantly reduced the levels of UREA and CREA in the serum of AKI mice, effectively alleviating AKI. ELISA results also showed that the 65 μg DIIIV1-RP81 treatment group significantly reduced serum KIM-1 and NGAL levels, thus slowing down kidney damage (as shown in Figure 57).

[0298] Example 23: Treatment of AKI mice by tail vein administration of DIIIV1-RP220

[0299] After establishing the RI-induced AKI model, mice were orally administered 1 μg of DIIIV1-RP220. Subsequently, 24 h after treatment, blood was collected from each group of mice. After incubation at 37°C for 1 h, the supernatant serum was separated by centrifugation at 4°C and 4000 rpm for 10 min. The levels of UREA and CREA in the serum were measured using an automated biochemical analyzer. The blood biochemistry results showed that, compared to the model group, the 1 μg treatment group effectively reduced the levels of UREA and CREA in the serum (as shown in Figure 58).

[0300] Example 24 Construction of a cisplatin-induced AKI model

[0301] Cisplatin was dissolved in 0.9% sodium chloride solution, wrapped in aluminum foil to protect from light, and shaken at 37°C for 2 hours to prepare a cisplatin solution. Male C57BL / 6J mice aged 8-10 weeks were randomly divided into a control group and a cisplatin group, with at least three mice in each group. The cisplatin group mice underwent intraperitoneal injection of 20 mg / kg cisplatin to establish an AKI model; the control group consisted of healthy mice under the same feeding conditions without any treatment. After 24 hours of feeding, serum samples were collected from all mice, and kidney tissue was isolated. The kidney tissue was fixed by immersion in 4% paraformaldehyde solution, and its histological morphology was subsequently observed using H&E staining. As shown in Figure 59, the UREA and CREA values ​​in the serum of the cisplatin group mice were significantly higher than those in the control group.

[0302] Example 25: Establishment of a mouse model of chronic kidney injury

[0303] A model of chronic kidney injury induced by unilateral ureteral obstruction:

[0304] After anesthetizing the mice, a 1-1.5 cm longitudinal incision was made along the midline of the abdomen (from the pubic symphysis to the xiphoid process). The intestines were gently pushed to the right with forceps to expose the left kidney and ureter. The ureter, located between the lower pole of the kidney and the bladder, appeared as a white, cord-like structure covered with adipose tissue. The periureteral fat was bluntly dissected with microforceps, freeing approximately 0.5 cm of the fat. The ureter was double-ligated with 5-0 silk suture at both the proximal end (approximately 2-3 mm from the renal pelvis) and the distal end (approximately 3 mm from the proximal end) of the ureter, with the ligation force sufficient to block urine flow without severing the ureter. Mouse weight was measured weekly during the modeling period. Serum samples were collected 14 days post-surgery to determine UREA and CREA levels. As shown in Figure 60, the UREA content in the serum of mice in the chronic kidney injury group was significantly higher than that in the control group. Simultaneously, mouse kidneys were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E, Masson's red, and Sirius red. Immunohistochemistry was used to observe the expression of myofibroblast marker α-SMA and fibrotic cytokine TGF-β1 in the kidney. H&E staining, Masson staining, Sirius red staining, and immunohistochemical results showed significant fibrosis in the kidney tissue of a mouse model of chronic kidney injury.

[0305] A model of chronic kidney injury induced by unilateral renal ischemia-reperfusion:

[0306] After anesthetizing the mice, the abdominal cavity was exposed through 0.5cm incisions below the ribs on both sides of the back. The right renal pedicle was dissected, and the right renal pedicle was clamped with an arterial clamp. After 30 minutes, the arterial clamp was released, and the restoration of renal blood supply was assessed based on the color change of the kidney. Finally, the abdomen was sutured closed. Twelve weeks postoperatively, mouse serum was collected to measure UREA and CREA. At the same time, the ischemic kidney was harvested for H&E staining, Masson staining, Sirius red staining, and immunohistochemistry (myofibroblast marker α-SMA, fibrotic cytokine TGF-β1).

[0307] Cisplatin-induced chronic kidney injury model

[0308] Male C57BL / 6J mice were intraperitoneally injected with 8 mg / kg cisplatin once a week for four consecutive weeks. After 12 weeks, mouse serum was collected to determine UREA and CREA levels; simultaneously, mouse kidneys were collected for H&E staining, Masson staining, Sirius red staining, and immunohistochemistry (for myofibroblast marker α-SMA and fibrotic cytokine TGF-β1).

[0309] Example 26: PK of DIIIV1-RP81 in a chronic kidney injury model

[0310] IR-780 was used as the labeling dye to observe the pharmacokinetics of DIIIV1-RP81 in a mouse model of chronic kidney injury. 100 μL of the DIIIV1-RP81@IR-780 complex was injected into C57BL / 6J mice via tail vein. Fluorescence was detected using a near-infrared II imaging system. Real-time imaging was performed within 0-30 min after administration (Figure 61). Fluorescence was acquired at 30 min, 1 h, 2 h, 4 h, 12 h, and 24 h after administration in prone, supine, and lateral positions (Figure 62). Sequence information.

[0311] Information on some of the sequences involved in this invention is provided in Table 4 below.

[0312] Table 4: Sequence Description

[0313] 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

A complex comprising a carrier protein and a functional molecule for the diagnosis, prevention, and / or treatment of kidney injury; 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. The complex of claim 1, compared with the wild type, 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 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. 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. 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. 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. 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:

5. 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: 39-43; Preferably, the DIIIV is selected from the amino acid sequences shown in SEQ ID NO:8-38; Preferably, the DIIIV has the amino acid sequence shown in SEQ ID NO:

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. 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. The complex according to any one of claims 1-9, wherein the kidney injury is selected from acute kidney injury and chronic kidney injury. 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 peptides, proteins, antibodies, nanobodies, nucleic acid drugs, or chemotherapeutic drugs; preferably, the peptide or protein is a peptide chain or a cyclic peptide; preferably, the antibody is a monoclonal antibody or its antigen-binding fragment; the nucleic acid drug is mRNA or a protein-nucleic acid complex; Preferably, the drug is selected from anti-inflammatory, anti-apoptotic or anti-oxidative stress drugs, such as: amifostine, cimetidine and glutathione, RNLS agonists (e.g. RP81, RP220); Preferably, the imaging agent is selected from cyanine dyes (such as IR-780, IR-783) and other photosensitizers; 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 therapeutic radionuclide is selected from... 64 Cu、 18 F, 68 Ga、 177 Lu、 125 I, 90 Y、 89 Sr、 32 P, 233 Ra. 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, and Fmoc-PEA. 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. A nucleic acid molecule encoding the complex according to any one of claims 1-13; Preferably, the complex is a fusion protein. 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). 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 HEK293F cells; Preferably, the prokaryotic cells are Escherichia coli cells, Bacillus subtilis cells, or any combination thereof. 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. 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 the treatment of kidney injury; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury. The complex according to any one of claims 1-13, the delivery combination or pharmaceutical composition according to claim 17, is used for treating kidney injury; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury. A method of treating kidney injury, 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 thereof; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury.

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