Diagnosis and treatment medicine for kidney injury

Abstract

The invention provides a medicine for diagnosing and treating kidney injury. Specifically provided is a complex comprising a carrier protein selected from the group consisting of a mutant DIIIbV of an albumin third domain substructure (e.g. DIIIb), an albumin third domain DIIIIV containing the mutant, and a multimer of the DIIIbV or DIIIIV, and a functional molecule for the diagnosis, prevention and / or treatment of renal injury. The carrier protein has improved affinity to FcRn and excellent transmucosal efficiency, and shows long-acting and slow-release characteristics. By means of fusion expression, chemical grafting and the like, the carrier protein is loaded with a kidney injury diagnosis and treatment drug and can be used for diagnosis, prevention and / or treatment of kidney injury.

Description

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

[0002] This application relates to the field of biological agents, specifically to diagnostic and therapeutic drugs for kidney damage. Background Art

[0003] Kidney injury refers to a clinical syndrome characterized by structural abnormalities or functional decline in the kidneys caused by various etiologies. Its core features include decreased renal excretion of metabolic waste products, disturbances in electrolyte / acid-base balance, and associated health risks. Based on the rate and duration of disease progression, it is clinically classified into two main subtypes: acute kidney injury (AKI) and chronic kidney injury. Both constitute significant contributing factors to end-stage renal disease (ESRD) and cardiovascular disease worldwide. As a core metabolic organ for maintaining homeostasis, rapid functional impairment of the kidneys can trigger a systemic chain reaction, leading to a significantly increased mortality rate in patients with kidney injury. This places a heavy burden on clinical diagnosis and treatment, thus necessitating in-depth research and technological optimization in the prevention and treatment of kidney injury.

[0004] Current treatment for kidney damage follows the core logic of "etiological control - functional maintenance - replacement support." The key principles of AKI treatment are timely identification and control of the cause, maintenance of hemodynamic stability, and avoidance of nephrotoxic drug exposure. If kidney function is severely impaired (e.g., severe uremia symptoms, uncorrectable electrolyte imbalances), renal replacement therapy is necessary to sustain life. The core of chronic kidney injury treatment is "long-term etiological control - slowing progression - complication prevention," to avoid progression to ESRD. However, there are currently no specific drugs for treating the pathological processes of kidney damage (e.g., renal tubular damage repair, reversal of renal fibrosis). Existing treatments are mostly "symptomatic supportive care," which can only relieve symptoms and maintain basic bodily functions, but cannot block the progression of kidney damage or promote renal function repair at the pathological mechanism level. Some patients still face the risk of kidney function deterioration, developing chronic kidney disease, or even end-stage renal disease.

[0005] Therefore, developing specific therapeutic drugs that can target the pathological characteristics of kidney damage, act directly on the lesion target, and block the pathological process has become the primary need to solve the clinical treatment dilemma of kidney damage.

[0006] In conclusion, identifying carriers that deliver drugs precisely, persistently, and efficiently to the kidneys is of great significance. Summary of the Invention

[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 grouped 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 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 the natural albumin having 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 having 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 having the amino acid sequence shown in SEQ ID NO: 1 is L;

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

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

[0031] (8) The amino acid at position 547 of natural albumin having 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 having 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 having the amino acid sequence shown in SEQ ID NO: 1 is E;

[0035] (12) The amino acid at position 512 of natural albumin having 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 having 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 position 528 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 position 512 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, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505N, T508L, F509L, H510R, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505Q, I523L, K524L, T527K, A528Y, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505Q, I523L, T527K, A528H, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505Q, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains the mutations E505T, I523L, T527K, A528Y, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528Y, E531L, and K573P.In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505Q, T508L, F509L, H510R, D512G, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500D, E505T, I523L, T527K, A528H, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528Y, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, I523L, T527K, A528Y, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505T, I523L, T527K, A528Y, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as K500L, E505N, I523L, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505N, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505T, A511T, I523L, T527K, A528H, E531L, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505N, I523L, K524L, T527K, A528H, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as E505Q, I523M, K524L, T527K, A528H, E531L, V547A, and K573P. In some embodiments, the DIIIbV or DIIIV contains mutations such as V498E, K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P.In some embodiments, the DIIIbV or DIIIV contains the mutations V498E, K500L, E505T, I523L, T527K, A528H, and 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 implementations, the DIIIbV or DIIIV contains mutations such as 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: 8~38.

[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、 89Sr、 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 a cleavable or non-cleavable linker; 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, MACglucuronide 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:

[0079] C-(LD) n

[0080] Wherein, C represents the carrier protein;

[0081] D represents the functional molecule;

[0082] 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;

[0083] 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.

[0084] In some implementations, C is selected from DIIIV1~DIIIV31, DIIIbV1, DIIIbV2, DIIIbV4, DIIIbV8 and DIIIbV22.

[0085] In some implementations, D is selected from amifostine, cimetidine, glutathione, and RNLS agonists (e.g., RP81, RP220).

[0086] In some implementation schemes, L is selected from , , , .

[0087] Nucleic acid molecules, vectors, and host cells

[0088] In another aspect, this application provides a nucleic acid molecule that encodes the complex described in any of the preceding claims.

[0089] In some embodiments, the complex is a fusion protein.

[0090] 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).

[0091] The techniques used to prepare the mutants or fusion proteins of this application are conventional in the art, for example, those disclosed in WO2009019314 (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.

[0092] 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.

[0093] Delivery of combination or pharmaceutical composition

[0094] In another aspect, this application provides a delivery combination or pharmaceutical composition comprising any of the complexes described above.

[0095] In some embodiments, the delivery combination or pharmaceutical composition is delivered intravenously.

[0096] 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.

[0097] 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).

[0098] 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.

[0099] In some embodiments, the delivery combination or pharmaceutical composition is an aerosol, powder inhaler, spray, or other dosage form suitable for inhalation administration.

[0100] In some embodiments, the pharmaceutical composition contains one or more pharmaceutically acceptable excipients.

[0101] Medical uses and methods

[0102] 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.

[0103] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0104] 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.

[0105] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0106] 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.

[0107] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0108] 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.

[0109] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0110] In another aspect, this application provides a carrier protein as defined in any of the preceding claims for the treatment of kidney injury.

[0111] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0112] 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.

[0113] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0114] 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.

[0115] In some implementations, the kidney injury is selected from acute kidney injury and chronic kidney injury.

[0116] Terminology Definition

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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).

[0124] 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.

[0125] 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 to both 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., TE Creighton 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.

[0126] 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.

[0127] The techniques used to prepare the mutants or fusion proteins of this application are conventional in the art, for example, those disclosed in WO2009019314 (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.

[0128] 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 identical at a position when the same base or amino acid monomeric subunit occupies the same location (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 identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0129] 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).

[0130] 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.

[0131] 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.

[0132] 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.

[0133] As used in this article, the term "subunit" refers to each polypeptide chain in the quaternary structure of a protein, which has an independent tertiary structure.

[0134] 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.

[0135] 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; Cosmids; 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.

[0136] 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.

[0137] 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)).

[0138] 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.

[0139] 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.

[0140] "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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] Beneficial effects of the invention

[0145] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0146] 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.

[0147] 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.

[0148] 3. This invention can enable the diagnosis, imaging, and / or treatment of kidney injury.

[0149] 4. This invention enables mucosal delivery, making the drug administration process more convenient, allowing patients to operate independently, and significantly improving patient compliance.

[0150] 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

[0151] Figure 1 The DIII mutant Saccharomyces cerevisiae plasmid vector pYD1 constructed as described in Example 1, wherein DIII specifically refers to the DIII mutant.

[0152] Figure 2 A to Figure 2 C represents the changes in fluorescence signals and their ratios of expression and binding of the initial library and different rounds of screening of yeast surface display as described in Example 1.

[0153] Figure 3 The DIIIV1 Pichia pastoris plasmid vector pPIC9K described in Example 2 refers to the DIII mutant.

[0154] Figure 4 The results show the isolation and purification of the Pichia pastoris expressing the DIII mutant described in Example 2.

[0155] Figures 5A to 5B This is a diagram showing 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 diagram are abbreviated. V1 corresponds to DIIIV27, V2 to DIIIV28, V3 to DIIIV29, V4 to DIIIV30, and V5 to DIIIV31.

[0156] Figure 6 A to Figure 6 B represents the fluorescence intensity results of samples from different DIII mutants and different cells detected by a near-infrared imager as described in Example 6; for ease of labeling, the sample names in the figure are abbreviated, with V1 corresponding to DIIIV27, V2 to DIIIV28, V3 to DIIIV29, V4 to DIIIV30, and V5 to DIIIV31.

[0157] Figure 7 The results of H&E staining of mouse kidneys of different grades as described in Example 7.

[0158] Figure 8 The result is the urine protein detection result among the AKI biochemical indicators described in Example 7.

[0159] Figure 9 The result is the blood urea nitrogen test result among the AKI biochemical indicators described in Example 7.

[0160] Figure 10 The result is the serum creatinine detection result among the AKI biochemical indicators described in Example 7.

[0161] Figure 11 The images show dynamic NIR-II imaging of mouse in vivo kidneys at different grades as described in Example 7.

[0162] Figure 12 The results are the correlation analysis results between the inflection point of the change in renal fluorescence intensity and GFR in mice with different grades of kidney injury as described in Example 7.

[0163] Figure 13 This is a flowchart illustrating the prediction of GFR using NIR-II imaging omics data combined with deep learning, as described in Example 7.

[0164] Figure 14 This is a map of the fusion expression plasmids of DIIIV1 and RP81 described in Example 8, where DIII in each plasmid diagram refers to DIIIV1.

[0165] Figure 15 The results of SDS-PAGE and WB analysis of DIIIV1-RP81 expressed using Pichia pastoris GS115 as described in Example 8 are shown.

[0166] Figure 16 The results are SDS-PAGE and WB of DIIIV1-RP81 expressed in HEK293F cells as described in Example 8.

[0167] Figure 17 This is the map of the fusion expression plasmid of DIIIV1 and RP220 described in Example 8, where DIII refers to DIIIV1.

[0168] Figure 18 The results are SDS-PAGE and WB of DIIIV1-RP220 expressed in HEK293F cells as described in Example 8.

[0169] Figure 19 The result is an SDS-PAGE of DIIIbV1 expressed in HEK293F cells as described in Example 8.

[0170] Figure 20 This is the map of the fusion expression plasmid of DIIIbV1 and RP81 described in Example 8.

[0171] Figure 21 This is the map of the fusion expression plasmid of DIIIbV1 and RP220 described in Example 8.

[0172] Figure 22 Synthesis of DIIIV1-amifostine as described in Example 9

[0173] Figure 23 The SDS-PAGE results of the synthesized DIIIV1-amifostine described in Example 9.

[0174] Figure 24Synthesis of DIIIV1-cimetidine as described in Example 9

[0175] Figure 25 This 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.

[0176] Figure 26 The results are NIR II fluorescence results obtained in mice in prone, supine, and lateral positions after administration of DIIIbV1@ICG via tail vein injection as described in Example 10.

[0177] Figure 27 This 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.

[0178] Figure 28 The results are NIR II fluorescence results of mice in prone, supine, and lateral positions obtained after administration of RP81@ICG via tail vein injection as described in Example 11.

[0179] Figure 29 This 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.

[0180] Figure 30 The results of NIR II fluorescence in mice in prone, supine, and lateral positions after administration of DIIIV1-RP81@IR780 via tail vein injection as described in Example 11 are shown.

[0181] Figure 31 This is a two-photon in vivo imaging of the kidney region in a small animal, as described in Example 12, by injecting Cy5-labeled DIIIV1 via the tail vein.

[0182] Figure 32 This is a two-photon in vivo imaging of the kidney region in a small animal, as described in Example 12, by injecting Cy5-labeled RP81 via the tail vein.

[0183] Figure 33 This is a two-photon in vivo imaging of the kidney region in a small animal, as described in Example 12, by injecting Cy5-labeled DIIIV1-RP81 via the tail vein.

[0184] Figure 34 The immunofluorescence results of co-localization with FcRn in the kidney site after tail vein injection of DIIIV1-RP81@IR780 as described in Example 13.

[0185] Figure 35The results are the blood biochemistry results of the AKI mouse model constructed by clamping both renal arteries for 30 min as described in Example 14.

[0186] Figure 36 The results are based on the blood biochemistry of patients treated with AKI by tail vein administration of 2 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 15.

[0187] Figure 37 The results are based on the ELISA of AKI treatment via tail vein administration of 2 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 15.

[0188] Figure 38 The H&E and TUNEL results for the kidneys of patients with AKI treated by tail vein administration of 2 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 15 are as follows.

[0189] Figure 39 The score results are those of kidney damage treated with 2 μg DIIIV1-RP81 via tail vein in an IRI-induced AKI model as described in Example 15.

[0190] Figure 40 The results are based on the blood biochemistry of AKI treated by tail vein administration of DIIIV1, RP81, and DIIIV1-RP81 in an IRI-induced AKI model as described in Example 16.

[0191] Figure 41 The results are based on the ELISA of AKI treatment via tail vein administration of DIIIV1, RP81, and DIIIV1-RP81 in an IRI-induced AKI model as described in Example 16.

[0192] Figure 42 The H&E and TUNEL results of the kidneys treated with DIIIV1, RP81, and DIIIV1-RP81 via tail vein in an IRI-induced AKI model, as described in Example 16, are as follows.

[0193] Figure 43 The results are the scores of kidney damage in the IRI-induced AKI model described in Example 16, obtained by tail vein administration of DIIIV1, RP81, and DIIIV1-RP81 to treat AKI.

[0194] Figure 44 The results are NIR II fluorescence results of mice in prone, supine, and lateral positions obtained after administration of RP81@ICG via nasal drops as described in Example 17.

[0195] Figure 45 The results are NIR II fluorescence of mice in prone, supine, and lateral positions after administration of DIIIV1-RP81@IR780 via nasal drops as described in Example 17.

[0196] Figure 46 The results are immunofluorescence of the renal site of DIIIV1-RP81@IR780 administered via nasal drops as described in Example 18.

[0197] Figure 47 The results are based on the blood biochemistry of patients treated with 20 μg DIIIV1-RP81 via nasal drops in an IRI-induced AKI model as described in Example 19.

[0198] Figure 48 The results are based on the ELISA of AKI treatment via nasal administration of 20 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 19.

[0199] Figure 49 The H&E and TUNEL results for the kidneys of patients with AKI treated by intranasal administration of 20 μg DIIIV1-RP81 in an IRI-induced AKI model as described in Example 19 are as follows.

[0200] Figure 50 The results are the scores of kidney damage in the IRI-induced AKI model described in Example 19, obtained by administering 20 μg DIIIV1-RP81 via nasal drops to treat AKI.

[0201] Figure 51 The results of blood biochemistry in the IRI-induced AKI model described in Example 20, involving the treatment of AKI by intranasal administration of DIIIV1, RP81, and DIIIV1-RP81.

[0202] Figure 52 The H&E and TUNEL results of the kidneys treated with DIIIV1, RP81, and DIIIV1-RP81 via nasal drops in an IRI-induced AKI model, as described in Example 20.

[0203] Figure 53 The scores for kidney damage in an IRI-induced AKI model, as described in Example 20, are obtained by administering DIIIV1, RP81, and DIIIV1-RP81 via nasal drops.

[0204] Figure 54 The results are based on the blood biochemistry of patients treated with 65 μg DIIIV1-RP81 orally in an IRI-induced AKI model as described in Example 21.

[0205] Figure 55 The results are based on the ELISA of AKI treatment with 65 μg DIIIV1-RP81 administered orally in an IRI-induced AKI model as described in Example 21.

[0206] Figure 56 The results of blood biochemistry in the IRI-induced AKI model described in Example 22, obtained by treating AKI with oral administration of DIIIV1, RP81, and DIIIV1-RP81.

[0207] Figure 57 The results of ELISA for treating AKI with oral administration of DIIIV1, RP81, and DIIIV1-RP81 in an IRI-induced AKI model as described in Example 22.

[0208] Figure 58 The results are based on the blood biochemistry of AKI treated by tail vein administration of 1 μg DIIIV1-RP220 in an IRI-induced AKI model as described in Example 23.

[0209] Figure 59 The results are blood biochemistry results of the AKI mouse model induced by cisplatin as described in Example 24.

[0210] Figure 60 The blood biochemical results of the mouse model of chronic kidney injury induced by UUO as described in Example 25.

[0211] Figure 61 This is a schematic diagram of real-time imaging taken 0-30 min after UUO-induced chronic kidney injury mice were injected with DIIIV1-RP81 via the tail vein as described in Example 26.

[0212] Figure 62 The results obtained in Example 26 are NIR II fluorescence results of mice in prone, supine, and lateral positions after administration of DIIIV1-RP81 via tail vein injection into mice with UUO-induced chronic kidney injury. Detailed Implementation

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

[0214] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CELLCULTURE (edited by R.R. Freshney (1987)).

[0215] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0216] Example 1. Screening for FcRn high-affinity DIII mutants

[0217] 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 then fabricated into a gene chip. The chip was first processed by high-speed centrifugation (12000 rpm, 1 min), and then dissolved in TE buffer (Tris-EDTA buffer). The mutant sequences were ligated to the vector pYD1 using a conventional seamless cloning method. An example of DIIIV ligation to the vector plasmid is shown below. Figure 1 As shown, store at -20℃ for later use.

[0218] Saccharomyces cerevisiae EBY100 glycerol culture was streaked onto YPD plates and incubated at 30℃ for approximately 36 h. 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 1 M 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 an OD600 of 0.5-1 and incubate on a shaker for 48-72 h (20 ℃, 200-250 rpm). Transfer to activation medium again using the same method 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, and keep them in 1 mL volume for later use.

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

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

[0221] (2) Sample groups: a. Single staining 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, 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 induced 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 at pH 6.0, wash the culture, centrifuge at 4200 rpm for 2 min, discard the supernatant, add 90 μL PBS pH 6.0 to resuspend the culture, add 10 μL FcRn-β2M protein (Biotinylated, His-Avi, 100 μg / mL), incubate at room temperature for 1 h, add 2 μL HA-Tag Mouse mAb (Alexa Fluor-488 Conjugate) antibody and 0.3 μL Streptavidin APC, incubate at room temperature for 1 h, add 1 mL PBS 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 PBS pH 6.0 to resuspend the culture for later use.

[0222] After all samples were prepared, they were sequentially tested using flow cytometry and FACS sorting. The sorting results are as follows: Figure 2 A-to Figure 2 As shown in C, after four to five rounds of sorting, it can be seen that both the fluorescence signal of the displayed mutant and the fluorescence signal of the mutant binding to FcRn are significantly enhanced, suggesting 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. Figure 2 B and Figure 2 In C, R.1 to R.5 represent the first to fifth rounds of sorting, respectively, ultimately resulting in a high-affinity mutant library, which then proceeds to the next stage of validation.

[0223] Example 2. Expression of high-affinity DIII mutant in Pichia pastoris

[0224] 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.

[0225] The mutant was used to synthesize the pPIC9K vector plasmid (example plasmid map shown). Figure 3As shown in the figure, firstly, GS115 Pichia pastoris competent cells were prepared. GS115 was streaked on YPD plates and incubated at 30℃ for 2-3 days until single colonies grew. Single colonies were picked and transferred to 50 mL centrifuge tubes containing 5 mL of YPD medium. The culture was incubated overnight at 30℃ with shaking until the OD600 value reached 1. The overnight culture was then transferred at a ratio of 1:100 to 250 mL Erlenmeyer flasks containing 50 mL of YPD medium. The culture was incubated overnight at 30℃ with shaking until the Pichia pastoris OD600 reached 0.8-1.0. The yeast pellet was collected by centrifugation at 1500 g for 10 min at room temperature, washed twice with 25 mL of sterile water, and centrifuged again at 1500 g for 10 min at room temperature. The supernatant was discarded. The cells were resuspended in 0.1 M LiCl solution and centrifuged at 10000 g for 15 s. The supernatant was discarded, and the yeast was resuspended in 0.1 M LiCl solution. The mixture was transferred to 1.5 mL centrifuge tubes, mixed well, and then aliquoted. The culture was then centrifuged at 1500 g for 15 s at room temperature. Collect competent yeast cells by centrifugation for 5 min. Linearization of plasmids: Linearize the plasmid DNA by single-enzyme digestion using standard methods. Preparation of 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. Preparation of transformation buffer: For each yeast transformation tube, add the following reagents in the following volumes to prepare the transformation buffer: 240 μL 50% PEG3350, 36 μL 1 M 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 6000 g for 1 min at room temperature, removing as much supernatant as possible, and collect the yeast precipitate to prevent the high concentration of LiCl solution from continuously toxicizing 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 6000 g for 1 min at room temperature, removing as much supernatant as possible, add 1 mL of YPD liquid medium and pipette until the yeast precipitate is dispersed and mixed, seal the centrifuge tube tightly with sealing film, and shake on a horizontal shaker at 30°C for 1-4 h (place the centrifuge tube horizontally, never vertically); take 25-100 Spread μL of bacterial culture onto MD plates and incubate at 30°C for 2-3 days (to avoid difficulty in adsorbing suspended cells onto the plates, the plates can be prepared 1-2 weeks in advance and placed in a 4°C refrigerator); after single colonies grow in MD culture for 2-3 days, add 1 mL of sterile water to each plate, mix the single colonies, and transfer them to 1.5 mL centrifuge tubes for later use. Add 200 μL of each plate to prepared YPD plates with different gradients of G418 antibiotics for a second screening of high-copy transformants. Culture at 30℃ for 3-4 days. Once single colonies have grown, pick a single colony and add it to a 250 mL Erlenmeyer flask containing 25 mL of BMGY medium. Incubate on a shaker at 30℃ and 250 rpm until the OD600 reaches 2-6. Centrifuge at 3000 g for 5 min at room temperature to collect the yeast. Add the yeast to a 1L shaker flask containing 100 mL 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 235 rpm for 1-5 days. Add 1% methanol every 12-24 h to induce expression. After induction, centrifuge at 9000 g for 20 min to collect the supernatant. Filter twice with a 0.8 μm vacuum pump and once with a 0.22 μm vacuum pump. Collect the filtrate for later use.

[0226] Protein purification was performed using Albu purification matrix, following the manufacturer's recommended method. First, the purification column was equilibrated to 5 column volumes (5 CV) using equilibration buffer. The filtered protein was then loaded, and the column was equilibrated again to 5 CV using equilibration buffer. Buffer 1 was used to wash away impurities, followed by equilibration again to 5 CV using equilibration buffer or UV back to baseline. Buffer 2 was used to wash away impurities, and the column was equilibrated again to 5 CV using equilibration buffer or UV back to baseline. Buffer 3 was used to wash away impurities, and the column was equilibrated again to 5 CV using equilibration buffer or UV back to baseline. Buffer 4 was used to elute the target protein, and the column was equilibrated again to 5 CV using equilibration buffer or UV back to baseline. The column was then washed with 0.5 M NaOH. Finally, the column was stored at 4°C using 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.

[0227] Purification results are as follows Figure 4 As shown in the figure, the purified target protein band is clearly visible at the black dotted line.

[0228] Table 1. Mutation sites of exemplary mutants

[0229] Example 3. Affinity detection of DIII mutant FcRn

[0230] 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.

[0231] 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 h. 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 h. 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 h. 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 min. 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.

[0232] 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).

[0233] Table 2. Affinity results of representative mutants

[0234] Example 4. Determination of the affinity between the DIIIb mutant and FcRn

[0235] 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 containing SEQ ID NO: 5 in Table 3) and omitting the DIIIa portion (i.e., SEQ ID NO: 4 in Table 3) with FcRn. This sequence corresponds to the fragment of amino acids 467 to 585 of SEQ ID NO: 1.

[0236] 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.

[0237] 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 h. 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 h. 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 h. 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 min. 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.

[0238] 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).

[0239] Table 3. Affinity results of representative mutants

[0240] Example 5. DIIIV transport and circulation mediated by FcRn

[0241] 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 by the BCA method to be 2 mg / mL, 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.

[0242] First, 300,000 293T cells per well were seeded in 35 mm glass-bottomed culture dishes (15 mm diameter) and cultured at 37 °C with 5% CO2 for 24 h. 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 1 μM) and kept in the dark. The cell culture medium was aspirated, and the cells were washed three times with 500 μL PBS (pH 7.4). 250 μL of the protein and cyanine dye complex was added to each group, and the cells were incubated at 37 °C for 1 h. For the control groups V4-Ab and V4-Ba A1, in addition to V4-IR-780, a DIII-specific polyclonal antibody (0.5 μM) 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 RedDND-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.

[0243] Experimental results showed that, compared with wild-type HSA and unmutated DIII, the high-affinity mutants DIIIV28 and DIIIV30 showed significantly stronger signals in cells, and their co-localization signals with FcRn were also significantly better than those in the control group (see [link to study]). Figure 5A (Bottom right corner). The Pearson product-moment correlation coefficient (PCCs) and overlap coefficient (O1R) of the mutant colocalization signal distribution with FcRn were significantly better than those of the control group. Meanwhile, 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 colocalization data (Figure 5B).

[0244] 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.

[0245] Example 6. Pharmacokinetic determination of mutant protein administered intranasally in mice

[0246] 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 post-administration.

[0247] The results are as follows Figure 6 As shown, after tail vein injection of DIIIV1, DIIIV1 exhibits specific enrichment in the kidney region. Replacing DIIIV1 with any other DIII mutant yields similar results for the resulting complex.

[0248] Example 7: The complex of the DIII mutant and cyanine dye IR-783 was used to detect a cisplatin-induced AKI mouse model.

[0249] I. Construction of a cisplatin-induced AKI mouse model

[0250] 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 h 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 h, fixed by immersion in 4% paraformaldehyde, and histological morphology was observed by hematoxylin and eosin (H&E) staining.

[0251] The results are as follows Figure 7 As shown, with increasing cisplatin dosage, H&E staining revealed an increasing number of aggregated, elongated vacuoles, and a significant decrease in the number of nephrons. These pathological findings indicate that higher cisplatin-induced doses lead to more severe kidney damage, reflecting the successful establishment of a kidney injury model.

[0252] II. Detection of AKI Biochemical Indicators

[0253] Urinary protein detection: Urine samples from each mouse before modeling were used as a 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. Figure 8 As the level of kidney injury increased, there was no obvious pattern or difference in the ratios among the healthy mice and the four groups G1, G2, and G3. Urinary protein increased slightly in G4, G5, and G6, while only G6 showed a significant increase. Here, G represents the grade of kidney injury, and the numbers 1, 2, 3, 4, 5, and 6 represent the degree of kidney injury. For example, G1 represents grade I kidney injury, which is mild kidney injury.

[0254] 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, and the changes in blood urea nitrogen before and after kidney injury induction were compared. Figure 9 There were no obvious patterns or differences between the groups.

[0255] 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, and the changes in serum creatinine before and after kidney injury induction were compared. Figure 10 The changes in serum creatinine values ​​in healthy mice and the six groups (G1, G2, G3, G4, and G5) were all less than 1.5, which did not meet the clinical criteria for identifying AKI (a surge in serum creatinine value of more than 1.5 times). This demonstrates that the protocol can more sensitively reflect the degree of kidney damage.

[0256] III. Dynamic NIR-II Imaging of Mouse Kidneys with Different Grades of Injury

[0257] Cyanide dye IR-783 and DIII mutant were mixed at a molar ratio of 1:1 to prepare a 10 μM mixture of IR-783 and DIII, which was then incubated in a 60℃ 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. 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. Figure 11 A. The first 30 minutes of video footage were used as the NIR-II imaging omics database for deep learning-based diagnosis of AKI. Simultaneously, fluorescence intensity in the kidneys of mice in a lateral decubitus position was analyzed to obtain characteristic curves for key time points in dynamic imaging of the kidneys at different grades. For example... Figure 11 As shown in B, the changes in renal fluorescence intensity in different groups were significantly different.

[0258] IV. Analysis of changes in renal fluorescence intensity in mice of different grades

[0259] Statistical analysis was performed on the changes in renal fluorescence intensity in different mice during the first 30 minutes of video recording. Figure 12 As shown in figure a, with increasing cisplatin-induced dose, the inflection point of renal fluorescence intensity gradually decreased, which could clearly distinguish the degree of renal injury. Furthermore, the glomerular filtration rate (GFR) of mice with different cisplatin-induced doses was measured using FITC-inulin. Figure 12 As shown in b, it was found that the GFR gradually decreased with increasing cisplatin induction dose. Correlation analysis revealed a strong correlation between the two, which can be used to evaluate or predict the degree of kidney damage.

[0260] V. Predicting GFR using NIR-II imaging omics data combined with deep learning

[0261] like Figure 13As shown in figure a, 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 a 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 priori to improve prediction accuracy. Figure 13 As shown in b, this study performed 10-fold cross-validation, calculated the accuracy of each fold, and found its average value as the final accuracy of the model, which was 0.8627. Simultaneously, the 10-fold cross-validation ROC curves for each GFR group were plotted, yielding an average AUC of 0.9377.

[0262] Example 8 Expression, isolation, and purification of fusion proteins

[0263] First, the DIII mutant and the RP81 fusion protein were combined to synthesize a vector plasmid (the composite plasmid map is shown below). Figure 14 As shown in the figure, the protein was expressed using both prokaryotic and eukaryotic systems (GS115 Pichia pastoris and HEK293F cells). The Pichia pastoris-expressed protein was then purified using an affinity column to obtain the purified DIIIV1 / RP81 fusion protein (as shown in the figure). Figure 15 (As shown). The protein expressed by HEK293F cells was purified and separated by nickel column chromatography to obtain the purified DIIIV1 / RP81 fusion protein (as shown). Figure 16 (As shown).

[0264] The DIIIV1 and RP220 fusion protein was used to synthesize a vector plasmid (complex plasmid map shown below). Figure 17 As shown in the figure, 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 The results are from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) of purified DIIIV1-RP220.

[0265] DIIIbV1 was expressed using the GS115 Pichia pastoris system, followed by purification using an albumin affinity column to obtain DIIIbV1 (e.g. Figure 19 ).

[0266] The DIIIbV1 and RP81 fusion protein was used to synthesize a vector plasmid (composite plasmid map shown below). Figure 20 (As shown), 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 / RP81 fusion protein.

[0267] The DIIIbV1 and RP220 fusion protein was used to synthesize a vector plasmid (composite plasmid map shown below). Figure 21 (As shown), 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.

[0268] Example 9 Synthesis of DIIIV1-Amifostine and DIIIV1-Cimitidine

[0269] Amifostine was grafted onto DIIIV1 via chemical synthesis (e.g.) Figure 22 (As shown). 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. The reaction was monitored by liquid chromatography until the starting material was completely consumed. The product Mc-VC-PAB-Amifostine was then obtained by semi-preparative HPLC purification. 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, and the mixture was stirred at 25°C for approximately 3 h. The filtrate was collected after desalting column chromatography. Add 5 eq. of TCEP in PBS to the filtrate and reduce at 25°C. Then add 10 eq. of Mc-VC-PAB-Amifostine in DMSO and stir overnight at 25°C. Next, add 10 eq. of N-acetyl-L-cysteine ​​in PBS and quench the reaction at 25°C. After centrifugation using a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Amifostine (e.g.). Figure 23 (As shown).

[0270] Cimetidine was grafted onto DIIIV1 via chemical synthesis (e.g., ... Figure 24(As shown). 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. The reaction was monitored by liquid chromatography until the reactants were completely consumed. The product Mc-VC-PAB-Cimetidine was then obtained by semi-preparative HPLC purification. 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 h, and 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 a desalting column, collect the filtrate and concentrate it by ultrafiltration to obtain DIIIV1-Cimetidine.

[0271] Example 10 Pharmacokinetics of DIIIbV1 in mice

[0272] 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 detection was performed using a near-infrared II imaging system. Real-time imaging was performed within 0–30 min after administration (e.g., [missing information - likely a typology]). Figure 25 (as shown in the figure), and fluorescence was obtained in prone, supine and lateral positions of mice at 30 min, 1 h, 2 h, 4 h, 12 h and 24 h after injection. Figure 25 and 26 Imaging results showed that DIIIV1b could accumulate efficiently and in large quantities in the kidneys.

[0273] Example 11 Pharmacokinetics of RP81 and DIIIV1-RP81 in mice

[0274] 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 r / min 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 detection was performed using a near-infrared II imaging system. Real-time imaging was performed within 0-30 min after administration (e.g., [image of RP81]). Figure 27 (as shown in the figure), and fluorescence was obtained in prone, supine and lateral positions of mice at 24 h and 72 h after injection. Figure 28 Imaging results showed that although RP81 is a small polypeptide, it does not accumulate in the kidneys, but rather in the liver.

[0275] 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, and real-time imaging was performed within 0-30 min after administration (e.g., [missing information]). Figure 29 As shown in the figure, fluorescence was observed in mice in prone, supine, and lateral positions at 24 h, 48 h, 72 h, 4 d, 5 d, 6 d, 7 d, and 10 d after drug administration. NIR II imaging results showed that DIIIV1-RP81 was enriched in the kidneys, and the enrichment time could reach 10 d (as shown in the figure). Figure 30 (As shown).

[0276] Example 12: Distribution of DIII, RP81, and DIIIV1-RP81 in the kidneys

[0277] 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 r / min 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. Then, 100 μL of FITC-dextran (mean relative molecular mass 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. DIIIV1 (e.g., ...) was clearly visible in the lumen of the renal tubules. Figure 31 (as shown) or DIIIV1-RP81 (as shown) Figure 32 The fluorescence signal (as shown) indicates that FITC-dextran is distributed in the capillaries surrounding the renal tubules; while RP81 does not appear in the kidneys (as shown). Figure 33 (As shown).

[0278] Example 13 Co-location of DIIIV1-RP81 and FcRn

[0279] 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 immunofluorescently stained with lotus tetragonolobus lectin (LTL), mouse FcRn antibody, and DAPI. The co-localization of DIIIV1-RP81 and FcRn was observed using confocal microscopy. Figure 34 The results showed that DIIIV1-RP81 was distributed in LTL-labeled proximal tubules and co-localized with FcRn expressed in proximal tubular cells.

[0280] Example 14: Establishment and Evaluation of AKI

[0281] Establishment of a mouse model of acute kidney injury (AKI) induced by bilateral renal ischemia / reperfusion (IRI): After anesthesia, mice underwent a 0.5 cm incision below the costal margin on both sides of the back to expose the abdominal cavity. The renal pedicles were dissected, and each pedicle was clamped with an arterial clamp. After 30 minutes, the clamps were released, and the restoration of renal blood supply was assessed based on changes in kidney color. Finally, the abdomen was sutured closed. The sham-operated group underwent the same procedure, except for dissecting the renal pedicles. Blood was collected from the mice 24 hours later, and serum UREA and CREA levels were measured using an automated biochemical analyzer. The blood biochemistry results showed that the UREA and CREA levels in the model group were more than 1.5 times higher than those in the sham-operated group, meeting the clinical criteria for AKI (e.g., ...). Figure 35 (As shown).

[0282] Example 15 Treatment of AKI mice with DIIIV1-RP81 via tail vein administration

[0283] 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 The results showed that 2 μg 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 mouse serum were measured by ELISA. The results also showed that 2 μg DIIIV1-RP81 effectively reduced the levels of KIM-1 and NGAL in the serum of AKI mice. Figure 37 (As shown). In addition, collected kidney tissue was subjected to H&E staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to observe the pathology and morphology of the kidney tissue. The results of H&E and kidney pathological scoring showed that 2 μg DIIIV1-RP81 effectively slowed kidney damage (e.g., Figure 38 As shown); similarly, TUNEL staining results showed that 2 μg DIIIV1-RP81 could reduce kidney cell apoptosis, thereby slowing down AKI (as shown). Figure 39 (As shown).

[0284] Example 16: Comparison of AKI relief in different treatment groups via tail vein administration.

[0285] After confirming that 2 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 (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 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. 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 the serum (e.g., ...). Figure 40 , 41 As shown), the pathological results of the kidneys also showed that DIIIIV-RP81 protects the kidneys by reducing renal tubular cell apoptosis (as shown). Figure 42 , 43 (As shown).

[0286] Example 17: PK of RP81 and DIIIV1-RP81 in mice via intranasal administration.

[0287] Fluorescence was detected in C57BL / 6J mice by intranasal instillation of 30 μL of the RP81@ICG complex 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 days 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 (e.g., [missing information]). Figure 44 (As shown).

[0288] 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 lung tissue and then accumulated in the kidneys, with an accumulation period of up to 9 days (e.g., ...). Figure 45 (As shown).

[0289] Example 18 Distribution of DIIIV1-RP81 in the kidney

[0290] Kidney tissues were obtained from C57BL / 6J mice 24 h and 48 h after administration by intranasal instillation of 30 μL of the DIIIV1-RP81@IR-783 complex. The tissues were frozen at -80°C and frozen sections were prepared. Subsequently, the kidney tissues were stained with LTL and DAPI for immunofluorescence, and the distribution of DIIIV1-RP81 in the kidneys was observed using confocal microscopy. Figure 46 Immunofluorescence results showed that after intranasal administration, DIIIV1-RP81 accumulated in the proximal renal tubules of the kidneys.

[0291] Example 19 Treatment of AKI mice with DIIIV1-RP81 via intranasal administration

[0292] 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 separate 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 reduced renal tubular cell apoptosis, effectively slowing down kidney damage (e.g., Figures 47-50 (As shown).

[0293] Example 20: Comparison of AKI relief in different treatment groups via nasal drops.

[0294] After confirming that 20 μg DIIIV1-RP81 effectively alleviated 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 and, after being left at room temperature for 1 h, centrifuged at 4000 rpm for 10 min at 4°C to separate the supernatant serum. 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 could reduce the levels of UREA and CREA in the serum (e.g., ...). Figure 51 As shown), the pathological results of the kidney also showed that DIIIV1-RP81 protects the kidney by reducing renal tubular cell apoptosis (as shown). Figure 52 , 53 (As shown).

[0295] Example 21 Treatment of AKI mice with oral administration of DIIIV1-RP81

[0296] 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 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 65 μg treatment group effectively reduced the levels of UREA and CREA in the serum (e.g., ...). Figure 54 As shown). ELISA results also showed that the 65 μg treatment group significantly reduced serum NGAL levels, i.e., slowed kidney damage (e.g.). Figure 55 (As shown).

[0297] Example 22: Comparison of AKI relief in different treatment groups via oral administration.

[0298] After confirming that oral administration of 65 μg DIIIV1-RP81 effectively alleviated AKI, the treatment effect was compared with all control groups in IRI-induced AKI. After establishing the AKI model, mice were injected via tail vein with DIIIV1 (50.82 μg), RP81 (8.86 μg), and DIIIV1-RP81 (65 μg), respectively. Subsequently, 24 h after treatment, 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. Figure 56 The results showed that, compared with oral administration of DIIIV1 and RP81, oral administration of 65 μg DIIIV1-RP81 significantly reduced UREA and CREA levels 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, i.e., slowed down kidney damage (e.g., Figure 57 (As shown).

[0299] Example 23 Treatment of AKI mice by tail vein administration of DIIIV1-RP220

[0300] 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 (e.g., ...). Figure 58 (As shown).

[0301] Example 24 Construction of a cisplatin-induced AKI model

[0302] Cisplatin was dissolved in 0.9% sodium chloride solution, wrapped in aluminum foil to protect from light, and shaken at 37 ℃ for 2 h 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 3 mice in each group. Mice in the cisplatin group 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 h 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. Figure 59As shown, the serum UREA and CREA levels in the cisplatin group were significantly higher than those in the control group.

[0303] Example 25: Establishment of a mouse model of chronic kidney injury

[0304] A model of chronic kidney injury induced by unilateral ureteral obstruction:

[0305] 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, appears as a white cord 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), 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. Figure 60 As shown, the serum UREA level in mice with chronic kidney injury was significantly higher than that in the control group. Simultaneously, mouse kidneys were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining, Masson staining, and Sirius red staining. Immunohistochemistry was used to observe the expression of myofibroblast marker α-SMA and fibrotic cytokine TGF-β1 in the kidneys. The results of H&E staining, Masson staining, Sirius red staining, and immunohistochemistry showed significant fibrosis in the kidney tissue of the chronic kidney injury mouse model.

[0306] A model of chronic kidney injury induced by unilateral renal ischemia-reperfusion:

[0307] After anesthetizing the mice, the abdominal cavity was exposed through 0.5 cm incisions below the costal margins 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. Simultaneously, the ischemic kidney was harvested for H&E staining, Masson staining, Sirius red staining, and immunohistochemistry (for myofibroblast marker α-SMA and fibrotic cytokine TGF-β1).

[0308] Cisplatin-induced chronic kidney injury model

[0309] 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; at the same time, mouse kidneys were collected for H&E staining, Masson staining, Sirius red staining, and immunohistochemistry (myofibroblast marker α-SMA, fibrotic cytokine TGF-β1).

[0310] Example 26: PK of DIIIV1-RP81 in a chronic kidney injury model

[0311] IR-780 was used as a 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, and real-time imaging was performed within 0–30 min after administration (e.g., [missing information]). Figure 61 As shown in the figure, fluorescence was observed in mice in prone, supine, and lateral positions at 30 min, 1 h, 2 h, 4 h, 12 h, and 24 h after drug administration. Figure 62 (As shown). Sequence information

[0312] Information on some of the sequences involved in this invention is provided in Table 4 below.

[0313] Table 4: Sequence Description

[0314] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A complex comprising a carrier protein and a functional molecule for the diagnosis, prevention, and / or treatment of kidney injury; in, 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.

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

1.

3. The complex of claim 1 or 2, wherein the DIIIbV or DIIIV: (1) Compared with the wild type, it has a higher FcRn affinity under acidic or weakly acidic conditions; preferably, the DIIIV has an FcRn affinity Kd value 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; (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) Contains a mutation at one or more amino acid sites selected from the following: corresponding to amino acids 500, 505, 523, 524, 527, 528, 531, 547, 509, 510, 498, 512 and 573 of natural albumin having the amino acid sequence shown in SEQ ID NO: 1; (4) The mutation is a conserved mutation; (5) The DIII portion comprises a fragment of natural albumin corresponding to amino acids 467 to 585 of SEQ ID NO: 1; Preferably, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 523 and 573; Preferably, the amino acid mutations contained in DIIIbV or DIIIV are located at positions 505, 523, 547, and 573; Preferably, the amino acid mutations in DIIIbV or DIIIV are located at positions 500, 505, 523, 524, 527, 528, 531, 547, and 573, or... (6) Any combination of the above.

4. The complex according to any one of claims 1-3, wherein the DIIIbV or DIIIV: (1) The 500th amino acid in natural albumin having the amino acid sequence shown in SEQ ID NO: 1 is L or D; (2) The amino acid at position 505 of the natural albumin having 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 having the amino acid sequence shown in SEQ ID NO:1 is L or M; (4) The amino acid at position 524 of natural albumin having the amino acid sequence shown in SEQ ID NO:1 is L; (5) The amino acid at position 527 of natural albumin having 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 having the amino acid sequence shown in SEQ ID NO: 1 is L; (8) The amino acid at position 547 of natural albumin having the amino acid sequence shown in SEQ ID NO: 1 is A or C; (9) The amino acid at position 509 of natural albumin having 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 having the amino acid sequence shown in SEQ ID NO: 1 is E; (12) The amino acid at position 512 of natural albumin having 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 having the amino acid sequence shown in SEQ ID NO: 1 is L; (15) Any combination of the above.

5. The complex of claim 4, wherein the DIIIbV or DIIIV: (1) The amino acid at the 500th position is mutated from K to L or D; (2) The amino acid at position 505 is mutated from E to Q, N or T; (3) The amino acid at position 523 is mutated from I to L or M; (4) The amino acid at position 524 is mutated from K to L; (5) The amino acid at position 527 is mutated from T to K; (6) The amino acid at position 528 is mutated from A to H or Y; (7) The amino acid at position 531 is mutated from E to L; (8) The amino acid at position 547 is mutated from V to A or C; (9) The amino acid at position 509 is mutated from F to L; (10) The amino acid at 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 position 512 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 such as K500L, E505Q, I523L, K524L, T527K, A528H, E531L, V547A, and K573P.

6. The complex according to any one of claims 1-5, wherein, The wild type of DIII or DIIIb is derived from natural serum albumin of mammals; preferably, the mammals are selected from humans, chimpanzees, gorillas, rhesus monkeys, rabbits, mice, rats, hamsters, cattle, horses, donkeys, goats, sheep, dogs, guinea pigs and pigs; preferably, the wild type of DIII or DIIIb is derived from natural human serum albumin. Preferably, the natural human serum albumin comprises, 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.

7. The complex according to any one of claims 1-6, wherein the DIIIbV is selected from the amino acid sequences shown in SEQ ID NO: 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.

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

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

10. The complex according to any one of claims 1-9, wherein the kidney injury is selected from acute kidney injury and chronic kidney injury.

11. The complex according to any one of claims 1-10, wherein the functional molecule is selected from diagnostic reagents, imaging agents, therapeutic radionuclides, or macromolecular or small molecule drugs, such as polypeptides, proteins, antibodies, nanobodies, nucleic acid drugs, or chemotherapeutic drugs; preferably, the polypeptide or protein is a peptide chain or a cyclic peptide; preferably, the antibody is a monoclonal antibody or its antigen-binding fragment; and the nucleic acid drug is mRNA or a protein-nucleic acid complex. Preferably, the drug is 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.

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

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

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

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

16. A host cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15; Preferably, the cells are eukaryotic cells or prokaryotic cells; Preferably, the eukaryotic cells are yeast cells (e.g., Saccharomyces cerevisiae, Pichia pastoris) and HEK293F cells; Preferably, the prokaryotic cells are Escherichia coli cells, Bacillus subtilis cells, or any combination thereof.

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

18. Use of the complex of any one of claims 1-13, the delivery combination of claim 17, or the pharmaceutical composition in the preparation of a medicament for treating kidney injury; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury.

19. The complex according to any one of claims 1-13, the delivery combination or pharmaceutical composition according to claim 17, for treating kidney injury; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury.

20. 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; Preferably, the kidney injury is selected from acute kidney injury and chronic kidney injury.

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