Preparation method for radionuclide conjugate

Abstract

Provided is a preparation method for a radionuclide conjugate.

Description

A method for preparing a radionuclide conjugate TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, and in particular to a method for preparing a radionuclide conjugate. BACKGROUND

[0002] Globally, malignant tumors are one of the major malignant diseases that seriously endanger human health, and the incidence is still rising. Nuclear medicine has the potential and advantage of integration of diagnosis and treatment, and therefore plays an increasingly significant role in the diagnosis and treatment of cancer. According to the different functions, nuclear drugs can be divided into diagnostic and therapeutic nuclear drugs. The former mainly includes single photon (gamma ray) drugs and positron (beta + ray) drugs, which are used for single photon emission computed tomography (SPECT / CT) and positron emission tomography (PET / CT), respectively; the function and metabolic process of nuclear drugs in the human body can be studied at the molecular level to achieve rapid and real-time imaging. Therapeutic nuclear drugs are a class of drugs that release radioactivity (mainly alpha rays, beta rays and Auger electrons) through radionuclide decay for treatment. Among them, radionuclide drug conjugates (RDC) combine precise targeting and real-time sensitive imaging or strong killing, bringing more obvious clinical benefits to patients, and better achieving precise and true treatment, and are a form of nuclear drugs that have received widespread attention in recent period.

[0003] Nucleus conjugate drugs are usually composed of four parts: targeting ligand, linker, radionuclide and chelating agent. As a class of precise targeted drugs, the core of radionuclide conjugate drugs is the targeting carrier (i.e. targeting ligand). The advantages of small molecules and polypeptides as targeting carriers mainly include: small molecular weight, strong tissue and tumor penetration ability, rapid tumor enrichment, low immunogenicity, fast blood clearance, low blood toxicity and simple synthesis, etc., so currently the development of nuclear drugs mainly focuses on small molecules and polypeptides as targeting carriers. However, using small molecules and polypeptides as targeting carriers also faces some challenges, for example: 1) blood clearance is too fast, and it is discharged out of the body before being fully enriched in the tumor; 2) the residence time in the tumor is too short, and the therapeutic effect is not long-lasting; 3) it is difficult to obtain a small molecule or polypeptide with high affinity for any target, and the high-affinity molecules screened in vitro often have poor drug properties in in vivo tests; 4) most polypeptide-based RDC drugs are discharged through the kidney-bladder pathway, so there is a high risk of kidney uptake and retention.

[0004] Monoclonal antibodies are also widely concerned and researched in the field of radionuclide conjugate drugs due to their easy availability, high tumor specificity, long intratumoral residence time and high absolute uptake, etc.; for example, monoclonal antibody-based RDC drugs In 2002, it was approved for the treatment of relapsed refractory non-Hodgkin's lymphoma (NHL). However, due to the large molecular weight of the monoclonal antibody (usually 150 KDa), the slow penetration speed of the tissue and tumor, most of the monoclonal antibody entering the body is retained in the reticuloendothelial cells and liver cells, and only a small amount can bind to the target protein. In addition, due to the long blood circulation half-life of the monoclonal antibody (usually more than 1 week), it is also exposed in the blood circulation and normal tissues for a long time, so it will produce more blood toxicity and off-target toxicity. These problems are the main factors restricting the clinical application of monoclonal antibody RDC drugs. Monoclonal antibodies can be modified into various types of smaller antibody fragments, including, for example, antigen-binding fragments (Fab), single-chain variable fragments (scFv), nanobodies (Nb) or single-domain antibodies (sdAb), and protein scaffolds; due to the diversity of antibody fragment structure, the molecular size is between monoclonal antibody and small molecule and polypeptide, and the unique pharmacokinetic characteristics, it is also widely used in the development of targeted nuclear drugs, especially for in vivo imaging and disease diagnosis. However, due to their rapid clearance in the blood circulation, limited enrichment in tumors, and relatively short residence time in tumors, tumor therapy is difficult to achieve good efficacy. Although various strategies have been developed to address these issues with small antibody fragments, such as the introduction of PEG, polypeptides, Fc fragments, albumin, or the introduction of Fc, albumin binding fragments, etc., these strategies have certain limitations and cannot be used as a general means to solve various problems.

[0005] There is an urgent need in the art for new radionuclide conjugate drugs (RDC) that can solve the above problems. SUMMARY

[0006] The present application provides a conjugate comprising the following formula (I):

[0007] wherein,

[0008] Q is an albumin binding unit;

[0009] D and D' are each independently a chelating group of a radionuclide, and D and D' are each chelated with a radionuclide;

[0010] A is a single-domain antibody or a single-chain antibody, or an antigen-binding fragment thereof;

[0011] Ld is selected from a chemical bond or C 1-60 alkylene, wherein the alkylene is optionally interrupted by at least one substituent selected from -O-, -NH- and -(CO)-;

[0012] each L1, L2, L 1’ and L 2’each independently is a chemical bond, a polymeric segment of 1-10 amino acids, or is selected from one or a combination of the following divalent groups: C 1-10 alkylene, -NH-, and -(CO)-, wherein the alkylene is optionally substituted with at least one substituent selected from the group consisting of hydroxyl, halogen, amino, nitro, cyano, and C 1-10 alkyl;

[0013] m is an integer selected from 0-20;

[0014] n is an integer selected from 2-20;

[0015] z is an integer selected from 1-20.

[0016] In another aspect, the present application also provides a method for preparing a radionuclide conjugate comprising a targeting moiety and a payload unit covalently linked, the payload unit comprising an albumin binding unit, a linker, a radionuclide, and a chelator group; the method comprising the steps of: catalyzing the formation of a covalent linkage between the targeting moiety and a moiety comprising the linker by an immobilized ligase or a free ligase.

[0017] In some embodiments, the targeting moiety and the linker are catalyzed to form a covalent linkage by an immobilized ligase. In other embodiments, the targeting moiety and the payload unit are catalyzed to form a covalent linkage by an immobilized ligase.

[0018] In some embodiments, the method for preparing comprises the steps of:

[0019] the albumin binding unit, the linker, and the chelator group are covalently linked first, and then the conjugate is formed by an immobilized ligase with the targeting moiety, and the conjugate is chelated with the radionuclide to obtain the radionuclide conjugate (as shown in FIG. 15A); or

[0020] the albumin binding unit, the linker, and the chelator group are covalently linked first, and then the conjugate is formed by an immobilized ligase with the targeting moiety, and the conjugate is chelated with the radionuclide to obtain the radionuclide conjugate (as shown in FIG. 15A); or

[0021] In other embodiments, the method for preparing comprises the steps of:

[0022] the intermediate compound I comprising the covalently linked albumin binding unit, the linker, and the chelator group is formed by an immobilized ligase with the targeting moiety;

[0023] the conjugate is chelated with the radionuclide to obtain the radionuclide conjugate; or

[0024] The targeting moiety is covalently linked to the payload unit via an immobilized ligase.

[0025] In some embodiments, the ligase comprises a Sortase enzyme, a transglutaminase, a formylglycine-generating enzyme, a tyrosinase, or an asparagine ligase; preferably a Sortase enzyme; more preferably Sortase A or a functional variant thereof.

[0026] In some embodiments, the ligase is covalently linked to the Halo, and is immobilized on a support comprising a haloalkyl linker via the Halo, which is a dehalogenase or a variant thereof or a functionally active portion thereof that is truncated.

[0027] The present application also relates to a fusion protein comprising the ligase of the present application and the Halo.

[0028] In some embodiments, the fusion protein comprising the ligase and the Halo comprises the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto.

[0029] In some embodiments, the albumin binding unit is a small molecule.

[0030] In some embodiments, the antibody is selected from a single domain antibody or a single chain antibody. In some embodiments, the method of preparing the radionuclide conjugate comprises the steps of covalently linking an albumin binding unit, a linker, a chelator group, and forming a conjugate with a targeting moiety via an immobilized ligase, and chelating the radionuclide to the conjugate to obtain the radionuclide conjugate of formula (I); or

[0031] covalently linking an albumin binding unit, a linker, a chelator group, and chelating the radionuclide to form a payload unit, and linking the payload unit to a targeting moiety via an immobilized ligase to obtain the radionuclide conjugate of formula (I);

[0032] wherein,

[0033] Q is an albumin binding unit;

[0034] D and D’ are each independently a chelator group for a radionuclide, and D and D’ each chelate a radionuclide;

[0035] A is a single domain antibody or a single chain antibody, or an antigen binding fragment thereof;

[0036] Ld is selected from a chemical bond or C 1-60alkylene, wherein the alkylene is optionally interrupted by at least one substituent selected from the group consisting of -O-, -NH- and -(CO)-;

[0037] each L1, L2, L 1’ and L 2’ each independently is a chemical bond, a polymeric fragment of 1-10 amino acids, or is selected from one or a combination of the following divalent groups: C 1-10 alkylene, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, nitro, cyano and C 1-10 alkyl;

[0038] m is an integer selected from 0-20;

[0039] n is an integer selected from 2-20;

[0040] z is an integer selected from 1-20.

[0041] The radionuclide conjugate of the present application employs an enzyme site-directed conjugation targeting moiety and a linker, which not only reduces the influence of chemical reagents on the activity of the targeting moiety, but also ensures the high homogeneity of the RDC. The targeting moiety can employ a modular ligand, a polypeptide, a single-domain antibody or a nanobody, providing an RDC platform technology: the screened targeting molecule can be quickly assembled and prepared into an RDC by using the preparation method of the present application, which is conducive to the popularization of RDC industrialization. The present application also provides a one-step catalytic reaction to efficiently conjugate a support (containing a radionuclide) to a specific site of a targeting molecule (especially an antibody fragment such as VHH and scFV), avoiding the influence of harsh reaction conditions on the activity of the targeting moiety. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 shows the HIC-HPLC detection results of the conjugate Ab62-LP1.

[0043] Figure 2 shows the SEC-HPLC detection results of the conjugate Ab62-LP1.

[0044] Figure 3 shows the detection results of the binding force of the conjugate to cells.

[0045] Figure 4 shows the intracellular internalization detection results of the conjugate.

[0046] Figure 5 shows the radio-HPLC chemical purity detection results of the radionuclide conjugate 68 Ga-Ab62-LP1.

[0047] Figure 6 shows the radio-HPLC radioactivity purity detection results of the radionuclide conjugate 68 Ga-Ab62-LP1.

[0048] Figure 7 shows radionuclide conjugates prepared by the method of Example Twenty-Four Preparation One 177 Results of radio-HPLC chemical purity testing of Lu-Ab62-LP1.

[0049] Figure 8 shows radionuclide conjugates prepared by the method of Example Twenty-Four Preparation One 177 Results of radio-HPLC radioactivity purity testing of Lu-Ab62-LP1.

[0050] Figure 9 shows radionuclide conjugates prepared by the method of Example Twenty-Four Preparation Two 177 Results of radio-HPLC chemical purity testing of Lu-Ab62-LP1.

[0051] Figure 10 shows radionuclide conjugates prepared by the method of Example Twenty-Four Preparation Two 177 Results of radio-HPLC radioactivity purity testing of Lu-Ab62-LP1.

[0052] Figure 11 shows radionuclide conjugates 64 Results of radio-HPLC chemical purity testing of Cu-Ab62-LP2.

[0053] Figure 12 shows radionuclide conjugates 64 Results of radio-HPLC radioactivity purity testing of Cu-Ab62-LP2.

[0054] Figure 13 shows stability results for radionuclide conjugates.

[0055] Figure 14 shows cell uptake and internalization results for radionuclide conjugates. The four bar graphs of uptake and internalization from left to right correspond to 64 Cu-PSMA-617, 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2 and 64 Cu-Ab62-LP3.

[0056] Figure 15 shows methods of preparing radionuclide conjugates. Figure 15A shows a flowchart of the strategy 1 for preparing radionuclide conjugates; Figure 15B shows a flowchart of the strategy 2 for preparing radionuclide conjugates. DETAILED DESCRIPTION

[0057] General Definitions and Terms

[0058] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the chemical synthetic, molecular biology and other related terms and laboratory operation procedures used herein are those widely used in the corresponding fields. Meanwhile, in order to better understand the present application, the definitions and explanations of the related terms are provided as follows.

[0059] As used herein, "at least one" or "one or more" can mean 1, 2, 3, 4, 5, 6, 7, 8, or more.

[0060] As used herein, the expressions "comprise", "include", "contain", and "have" are open-ended, meaning including but not limited to the listed elements, steps, or components. The expression "consist of" excludes any element, step, or component not specified. The expression "consist essentially of" means limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. It is understood that the expressions "consist essentially of" and "consist of" are encompassed within the meaning of the expression "comprise".

[0061] As used herein, the joining term "and / or", between elements of a more than one recited element, is to be understood as including the individual options as well as combinations thereof. In other words, "and / or" includes "and" as well as "or". For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements defined with "and / or" are understood in a similar manner and include any of them and any combination thereof.

[0062] Unless otherwise indicated, any numerical values or numerical ranges, such as concentrations or concentration ranges, are to be understood as being modified in all instances by the term "about". Thus, a numerical value typically includes ±10% of the stated value. As used herein, use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers and fractions, unless the context clearly indicates otherwise.

[0063] The term "optional" means that the event subsequently described can, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0064] The term "alkyl" refers to a straight or branched chain, saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is attached to the rest of the molecule by a single bond. Alkyl groups can have 1-60 carbon atoms, for example, having 1-20 carbon atoms means "C1-C20 alkyl". Alkyl groups can be substituted or unsubstituted. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, hexyl, isohexyl, and the like. 20 The term "alkyl(C 1-20"alkyl" groups, e.g., C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or isomers thereof.

[0065] The term "heteroalkyl" denotes a stable straight or branched chain alkyl radical, or combinations thereof, consisting of a number of carbon atoms and at least one heteroatom. In some embodiments, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized.

[0066] The term "alkynyl" denotes a straight or branched chain hydrocarbon group containing one or more carbon-carbon triple bonds, which can be located in any position on the group.

[0067] The term "cycloalkyl" includes any stable cyclic alkyl radical, including monocyclic, bicyclic, or tricyclic ring systems, wherein the bicyclic and tricyclic ring systems include spiro, fused, and bridged ring systems. The term "heterocycloalkyl" denotes a "heteroalkyl" that is cyclic, including monocyclic, bicyclic, and tricyclic ring systems, wherein the bicyclic and tricyclic ring systems include spiro, fused, and bridged ring systems. In some embodiments, the heterocycloalkyl is a 3-10 membered heterocycloalkyl; in other embodiments, the heterocycloalkyl is a 5-6 membered heterocycloalkyl. Examples of heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, 1H-pyrrole-2,5-dione, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl, and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl, or oxepanyl.

[0068] The term "aryl" denotes a polyunsaturated, carbocyclic ring system, which can be a monocyclic, bicyclic, or polycyclic ring system, wherein at least one ring is aromatic, and each ring in the bicyclic and polycyclic ring systems is fused together.

[0069] The term "heteroaryl" refers to an aryl group containing 1, 2, 3, or 4 heteroatoms independently selected from B, N, O, and S, which can be a monocyclic, bicyclic, or tricyclic ring system. In some embodiments, the heteroaryl group is a 5-10 membered heteroaryl group. In other embodiments, the heteroaryl group is a 5-6 membered heteroaryl group. Examples of heteroaryl groups include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl, and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl, and 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl, and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl, and 5-thiazolyl, etc.), furanyl (including 2-furanyl and 3-furanyl, etc.), thiophenyl (including 2-thiophenyl and 3-thiophenyl, etc.), pyridyl (including 2-pyridyl, 3-pyridyl, and 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.), benzothiazolyl (including 5-benzothiazolyl, etc.), purinyl, benzimidazolyl (including 2-benzimidazolyl, etc.), indolyl (including 5-indolyl, etc.), isoquinolinyl (including 1-isoquinolinyl and 5-isoquinolinyl, etc.), quinoxalinyl (including 2-quinoxalinyl and 5-quinoxalinyl, etc.), quinolinyl (including 3-quinolinyl and 6-quinolinyl, etc.), pyrazinyl, purinyl, phenyloxazolyl. Substituents on any one of the above heteroaryl ring systems are selected from the acceptable substituents described herein.

[0070] A divalent radical refers to a radical obtained by removing one hydrogen atom from a carbon atom having a free valence electron of the corresponding monovalent radical. A divalent radical has two attachment sites to the remainder of the molecule. For example, "alkylene" or "alkylidene" refers to a saturated, straight or branched chain divalent hydrocarbon radical. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), pentylene (-C5H 10 -), hexylene (-C6H 12 -), 1-methylethylene (-CH(CH3)CH2-), 2-methylethylene (-CH2CH(CH3)-), methylpropylene or ethylpropylene, and the like. "Cycloalkylene" refers to a divalent cyclic hydrocarbon radical of a cycloalkyl group. "Heterocycloalkylene" refers to a divalent radical of a heterocycloalkyl group. "Arylene" refers to a divalent radical of an aryl group, for example, phenylene. "Heteroarylene" refers to a divalent radical of a heteroaryl group.

[0071] As used herein, the term "substituted" with reference to each carbon chain unit replaced by a substituent means that a -CH2- in the carbon chain backbone is replaced by a divalent substituent, for example, -CH2-CH2-CH2-CH2- can be replaced by -O- to -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, or -O-CH2-O-CH2-.

[0072] As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope through at least one antigen binding site. Antibodies encompass antibody fragments. As used herein, the term "antibody" includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, single domain antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single chain Fv (scFv), single chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antibodies to any of the above. Antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass (e.g., IgG2a and IgG2b). In preferred embodiments, the antibodies of the application are single domain antibodies.

[0073] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which at least contains a portion of the variable region of the antibody (e.g., one or more CDRs and / or one or more antigen binding sites) that binds an antigen, and thus retains the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments can include multiple chains linked together, e.g., by disulfide bonds and / or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by substitution of the corresponding region), results in an antibody that immunospecifically binds an antigen.

[0074] As used herein, an "immunoglobulin single variable domain" or "single variable domain" refers to a single variable region (variable domain) having antigen-binding activity. Unlike the functional antigen-binding unit in a conventional antibody, which is composed of a pair of VH and VL, a single variable domain can form a functional antigen-binding unit by itself. Single variable domains can be derived from naturally occurring heavy chain antibodies, such as the variable domain of heavy chain of heavy-chain antibody (VHH) of camelids (e.g., camels and llamas) and the single variable domain of new antigen receptor (IgNAR variable single-domain, VNAR) of sharks, and can also be screened from full-length antibodies, such as light chain variable domains and heavy chain variable domains having antigen-binding activity in human antibodies. A VHH can generally comprise three highly variable "complementarity determining regions" (CDRs) and four relatively conserved "framework regions" (FRs), and is connected in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus.

[0075] As used herein, a "single domain antibody (sdAb)" or "nanobody" refers to an antibody comprising a single immunoglobulin variable domain (single variable domain) as a functional antigen binding fragment. Similar to the variable region of a full-length antibody, a single variable domain typically comprises CDR1, CDR2, and CDR3 forming an antigen binding site and a supporting framework region. Unlike a full-length antibody, which typically comprises two heavy chains and two light chains, a single domain antibody typically comprises a single peptide chain consisting of a single variable domain, with a molecular weight of only about 15 kDa. The single variable domain can be, for example, a variable domain of heavy-chain antibody (VHH) of a llama, an IgNAR variable domain of a shark, or a human light chain antibody variable domain.

[0076] As used herein, the term "single chain antibody", "single chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (V H ) and an antibody light chain variable domain (V L ) connected by a linker. Such scFv molecules can have the general structure NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0077] The amino acid sequences of the CDRs in the present application are shown according to the Kabat definition. However, it is well known in the art that CDRs of an antibody can be defined by various methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (see, e.g., Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (see, e.g., Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, A. C. R. and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95-118), Contact (MacCallum, R. M. et al., (1996) J. Mol. Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003)), and North CDR definition based on affinity propagation clustering using a large number of crystal structures. In this context, multiple CDR numbering systems can be used for the same variable region, such as Chothia, Abm, Kabat, Contact, and IMGT. It is understood by one skilled in the art that although the CDRs defined by different numbering systems can be different, the CDRs corresponding to the same numbering system represent the effective antigen binding sites capable of binding to the epitope of the antigen.Unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., a variable region) shall be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described herein. While the scope of the claims of the present application is based on the sequences shown according to the Kabat definition rules, the amino acid sequences corresponding to the other CDR definition rules shall also fall within the scope of the present application.

[0078] Thus, in connection with an antibody defined with respect to specific CDR sequences as defined herein, the scope of the antibody also encompasses antibodies whose variable region sequences comprise the specific CDR sequences recited, but whose recited CDR boundaries differ from the specific CDR boundaries defined herein due to application of a different scheme (e.g., different assignment system rules or combinations).

[0079] As used herein, the terms "framework region" and "complementarity determining region" can be used interchangeably. As used herein, the term "framework region," "complementarity determining region," or "FR" residues refer to those amino acid residues in a variable region of an antibody other than the CDR sequences as defined above.

[0080] The term "disulfide bond" as used herein includes a covalent bond formed between two sulfur atoms. Amino acid cysteines contain a thiol group that can form a disulfide bond or bridge with a second thiol group.

[0081] As used herein, "percent (%) sequence identity" of an amino acid sequence has the art-recognized definition that refers to the percentage of identical amino acids between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a publicly available algorithm). It can be determined using methods known to one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0082] Non-essential regions of a polypeptide can be modified, e.g., by substitution, addition and / or deletion of one or more amino acids, without changing the function of the polypeptide. Suitable conservative amino acid substitutions in a peptide or protein are known to one of skill in the art and can generally be made without changing the biological activity of the resulting molecule. In general, one of skill in the art recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. co., p. 224).

[0083] “Spacer” or “SP” refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a certain function or not, nor whether it can be cleaved or degraded in vivo or not. Examples of a spacer include, but are not limited to, amino acid and non-amino acid structures, which can be, but are not limited to, amino acid derivatives or analogs. “Spacer sequence” refers to an amino acid sequence that serves as a spacer, examples of which include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, for example, a sequence containing two amino acids, such as GA, or for example, GGGGS (SEQ ID NO: 14), GGGGSGGGGS (SEQ ID NO: 15), GGGGSGGGGSGGGGS (SEQ ID NO: 16), and the like.

[0084] As used herein, the term “amino acid” includes “natural amino acids” and “non-natural amino acids.”

[0085] The term “natural amino acid” refers to an amino acid that is a proteinogenic amino acid, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as the less common selenocysteine and pyrrolysine.

[0086] As used herein, the term “non-natural amino acid” refers to an amino acid that is not a proteinogenic amino acid. In particular, the term refers to an amino acid that is not a natural amino acid as defined above.

[0087] “Affinity” or “binding affinity” is used to measure the strength of the interaction between an antibody and an antigen by non-covalent forces. Affinity can be determined using routine techniques known in the art, such as bio-layer interferometry (which can employ, for example, the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance (SPR), enzyme-linked immunoassay (ELISA), or flow cytometry (FACS), among others. Binding affinity is typically measured by the equilibrium dissociation constant (KD), which is the ratio of the “off-rate” (koff) and the “on-rate” (kon) and is used to assess and rank the strength of a bimolecular interaction. The “on-rate” (Kon) characterizes the rate at which a ligand binds to its target, and the “off-rate” (Koff) characterizes the rate at which a ligand dissociates from its target. KD (Koff / Kon) and binding affinity are inversely proportional.

[0088] "Specifically binds" generally means that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, binds an epitope through its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. Under this definition, a binding molecule is said to "specifically bind" an epitope when it binds to that epitope through its antigen binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualitatively describe the relative avidity of an antibody to bind to an epitope. For example, a binding molecule "A" can be said to have a higher specificity for a given epitope than a binding molecule "B," or a binding molecule "A" can be said to bind to an epitope "C" with a higher specificity than it binds to a related epitope "D."

[0089] A binding molecule, e.g., an antibody or fragment, variant, or derivative thereof, can be said to competitively inhibit the binding of a reference antibody or antigen binding fragment to a given epitope if it preferentially binds that epitope to the extent that it blocks the binding of the reference antibody or antigen binding fragment to the epitope to some extent. Competitive inhibition can be determined by any method known in the art, e.g., a competition ELISA assay. A binding molecule can be said to competitively inhibit at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the binding of a reference antibody or antigen binding fragment to a given epitope.

[0090] The terms, e.g., "treat" or "treatment" or "to treat" or "to alleviate" or "alleviate" refer to therapeutic measures that cure, slow down, lessen the symptoms of, and / or halt the progression of an existing diagnosed pathologic condition or disorder. The terms, e.g., "prevent," "prevention," "avoid," "arrest," and the like refer to prophylactic or preventative measures that prevent the progression of an undiagnosed target pathologic condition or disorder. Accordingly, a "subject in need" can include a subject already suffering from a disease; a subject predisposed to a disease; and a subject in need of prophylaxis against a disease.

[0091] As used herein, "efficacy" means an effect resulting from the treatment of an individual that alters, usually improves or ameliorates, symptoms of a disease or disease state, or cures the disease or disease state.

[0092] As used herein, the term "effective amount" refers to the amount of an antibody, polypeptide, polynucleotide, small organic molecule, or other agent that is effective for "treating," "preventing," or "alleviating" a disease or condition in a subject or mammal. In the context of cancer, an effective amount of an agent can reduce the number of cancer cells; block or reduce tumor invasion; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse, cancer cell infiltration into peripheral organs including, e.g., the spread of cancer into soft tissue and bone; inhibit, e.g., suppress, retard, prevent, contract, stop, delay, or reverse, tumor metastasis; inhibit, e.g., suppress, retard, prevent, stop, delay, or reverse, tumor growth; relieve to some extent one or more of the symptoms associated with the cancer, reduce morbidity and mortality, improve quality of life, or a combination of these effects. It can refer to an agent that inhibits cell growth and / or is cytotoxic to the extent that it prevents growth and / or kills existing cancer cells.

[0093] As used herein, the term "subject," "patient," or "individual" generally includes humans and non-human animals, and preferably includes mammals (e.g., non-human primates, including marmosets, tamarins, spider monkeys, owl monkeys, black-and-white langurs, squirrel monkeys, and baboons, macaques, chimpanzees, orangutans, gorillas; cows; horses; sheep; pigs; chickens; cats; dogs; mice; rats; rabbits; guinea pigs; and the like), including chimeric and transgenic animals and disease models. The term "subject" preferably refers to a non-human primate or a human, most preferably a human.

[0094] As used herein, the term "radionuclide" (or "radioisotope") refers to an isotope of natural or artificial origin that has an unstable combination of neutrons and protons, which disintegrates with the emission of a particle (i.e., a proton (alpha radiation) or an electron (beta radiation) or electromagnetic radiation (gamma radiation). The radionuclide can preferably be used for cancer imaging or therapy.

[0095] The antibody numbers used herein (e.g., Ab60, Ab61, Ab62, and Ab63, etc.) are used only for the purpose of distinguishing or identifying antibodies or products, and are not intended to indicate that such identification is a characteristic of the antibodies or products of the present application. Those skilled in the art will understand that other antibodies or products can also use such identification for the purpose of distinguishing or identifying, but are not intended to refer to the same or equivalent antibodies or products. Similarly, the similar numbers or identifications used in the examples are only for the convenience of illustration, and the antibodies or products of the present application are defined by the characteristics described in the appended claims.

[0096] Conjugates of the present application

[0097] In one aspect, the present application relates to a conjugate comprising a targeting moiety A and a payload unit covalently linked, said targeting moiety A and said payload unit are covalently linked by a ligase to form a linker; said payload unit comprises an albumin binding unit (Q) and a chelating group of a radionuclide (D and / or D’).

[0098] In some embodiments, said albumin binding unit is a small molecule.

[0099] In some embodiments, said chelating group chelates a radionuclide.

[0100] In some embodiments, said ligase is a formylglycine-generating enzyme, a transglutaminase, a tyrosinase or a Sortase enzyme. In some embodiments, said ligase is a

[0101] In some embodiments, Sortase is sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE) or sortase F (SrtF), but not limited to. In other embodiments, Sortase also includes functional variants of Sortase, such as functional variants of sortase A (SrtA), functional variants of sortase B (SrtB), functional variants of sortase C (SrtC), functional variants of sortase D (SrtD), functional variants of sortase E (SrtE) or functional variants of sortase F (SrtF).

[0102] A “sortase” or “sortase enzyme” herein refers to an enzyme having sortase activity to catalyze a transpeptidation reaction, including, for example, Class A, Class B, Class C, Class D, Class E, and Class F sortases of the sortase enzyme superfamily (see, e.g., Dramsi, et al., Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria, Research in Microbiology, (2005), 156: 289-297; Bradshaw, et al., Molecular features of the sortase enzyme family, FEBS Journal, (2015), 282: 2097-2114; Malik and Kim, A comprehensive in silico analysis of sortase superfamily, J Microbiol., (2019), 57(6):431-443; and EP3647419A1), but is not limited thereto. Such an enzyme can be referred to as SrtA, SrtB, SrtC, SrtD, SrtE, or SrtF, but is not limited thereto. A sortase can be naturally occurring or engineered. A naturally occurring sortase can be found in a variety of Gram-positive bacteria, such as any strain, species, or subspecies of the following genera: Streptococcus (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Staphylococcus (e.g., Staphylococcus argenteus and Staphylococcus aureus), Bacillus (e.g., Bacillus anthracis), and Listeria (e.g., Listeria monocytogenes), but is not limited thereto. Engineered sortases, such as sortase variants having one or more amino acid residue substitutions, deletions, or insertions, can be obtained from their naturally occurring counterparts by methods known in the art, such as protein engineering and chemical synthesis. Other variants of any wild-type sortase known in the art (such as those having one or more active groups or labels) are also contemplated.Provided that the variant has the same or similar function as the wild-type sortase. One skilled in the art will be able to readily identify a sortase and classify it into a particular class according to its sequence and other characteristics. However, the definition of sortase is not limited to any classification method or nomenclature system.

[0103] In some embodiments, the targeting moiety is selected from a ligand, a polypeptide, an antibody or an antigen binding fragment thereof.

[0104] In some embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof; preferably, the antibody is selected from a single domain antibody or a single chain antibody. In some embodiments, the targeting moiety is a polypeptide. In some embodiments, the polypeptide is a cyclic peptide. In some embodiments, the targeting moiety comprises a polypeptide and a covalently bound molecular scaffold.

[0105] The present application also relates to a conjugate comprising the structure of formula (I):

[0106] wherein,

[0107] Q is an albumin binding unit;

[0108] D and D' are each independently a chelating group for a radionuclide, both D and D' chelating a radionuclide;

[0109] A is a targeting moiety comprising an antibody or an antigen binding fragment thereof; preferably, the antibody is selected from a single domain antibody or a single chain antibody;

[0110] Ld is selected from a chemical bond or C 1-60 alkylene, wherein the alkylene is optionally interrupted by at least one substituent selected from -0-, -NH- and -(CO)-;

[0111] each L1, L2, L 1’ and L 2’ are each independently a chemical bond, a polymeric fragment of 1-10 amino acids, or selected from one or a combination of the following divalent groups: C 1-10 alkylene, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one substituent selected from hydroxyl, halogen, amino, nitro, cyano and C 1-10 alkyl;

[0112] m is an integer selected from 0-20;

[0113] n is an integer selected from 2-20;

[0114] z is an integer selected from 1-20.

[0115] The present application can use ligase dependent conjugation (LDC) to couple A with the rest of the compound of formula (I) using a ligase. Here, the ligase refers to a sortase, including but not limited to various natural Sortase enzymes (including Sortases of A, B, C, D, L. plantarum, etc., see patents US20110321183A and WO2022160156A) and various novel sortases that have been preferably modified. The coupling reaction is achieved by means of biological enzyme catalysis, the reaction conditions are mild, which reduces the physical and chemical damage of the antibody in the coupling process, the preparation process and flow are more optimized, easy to industrialize and upgrade, and conducive to the quality control of the coupling product.

[0116] In some embodiments, the A terminus is coupled to the (Gly) n moiety in formula (I) under the action of a ligase through modification.

[0117] In some embodiments, the ligase is a Sortase enzyme. In some embodiments, A comprises an antibody with a C-terminal modification or an N-terminal modification. In some embodiments, the antibody, the spacer (SP), and the ligase donor substrate recognition sequence are sequentially linked. In some embodiments, the antibody and the ligase donor substrate recognition sequence are sequentially linked.

[0118] In some embodiments, the spacer is selected from GA, GGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGS; preferably, the spacer is GA.

[0119] In some embodiments, the ligase donor substrate recognition sequence is LPX1TGX2(SEQ ID NO: 17), wherein X1is any natural or unnatural amino acid, and X2is absent or an amino acid fragment comprising 1-10 amino acids. In particular, the ligase donor substrate recognition sequence is LPETGG (SEQ ID NO: 18).

[0120] In some embodiments, the antibody A is an anti-prostate specific membrane antigen (PSMA) antibody, an anti-epidermal growth factor receptor 2 (HER2) antibody, or an anti-Delta-like ligand 3 (DLL3) antibody; preferably, the antibody A is an anti-PSMA single domain antibody.

[0121] In some embodiments, the antibody A comprises a HCDR1 as shown in SEQ ID NO: 1, a HCDR2 as shown in SEQ ID NO: 2, and a HCDR3 as shown in SEQ ID NO: 3.

[0122] In some embodiments, antibody A comprises a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 5, and a HCDR3 as set forth in SEQ ID NO: 6.

[0123] In some embodiments, antibody A comprises a HCDR1 as set forth in SEQ ID NO: 7, a HCDR2 as set forth in SEQ ID NO: 8, and a HCDR3 as set forth in SEQ ID NO: 9.

[0124] In some embodiments, antibody A comprises a HCDR1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24, and a HCDR3 as set forth in SEQ ID NO: 25.

[0125] In some embodiments, antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or comprises an amino acid sequence as set forth in positions 1 to 127 of SEQ ID NO: 12, or comprises an amino acid sequence as set forth in positions 1 to 115 of SEQ ID NO: 22. In some embodiments, antibody A comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, antibody A comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in positions 1 to 127 of SEQ ID NO: 12. In other embodiments, antibody A comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in positions 1 to 115 of SEQ ID NO: 22. In some embodiments, antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, antibody A comprises an amino acid sequence as set forth in positions 1 to 127 of SEQ ID NO: 12. In other embodiments, antibody A comprises an amino acid sequence as set forth in positions 1 to 115 of SEQ ID NO: 22.

[0126] In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, when antibody A is attached to a Gly in formula (I), its C-terminal amino acid sequence GGHHHHHH (SEQ ID NO: 19) is cleaved off by Sortase enzyme. In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 12 from position 1 to position 133.

[0127] In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, when antibody A is attached to a Gly in formula (I), its C-terminal amino acid sequence GG is cleaved off by Sortase enzyme. In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 13 from position 1 to position 141.

[0128] In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 22. In some embodiments, when antibody A is attached to a Gly in formula (I), its C-terminal amino acid sequence GGHHHHHH is cleaved off by Sortase enzyme. In some embodiments, the modified antibody A comprises an amino acid sequence as set forth in SEQ ID NO: 22 from position 1 to position 121.

[0129] In some embodiments, Ld is selected from a chemical bond, -NH-C 1-20 alkylene-(CO)-, or -NH-(PEG) i -(CO)-, said (PEG) i comprising 1-20 structural units selected from -(O-C2H4)- or -(C2H4-O)-, and optionally attached to at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene. In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0130] In some embodiments, Ld is -NH-(PEG) i -(CO)-, said (PEG) i comprising 1-20 structural units selected from -(O-C2H4)- or -(C2H4-O)-, and optionally attached to at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene; preferably, Ld is -NH-(PEG) i -C 1-10alkylene-(CO)-; more preferably, Ld is -NH-PEG4-C2H4-(CO)-, -NH-PEG3-C2H4-(CO)-, or -NH-PEG4-C3H6-(CO)-; further preferably, Ld is -NH-(C2H4-O)4-C2H4-(CO)-. In some embodiments, Ld is -NH-(PEG) i alkylene-(CO)-, wherein i is an integer selected from 1-12, preferably, i is 2, 3, 4, 5, or 6; more preferably, i is 4. In some embodiments, Ld is -NH-(PEG) 1-10 alkylene-(CO)-, wherein i is an integer selected from 1-12, preferably, i is 2, 3, 4, 5, or 6; more preferably, i is 4. In some embodiments, Ld is -NH-(PEG) i -(CO)-, wherein i is an integer selected from 1-12, preferably, i is 2, 3, 4, 5, or 6; more preferably, i is 4. In some embodiments, Ld is -NH-PEG4-(CO)-.

[0131] In some embodiments, L1and L 1’ are each independently selected from any one of a bond, C 1-10 alkylene, -NH-, and -(CO)-, or any combination thereof; preferably, L1is selected from -(CH2)4-NH-, -CO-NH-C2H4-NH-, or -NH-, L 1’ is selected from a bond, -(CH2)4-NH-, -CO-NH-C2H4-NH-, or -NH-. In some embodiments, L1and L 1’ are the same. In some embodiments, L2and L 2’ are each independently selected from any one of a bond, a polymeric segment of 1-10 amino acids, -(CO)-, C 1-10 alkylene, and -NH-, or any combination thereof; preferably, L2is selected from -(CO)-, -(CH2)4-NH-, -CO-polymeric segment of 1-10 amino acids-, or -CO-Lys-, L 2’ is selected from a bond, -(CO)-, -(CH2)4-NH-, -CO-polymeric segment of 1-10 amino acids-, or -CO-Lys-. In some embodiments, L2and L 2’ are the same.

[0132] In some embodiments, m is an integer selected from 0-10; preferably, m is 0, 1, or 2; more preferably, m is 0 or 1. In some embodiments, n is an integer selected from 2-10, preferably, n is 2, 3, or 4; more preferably, n is 3. In some embodiments, z is an integer selected from 1-10, preferably, z is 1, 2, 3, or 4; more preferably, z is 1.

[0133] In some embodiments, D and D’ are each independently selected from the group consisting of bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)- methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5- aminopentyl-(hydroxy)amino]-4-oxobutyryl]amino]pentyl]-N-hydroxyoxal diamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), a-(2- carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10- azacyclododecane-N,N',N",N"'-1,4,7,10-tetramethylene phosphonic acid (DOTMP), N,N'-dipyridooxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphonate) (DPDP), diethylenetriamine N,N',N"-pentamethylene phosphonic acid (DTMP), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra 3-hydroxy-N-methyl-2-pyridinone chelator abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2- (bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridyl-bis(methylene amine tetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1-yl]methyl-hydroxy-phosphoryl]propanoic acid, and triethylenetetraminehexaacetic acid (TTHA); preferably, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid; D' is 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid.

[0134] In some embodiments, the radionuclide is selected from any one of the radioactive cations or anions of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At and Er. Preferably, the radionuclide is selected from 18 F, 77 Br, 131 I, 125 I, 43 Sc, 44 Sc, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 90 In, 111 In, 177 Lu, 94 Tc, 99 Tc, 153 Sm, 89 Sr, 223 Ra, 151 Tb, 166 Ho, 186 Re, 188 Re, 212 Pb, 213 Bi, 212 Bi, 225 Ac, 227 Th, 55 Co, 57 Co, 152 Gd, 153 Gd,157 Gd, 166 Dy, 89 Zr or 211 At; preferably 68 Ga, 64 Cu or 177 Lu.

[0135] In some embodiments, Q is selected from

[0136] the wavy line indicates the site of attachment to L2in formula (I);

[0137] wherein,

[0138] R 1 is selected from H, C 1-6 alkyl, halogen, methoxy, trifluoromethyl; preferably, halogen is fluorine, chlorine, bromine or iodine; preferably, R 1 is selected from methyl or iodine;

[0139] R a1 to R a11 are each independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino or hydroxy; preferably, halogen is fluorine, chlorine, bromine or iodine; preferably, Q is selected from

[0140] In some embodiments, m is 0 and the conjugate of formula (I) comprises the structure shown in formula (I’):

[0141] wherein the groups are as defined for formula (I) of the present application.

[0142] In a particular embodiment, m is 0, n is 3, z is 1, Ld is -NH-(C2H4-O)4-C2H4-(CO)-, Li is -(CH2)4-NH-, L2 is -(CO)-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid when not chelating a radionuclide, and Q is In a particular embodiment, the compound of formula (I’) comprises the following structure:

[0143] In a particular embodiment, m is 1, n is 3, z is 1, Ld is -NH-(C2H4-O)4-C2H4-(CO)-, Li and L 1’ are -(CH2)4-NH-, L2 and L 2’is -(CO)-, D and D' are 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a particular embodiment, the compound of formula (I) comprises the following structure:

[0144] In a particular embodiment, m is 0, n is 3, z is 1, Ld is -NH-(C2H4-0)4-C2H4-(CO)-, L1 is -CO-NH-C2H4-NH-, L2 is -(CH2)4-NH-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a particular embodiment, the compound of formula (I) comprises the following structure:

[0145] In a particular embodiment, m is 0, n is 3, z is 1, Ld is -NH-(C2H4-0)4-C2H4-(CO)-, L1 is -CO-NH-C2H4-NH-, L2 is -(CH2)4-NH-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a particular embodiment, the compound of formula (I) comprises the following structure:

[0146] Another aspect of the present application provides a compound comprising the following structure of formula (II):

[0147] wherein,

[0148] Q is an albumin binding unit;

[0149] D and D' are each independently a chelating group for a radionuclide, both D and D' chelate a radionuclide;

[0150] Ld is selected from the group consisting of a chemical bond or C 1-60 alkylene, wherein said alkylene is optionally interrupted by a substituent selected from the group consisting of -0-, -NH- and -(CO)-;

[0151] L1, L2, each L 1’ and L 2’ are each independently a chemical bond, a polymeric fragment of 1-10 amino acids, or one or a combination of the following divalent groups: C 1-10 alkylene, -NH- and -(CO)-, wherein said alkylene is optionally interrupted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, nitro, cyano and C 1-10substituted by substituents of alkyl;

[0152] m is an integer selected from 0 to 20;

[0153] n is an integer selected from 2 to 20.

[0154] In some embodiments, Ld is selected from a chemical bond, -NH-C 1-20 alkylene-(CO)-, or -NH-(PEG) i -(CO)-, said (PEG) i comprising 1 to 20 structural units selected from -(O-C2H4)- or -(C2H4-O)-, and optionally attached at at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene. In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0155] In some embodiments, Ld is -NH-(PEG) i -(CO)-, said (PEG) i 1 to 20 consecutive -(O-C2H4)- or -(C2H4-O)- structural units, and optionally attached at at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene; preferably, Ld is -NH-(PEG) i -C 1-10 alkylene-(CO)-; more preferably, Ld is -NH-PEG4-C2H4-(CO)-, -NH-PEG3-C2H4-(CO)-, or -NH-PEG4-C3H6-(CO)-; further preferably, Ld is -NH-(C2H4-O)4-C2H4-(CO)-. In some embodiments, Ld is -NH-(PEG) i -C 1-10 alkylene-(CO)-, wherein i is an integer selected from 1 to 12, preferably i is 2, 3, 4, 5, or 6; more preferably, i is 4. In some embodiments, Ld is -NH-PEG4-(CO)-.

[0156] In some embodiments, L1 and L 1’ are each independently selected from any one or any combination of a chemical bond, C 1-10 alkylene, -NH-, and -(CO)-; preferably, L1 is selected from -(CH2)4-NH-, -CO-NH-C2H4-NH-, or -NH-, L 1’is selected from the group consisting of a chemical bond, -(CH2)4-NH-, -CO-NH-C2H4-NH-, or -NH-. In some embodiments, L1and L 1’ are the same.

[0157] In some embodiments, L2and L 2’ are each independently selected from the group consisting of a chemical bond, a polymeric fragment of 1-10 amino acids, -(CO)-, C 1-10 alkylene, and -NH-; preferably, L2is selected from -(CO)-, -(CH2)4-NH-, -CO-polymeric fragment of 1-10 amino acids-, or -CO-Lys-, L 2’ is selected from the group consisting of a chemical bond, -(CO)-, -(CH2)4-NH-, -CO-polymeric fragment of 1-10 amino acids-, or -CO-Lys-. In some embodiments, L1and L 1’ are the same.

[0158] In some embodiments, m is an integer selected from 0-10; preferably, m is 0, 1, or 2; more preferably, m is 0 or 1. In some embodiments, n is an integer selected from 2-10, preferably, n is 2, 3, or 4; more preferably, n is 3. In some embodiments, z is an integer selected from 1-10, preferably, z is 1, 2, 3, or 4; more preferably, z is 1.

[0159] In some embodiments, D and D’ are each independently selected from the group consisting of bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)- methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5- aminopentyl-(hydroxy)amino]-4-oxobutyryl]amino]pentyl]-N-hydroxyoxal diamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), a-(2- carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10- azacyclododecane-N,N',N",N"'-1,4,7,10-tetramethylene phosphonic acid (DOTMP), N,N'-dipyridooxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphonate) (DPDP), diethylenetriamine N,N',N"-pentamethylene phosphonic acid (DTMP), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra 3-hydroxy-N-methyl-2-pyridinone chelator abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2- (bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,11-Tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), Tris(hydroxypyridinone) (THP), Terpyridyl-bis(methylene amine tetraacetic acid (TMT), 1,4,7-Triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-Tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1-yl]methyl-hydroxy-phosphoryl]propanoic acid, and triethylenetetraminehexaacetic acid (TTHA); preferably, D is 1,4,7,10-Tetraazacyclododecane-N,N',N",N"'-tetraacetic acid; D' is 1,4,7,10-Tetraazacyclododecane-N,N',N",N"'-tetraacetic acid.

[0160] In some embodiments, the radionuclide is selected from any one of the radioactive cations or anions of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At and Er; preferably, the radionuclide is selected from 18 F, 77 Br, 131 I, 125 I, 43 Sc, 44 Sc, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 90 In, 111 In, 177 Lu, 94 Tc, 99 Tc, 153 Sm, 89 Sr, 223 Ra, 151 Tb, 166 Ho, 186 Re, 188 Re, 212 Pb, 213 Bi, 212 Bi, 225 Ac, 227 Th, 55 Co, 57 Co, 152 Gd, 153 Gd,157 Gd, 166 Dy, 89 Zr or 211 At; preferably 68 Ga, 64 Cu or 177 Lu.

[0161] In some embodiments, Q is selected from

[0162] the wavy line indicates the site of attachment to L2in formula (II);

[0163] wherein,

[0164] R 1 is selected from H, C 1-6 alkyl, halogen, methoxy, trifluoromethyl; preferably, halogen is fluorine, chlorine, bromine or iodine; preferably, R 1 is selected from methyl or iodine;

[0165] R a1 to R a11 are each independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino or hydroxy; preferably, halogen is fluorine, chlorine, bromine or iodine;

[0166] Preferably, Q is selected from

[0167] In some embodiments, m is 0 and the compound of formula (II) comprises the structure shown in formula (II'):

[0168] wherein the groups are as defined for formula (II) of the present application.

[0169] In a particular embodiment, m is 0, n is 3, Ldis -NH-(C2H4-0)4-C2H4-(CO)-, Li is -(CH2)4-NH-, L2is -(CO)-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, and Q is In a particular embodiment, the compound of formula (II') comprises the following structure:

[0170] In a particular embodiment, m is 1, n is 3, Ldis -NH-(C2H4-0)-C2H4-(CO)-, Li and L 1’ are -(CH2)4-NH-, L2and L 2’is -(CO)-, D and D' are 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a specific embodiment, the compound of formula (II') comprises the following structure:

[0171] In a specific embodiment, m is 0, n is 3, Ld is -NH-(C2H4-O)4-C2H4-(CO)-, L1 is -CO-NH-C2H4-NH-, L2 is -(CH2)4-NH-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a specific embodiment, the compound of formula (II') comprises the following structure:

[0172] In a specific embodiment, m is 0, n is 3, Ld is -NH-(C2H4-O)4-C2H4-(CO)-, L1 is -CO-NH-C2H4-NH-, L2 is -(CH2)4-NH-, D is 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid when not chelating a radionuclide, Q is In a specific embodiment, the compound of formula (II') comprises the following structure:

[0173] Another aspect of the present application provides a compound comprising the following structure of formula (III):

[0174] wherein,

[0175] is a targeting moiety, the rest is a payload unit, wherein the targeting moiety and the payload unit form a covalent bond by way of an enzymatic coupling;

[0176] each Q is independently an albumin binding unit;

[0177] each D is independently a chelating group chelating a radionuclide;

[0178] L a is a coupling unit linking the targeting moiety and G, each L a is independently selected from 1), 2) or a combination thereof:

[0179] 1) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids having a degree of polymerization of 2-20;

[0180] 2) a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -S-, -NH-, -(CO)-, C 2-6 alkynyl, C 3-10 cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 arylene and 5-10 membered heteroarylene, wherein the alkylene, alkynyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene are optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl and 3-10 membered heterocycloalkyl;

[0181] each L b and each L c is, when occurring, independently a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -(CO)-, -NH-, -(C=S)-, C 6-10 arylene and 5-10 membered heteroarylene, wherein the alkylene, arylene and heteroarylene are optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1- 10 alkyl and 3-10 membered heterocycloalkyl;

[0182] G is a branching moiety having a branching function, directly or indirectly connected to Q and D; wherein each G is independently selected from 3), 4) or a combination thereof of the following:

[0183] 3) one or more natural or unnatural amino acid or an oligomer of natural or unnatural amino acids having a degree of polymerization of 2-20;

[0184] 4) a chemical bond or C 1-60 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl and 3-10 membered heterocycloalkyl;

[0185] j is an integer selected from 1-30;

[0186] k is an integer selected from 1-20;

[0187] o is an integer or non-integer greater than 0 and less than 20.

[0188] In some embodiments, the radionuclide conjugate comprises the following structure of Formula (IV):

[0189] wherein,

[0190] is a targeting moiety, and the rest is a payload unit, wherein the targeting moiety and the payload unit form a covalent bond by way of an enzymatic conjugation;

[0191] each Q is independently an albumin binding unit;

[0192] each D is independently a chelating group chelating a radionuclide;

[0193] L a is a conjugation unit linking the targeting moiety and G, each L a is independently selected from 1), 2), or a combination thereof:

[0194] 1) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids having a degree of polymerization of 2-20;

[0195] 2) a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -S-, -NH-, -(CO)-, C 2-6 alkynyl, C 3-10 cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 arylene, and 5-10 membered heteroarylene, wherein the alkylene, alkynyl, cycloalkylene, heterocycloalkylene, arylene, and heteroarylene are optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, amido, sulfonyl-C 1-10 alkyl, and 3-10 membered heterocycloalkyl;

[0196] each L b and each L c is independently a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -(CO)-, -NH-, -(C=S)-, C 6-10substituents of alkylene, arylene and heteroarylene are optionally substituted with at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C1-10alkyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1- 10 alkyl;

[0197] each G 1 or G 3 is independently selected from a bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced with at least one substituent selected from the group consisting of -0-, -NH- and -(CO)-, wherein the alkylene is optionally substituted with at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C1-10alkyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl; preferably, each G 1 or G 3 is independently selected from a bond, optionally substituted -NH-(C 1-10 alkylene)-CO-, optionally substituted -NH-PEG-CO-, optionally substituted -NH-PEG-(C 1-10 alkylene)-CO-, optionally substituted -NH-(C 1-10 alkylene)-PEG-CO-; the substituents of the substitutions are selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C1-10alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl-; the PEG is -(CH2CH20) x - or -(OCH2CH2) y -, x or y is an integer from 1 to 20;

[0198] each G 2 or G 4 is independently a branching unit; preferably, it is selected from one or more combinations of the following groups: 1) one or more branched natural or unnatural amino acid fragments; preferably, the branched natural or unnatural amino acid fragments have the following structure: -NH-(CR 2 R 3 )-CO-, wherein, R 2 and R 3 are each independently selected from hydrogen, optionally substituted -(C 1-10 alkylene)-NH-, optionally substituted -(C 1-10 alkylene)-CO-; wherein R2 and R 3 are not simultaneously hydrogen; more preferably, the branched natural or unnatural amino acid is a glutamic acid moiety, an aspartic acid moiety, a lysine moiety; the substituent of the substituent is selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1- 10 alkyl, C 1-10 alkoxy, amine and sulfonyl-C 1-10 alkyl-; 2) C 1-20 straight chain or branched alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, amine and sulfonyl-C 1-10 alkyl;

[0199] n1, n2 are independently an integer from 0 to 10;

[0200] j1, j2, k1, k2 are independently an integer from 0 to 10;

[0201] o is an integer or non-integer greater than 0 and less than or equal to 8.

[0202] targeting moiety A or Ab

[0203] In the conjugate of the present application, A is a targeting moiety targeting a specific target. By including a targeting moiety in the conjugate of the present application, an excellent cell / tissue targeting function can be achieved. The “targeting moiety” refers to having affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.). The targeting molecule can deliver the payload to a specific site in the body through targeted delivery. The targeting moiety can recognize one or more targets. The specific target is defined by the target it recognizes. For example, a targeting moiety targeting a receptor can deliver a moiety chelating a radionuclide to a site containing a large amount of the receptor.

[0204] In an embodiment, A is a targeting moiety targeting prostate-specific membrane antigen (PSMA). PSMA is expressed on malignant cancer cells.

[0205] As used herein, the term "cancer" refers to a neoplasm characterized by cells that are uncontrolled and usually rapidly proliferating that tend to invade surrounding tissues and metastasize to distant body sites; it includes both benign and malignant neoplasms. Malignant tumors of cancer are usually characterized by anaplasia, invasion, and metastasis; while benign malignant tumors usually do not have those characteristics. In particular, PSMA can be optionally highly expressed in prostate cancer cells, pancreatic cancer cells, kidney cancer cells, or bladder cancer cells. The presence of cells or tissues expressing PSMA can be indicative of a prostate tumor (cell), a metastasized prostate tumor (cell), a kidney tumor (cell), a pancreatic tumor (cell), a bladder tumor (cell), and combinations thereof. Accordingly, the radionuclide conjugates, pharmaceutical compositions, and kits of the present application can be used in the diagnosis and optionally in the retardation or treatment of prostate cancer, kidney cancer, pancreatic cancer, or bladder cancer. In other embodiments, A is a targeting moiety that targets the human epidermal growth factor receptor 2 (HER2). In yet other embodiments, A is a targeting moiety that targets Delta-like ligand 3 (DLL3).

[0206] In one embodiment, A is a targeting moiety comprising an antibody or antigen binding fragment thereof, the antibody being a single domain antibody.

[0207] In some embodiments, the targeting moiety represents a targeting moiety that is unmodified or modified. In some embodiments, represents a modified targeting moiety, the modification being such that, under the action of a ligase, can be formed by reaction of L a’ with L a In some embodiments, contains a ligase recognition substrate, and L a’ contains a ligase recognition substrate. In some embodiments, contains a ligase donor recognition substrate, L a’ contains a ligase acceptor recognition substrate. In some embodiments, L a’ contains a ligase donor recognition substrate, contains a ligase acceptor recognition substrate. In some embodiments, contains a spacer (SP) and a ligase donor substrate recognition sequence, in that order. In some embodiments, the spacer is selected from GA, GGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGS; preferably, the spacer is GA.

[0208] In some embodiments, the targeting moiety naturally contains a moiety, such as Cys, Lys, Gin, that can react with L a’ to form a payload unit. In some embodiments, a the moiety is Cys.

[0209] In some embodiments, the ligase is a Sortase enzyme. In some embodiments, the targeting moiety is modified at the C-terminus or N-terminus, in turn comprising a spacer (SP) and a ligase donor substrate recognition sequence.

[0210] In some embodiments, the spacer is selected from GA, GGGGS, GGGGSGGGGS or GGGGSGGGGSGGGGS; preferably, the spacer is GA.

[0211] In some embodiments, the ligase donor substrate recognition sequence is LPX1TGX2(SEQ ID NO: 17), wherein X1is any one of natural or unnatural amino acid, and X2is absent or an amino acid fragment comprising 1-10 amino acids. In particular, the ligase donor substrate recognition sequence is LPETGG (SEQ ID NO: 18).

[0212] The ligase used in the present application can also include transglutaminase, formylglycine-generating enzyme, tyrosinase and asparagine ligase.

[0213] In some embodiments, the transglutaminase (TGase) catalyzes the reaction of glutamine with lysine and its derivatives, and the site-specific coupling of the targeting moiety and the payload unit can be achieved by TGase. TGase cannot recognize the naturally occurring glutamine in the constant region of glycosylated antibodies, and has high specificity. TGase transfers the transglutaminase acceptor substrate recognition structure contained in the payload unit to the containing the transglutaminase donor substrate recognition structure. In some embodiments, the targeting moiety contains the LLQG (SEQ ID NO: 21) peptide segment, and TGase can specifically recognize the glutamine in the sequence of the LLQG peptide segment, so that the coupling of the targeting moiety and the payload unit is achieved. In some embodiments, under the action of the ligase, the targeting moiety of formula (I) reacts with L a’ to form the L a targeting moiety and L a’ respectively contain the transglutaminase donor substrate recognition structure and the transglutaminase acceptor substrate recognition structure. In some embodiments, the transglutaminase donor substrate recognition structure comprises glutamine. In some embodiments, the targeting moiety contains glutamine. In some embodiments, the targeting moiety is modified by introducing glutamine. In some embodiments, the modified targeting moiety contains the peptide LLQG. In some embodiments, the transglutaminase receptor substrate recognition structure is -NH2. In some embodiments, L a’ contains -NH2, e.g., L a’ contains -C 1-10 alkylene-NH2or lysine. In other embodiments, the targeting moiety contains glutamine.

[0214] Formylglycine-generating enzyme (FGE) can specifically recognize a CX3PX4R pentapeptide sequence, where X3and X4are any natural or unnatural amino acid, replacing the cysteine residue with an aldehyde group. The aldehyde group can further react with a formylglycine-generating enzyme donor substrate recognition structure to form a stable structure. For example, the aldehyde group reacts with dimethylated 2-(hydrazinomethyl)-3-indole to form a stable carbon-carbon bond via a Hantzsch imidazole synthesis (HIPS) reaction at near neutral pH. In some embodiments, the targeting moiety of Formula (I) forms L a’ , the payload unit, by reacting with L a , the targeting moiety contains a C-terminal or N-terminal modification comprising, in order, a spacer (SP) and a formylglycine-generating enzyme donor substrate recognition sequence. In some embodiments, the targeting moiety of Formula (I) and L a’ comprise a formylglycine-generating enzyme donor substrate recognition structure and a formylglycine-generating enzyme acceptor substrate recognition structure, respectively. In some embodiments, the formylglycine-generating enzyme donor substrate recognition structure comprises the recognition sequence CX3PX4R, where X3and X4are any natural or unnatural amino acid. In some embodiments, the formylglycine-generating enzyme acceptor substrate recognition structure comprises a structure that can form a stable reaction product with an aldehyde group. In some embodiments, L a’ comprises where the wavy line indicates the site of attachment to the other structure of the payload unit. In other embodiments, the targeting moiety of Formula (I) comprises the recognition sequence CX3PX4R, where X3and X4are any natural or unnatural amino acid.

[0215] Tyrosinase oxidizes tyrosine to 1,2-quinone, which can undergo cycloaddition reactions with a variety of structures. For example, cycloaddition reactions with various bicyclo[6.1.0]nonyne (BCN) derivatives. In some embodiments, the targeting moiety of Formula (I) forms L a’ , the payload unit, by reacting with L a , the targeting moiety is modified to comprise a tyrosinase donor substrate recognition structure. In some embodiments, the tyrosinase donor substrate recognition structure comprises a tyrosine. In some embodiments, the targeting moiety and L a’ comprise a tyrosinase donor substrate recognition structure and a tyrosinase acceptor substrate recognition structure, respectively. In some embodiments, the tyrosinase donor substrate recognition structure comprises a tyrosine. In some embodiments, the targeting moiety comprises a tyrosine. In some embodiments, the targeting moiety is modified by the introduction of a tyrosine. In some embodiments, the modified targeting moiety comprises a tyrosine. In some embodiments, the tyrosinase acceptor substrate recognition structure comprises any structure that can undergo a cycloaddition reaction with a 1,2-quinone to generate a stable product. In some embodiments, L a’ comprises a bicyclo[6.1.0]nonyne structure. In other embodiments, the targeting moiety comprises a tyrosine.

[0216] Asparagine ligases of the present application include Singzyme and butelase. Singzyme specifically recognizes a ligase donor substrate recognition sequence NX5L, wherein X5 is any natural or unnatural amino acid, for ligation to a ligase acceptor substrate recognition sequence GI, under the mediation of which ligation occurs.

[0217] Butelase is an asparagine ligase derived from butterfly pea that specifically recognizes an Asn-His-Val (NHV) amino acid sequence at the carboxy terminus of a polypeptide and catalyzes ligation of the Asn residue in the sequence to an amino-terminal amino acid residue of the same or another polypeptide to form a peptide bond.

[0218] In some embodiments, the targeting moiety of Formula (I) is modified to comprise a ligase donor substrate recognition sequence. In some embodiments, the modified targeting moiety a’ reacts with L a to form L a’ , the targeting moiety and L a’ comprise an asparagine ligase donor substrate recognition structure and an asparagine ligase acceptor substrate recognition structure, respectively. In some embodiments, the targeting moiety is modified at its C-terminus or N-terminus to comprise, in order, a spacer (SP) and a ligase donor substrate recognition sequence. In some embodiments, the asparagine ligase donor substrate recognition structure comprises a recognition sequence NX5L, wherein X5 is any natural or unnatural amino acid, and a recognition sequence NHV. In other embodiments, the asparagine ligase acceptor substrate recognition structure comprises an amino acid fragment GI. In some embodiments, La’ comprises the amino acid fragment GI. In further embodiments, the targeting moiety comprises the recognition sequence NX5L and NHV, wherein X5 is any natural or non-natural amino acid.

[0219] In some embodiments, the targeting moiety is selected from a ligand, a polypeptide, an antibody or an antigen binding fragment thereof that specifically binds to the target; preferably, the targeting moiety is an antibody or an antigen binding fragment thereof; more preferably, the targeting moiety is selected from a single domain antibody or a single chain antibody. In a particular embodiment, the targeting moiety is an anti-prostate specific membrane antigen (PSMA) antibody; preferably, is an anti-PSMA single domain antibody. In a particular embodiment, the targeting moiety is an anti-epidermal growth factor receptor 2 (HER2) antibody. In another particular embodiment, the targeting moiety is an anti-Delta like ligand 3 (DLL3) antibody.

[0220] In some embodiments, the targeting moiety comprises a HCDR1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2 and a HCDR3 as set forth in SEQ ID NO: 3.

[0221] In some embodiments, the targeting moiety comprises a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 5 and a HCDR3 as set forth in SEQ ID NO: 6.

[0222] In some embodiments, the targeting moiety comprises a HCDR1 as set forth in SEQ ID NO: 7, a HCDR2 as set forth in SEQ ID NO: 8 and a HCDR3 as set forth in SEQ ID NO: 9.

[0223] In some embodiments, the targeting moiety comprises a HCDR1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24 and a HCDR3 as set forth in SEQ ID NO: 25.

[0224] In some embodiments, the targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 11, or an amino acid sequence as set forth in positions 1-127 of SEQ ID NO: 12, or an amino acid sequence as set forth in positions 1-115 of SEQ ID NO: 22. In other embodiments, the targeting moiety comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 11. In yet other embodiments, the targeting moiety comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in positions 1-127 of SEQ ID NO: 12. In other embodiments, the targeting moiety comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence as set forth in positions 1-115 of SEQ ID NO: 22.

[0225] In some embodiments, the targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the targeting moiety comprises an amino acid sequence as set forth in positions 1-127 of SEQ ID NO: 12. In other embodiments, the targeting moiety comprises an amino acid sequence as set forth in positions 1-115 of SEQ ID NO: 22.

[0226] In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, when the targeting moiety the C-terminal amino acid sequence GGHHHHHH (SEQ ID NO: 19) is cleaved off by Sortase enzyme when attached to Gly in formula (I). In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in positions 1-133 of SEQ ID NO: 12.

[0227] In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, when the targeting moiety is linked to Gly in Formula (I) or (II), the C-terminal amino acid sequence GG is cleaved off by Sortase enzyme. In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 13 from position 1 to position 141.

[0228] In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 22. In some embodiments, when the targeting moiety is linked to Gly in Formula (I) or (II), the C-terminal amino acid sequence GGHHHHHH (SEQ ID NO: 19) is cleaved off by Sortase enzyme. In some embodiments, the modified targeting moiety comprises an amino acid sequence as set forth in SEQ ID NO: 22 from position 1 to position 121.

[0229] Method for preparing radionuclide conjugate

[0230] The present application also provides a method for preparing a radionuclide conjugate, wherein the radionuclide conjugate comprises a targeting moiety and a payload unit covalently linked, and the payload unit comprises an albumin binding unit, a linker, a radionuclide and a chelator group; the method comprises the following steps: the targeting moiety and the moiety comprising the linker are catalytically covalently linked by an immobilized ligase or a free ligase.

[0231] In some embodiments, the targeting moiety and the linker are catalytically covalently linked by an immobilized ligase. In other embodiments, the targeting moiety and the payload unit are catalytically covalently linked by an immobilized ligase.

[0232] In some embodiments, the method for preparing comprises the following steps:

[0233] The albumin binding unit, the linker, the chelator group are covalently linked first, and then the conjugate is formed by the immobilized ligase with the targeting moiety, and the conjugate is chelated with the radionuclide to obtain the radionuclide conjugate; or

[0234] The albumin binding unit, the linker, the chelator group are covalently linked first, and then the conjugate is formed by the immobilized ligase with the targeting moiety, and the conjugate is chelated with the radionuclide to obtain the radionuclide conjugate; or

[0235] In other embodiments, the method of preparation comprises the following steps:

[0236] intermediate compound I comprising covalently linked albumin binding unit, linker, chelator group; and

[0237] the conjugate is chelated with a radionuclide to obtain a radionuclide conjugate; or

[0238] the payload unit is linked with a targeting moiety via an immobilized ligase to obtain a radionuclide conjugate.

[0239] In some embodiments, the method of preparation of the present application can directly couple the provided intermediate compound I with a targeting moiety via an immobilized ligase and then chelate with a radionuclide to obtain a radionuclide conjugate in only two steps. In other embodiments, the method of preparation of the present application can directly couple the provided payload unit with a targeting moiety via an immobilized ligase to obtain a radionuclide conjugate in only one step.

[0240] In some embodiments, each of the following steps of the method of preparation of the present application can optionally be followed by purification:

[0241] a) covalently linking albumin binding unit, linker, chelator group first;

[0242] b) forming a conjugate with a targeting moiety via an immobilized ligase;

[0243] c) chelating the conjugate with a radionuclide to obtain a radionuclide conjugate; or

[0244] a') covalently linking albumin binding unit, linker, chelator group;

[0245] b') chelating the product of a') with a radionuclide and forming a payload unit;

[0246] c') linking the payload unit with a targeting moiety via an immobilized ligase to obtain a radionuclide conjugate; or

[0247] a") forming a conjugate with a targeting moiety via an immobilized ligase for intermediate compound I comprising covalently linked albumin binding unit, linker, chelator group;

[0248] b") chelating the conjugate with a radionuclide to obtain a radionuclide conjugate; or

[0249] a'') linking the payload unit with a targeting moiety via an immobilized ligase to obtain a radionuclide conjugate.

[0250] In some embodiments, the purification methods of the present application include, but are not limited to, column chromatography, such as adsorption chromatography, distribution chromatography, ion-exchange chromatography, gel filtration chromatography, affinity chromatography.

[0251] In some embodiments, the catalytic coupling reaction of the ligase of the present application requires the addition of a calcium ion solution, preferably a CaCl2solution. In some embodiments, the intermediate compound I is coupled to the targeting moiety by the immobilized ligase, which comprises the step of mixing the immobilized ligase with a calcium ion solution, preferably CaCl2. In other embodiments, the support unit is coupled to the targeting moiety by the immobilized ligase to obtain the radionuclide conjugate, which comprises the step of mixing the immobilized ligase with a calcium ion solution, preferably CaCl2.

[0252] In some embodiments, the coupling reaction catalyzed by the ligase of the present application is followed by the addition of EDTA for incubation. In some embodiments, the intermediate compound I is coupled to the targeting moiety by the immobilized ligase, which comprises the step of centrifuging the system comprising the reaction of the intermediate compound I with the targeting moiety by the immobilized ligase, adding EDTA solution to the supernatant and incubating at room temperature. In other embodiments, the support unit is coupled to the targeting moiety by the immobilized ligase to obtain the radionuclide conjugate, which comprises the step of centrifuging the system comprising the reaction of the support unit with the targeting moiety by the immobilized ligase, adding EDTA solution to the supernatant and incubating at room temperature.

[0253] In some embodiments, the ligase is formylglycine-generating enzyme, transglutaminase, tyrosinase, or Sortase enzyme. In some embodiments, the ligase is a asparagine ligase (PAL). In some embodiments, the asparagine ligase is Singzyme or Butelase. In some embodiments, the asparagine ligase recognizes the amino acid sequence is N-X-L and GI, X is any amino acid. In some embodiments, the Sortase is sortase A (SrtA), sortase B (SrtB), sortase C (SrtC), sortase D (SrtD), sortase E (SrtE), or sortase F (SrtF). In other embodiments, the Sortase also includes functional variants of Sortase, such as functional variants of sortase A (SrtA), functional variants of sortase B (SrtB), functional variants of sortase C (SrtC), functional variants of sortase D (SrtD), functional variants of sortase E (SrtE), or functional variants of sortase F (SrtF). In some preferred embodiments, the Sortase is sortase A (SrtA) or a functional variant thereof.

[0254] In the present application, the ligase can be used in a variety of ways. In some embodiments, the ligase is covalently linked to Halo, which is a dehalogenase or a variant thereof or a truncated functional active portion thereof, and is immobilized on a support containing a haloalkyl linker through Halo.

[0255] In some embodiments, the present application provides a fusion protein comprising a ligase and Halo. In some embodiments, the fusion protein comprises one or more modifications, in which the ligase, Halo, and additional polypeptide (when applicable) are independently modified by, for example, substitution, deletion, addition, insertion of one or more amino acids or introduction of a moiety or active group at one or more suitable residues, as long as the desired biological activity or function of the modified fusion protein is substantially similar to that of the corresponding fusion protein. The fusion protein can further comprise one or more additional elements, such as additional polypeptides or tags. Preferably, the ligase fusion protein substantially retains the desired properties. Suitable elements can be selected by one skilled in the art according to the desired function or property of the fusion protein. Methods for introducing such elements are known in the art.

[0256] In some embodiments, the fusion protein comprising a ligase and Halo comprises the following amino acid sequence:

[0257] In some embodiments, the fusion protein comprising a ligase and a Halo comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to SEQ ID NO: 26.

[0258] In some embodiments, the fusion protein comprising a ligase and a Halo is packed in a pre-packed column. In other embodiments, the ligase is covalently linked to a Halo and immobilized on a support comprising a haloalkyl linker by the Halo, and the support is packed in a pre-packed column. In some embodiments, the support is a resin microsphere. In other embodiments, the fusion protein is covalently linked to a resin microsphere.

[0259] In some embodiments, the albumin binding unit is a small molecule.

[0260] In some embodiments, the targeting moiety is selected from a ligand, a polypeptide, an antibody or an antigen binding fragment thereof that specifically binds to a target; preferably an antibody or an antigen binding fragment thereof; more preferably a single domain antibody or a single chain antibody. In some embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof; preferably the antibody is selected from a single domain antibody or a single chain antibody. In some embodiments, the targeting moiety is a polypeptide. In some embodiments, the polypeptide is a cyclic peptide. In some embodiments, the targeting moiety comprises a polypeptide and a covalently bound molecular scaffold.

[0261] A method for preparing a radionuclide conjugate, the method comprising the steps of: covalently linking an albumin binding unit, a linker, a chelator group to form an intermediate compound I; covalently linking the intermediate compound I to a targeting moiety via an immobilized ligase to form a conjugate; and chelating the conjugate with a radionuclide to obtain the radionuclide conjugate of formula (III); or

[0262] covalently linking an albumin binding unit, a linker, a chelator group to form an intermediate compound I; covalently linking the intermediate compound I to a targeting moiety via an immobilized ligase to form a conjugate; and chelating the conjugate with a radionuclide to obtain the radionuclide conjugate of formula (III); or

[0263] covalently linking an albumin binding unit, a linker, a chelator group to form an intermediate compound I; covalently linking the intermediate compound I to a targeting moiety via an immobilized ligase to form a conjugate; and chelating the conjugate with a radionuclide to obtain the radionuclide conjugate of formula (III); or

[0264] covalently linking an albumin binding unit, a linker, a chelator group to form an intermediate compound I; covalently linking the intermediate compound I to a targeting moiety via an immobilized ligase to form a conjugate; and chelating the conjugate with a radionuclide to obtain the radionuclide conjugate of formula (III); or

[0265] wherein,

[0266] for the targeting moiety, the rest is a payload unit, wherein the targeting moiety and the payload unit form a covalent bond by means of an enzymatic coupling;

[0267] each Q is independently an albumin binding unit;

[0268] each D is independently a chelating group chelating a radionuclide;

[0269] L a for the coupling of the targeting moiety and G, each L a is independently selected from 1), 2) or a combination thereof:

[0270] 1) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids having a degree of polymerization of 2 to 20;

[0271] 2) a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -S-, -NH-, -(CO)-, C 2-6 alkynyl, C 3-10 cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 arylene and 5-10 membered heteroarylene, wherein the alkylene, alkynyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene are optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl, sulfonyl-C 1-10 alkyl and 3-10 membered heterocycloalkyl;

[0272] each L b and each L c is independently a chemical bond or C 1-20 alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -(CO)-, -NH-, -(C=S)-, C 6-10 arylene and 5-10 membered heteroarylene, wherein the alkylene, arylene and heteroarylene are optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1- 10 alkyl;

[0273] G is a branching moiety having a branching function, directly or indirectly connected to Q and D; wherein each G is independently selected from the following 3), 4) or a combination thereof:

[0274] 3) one or more natural or unnatural amino acid or an oligomeric natural or unnatural amino acid having a degree of polymerization of 2-20;

[0275] 4) a chemical bond or C 1-60 alkylene, wherein the carbon chain units of said alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -NH- and -(CO)-, wherein said alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, amine and sulfonyl-C 1-10 alkyl;

[0276] j is an integer selected from 1-30;

[0277] k is an integer selected from 1-20;

[0278] o is an integer or non-integer greater than 0 and less than 20.

[0279] In some embodiments, the radionuclide conjugate comprises the following structure of Formula (IV):

[0280] wherein,

[0281] is a targeting moiety, and the rest is a payload unit, wherein the targeting moiety and the payload unit form a covalent bond by means of an enzymatic conjugation;

[0282] each Q is independently an albumin binding unit;

[0283] each D is independently a chelating group chelating a radionuclide;

[0284] L a is a conjugation unit connecting the targeting moiety and G, each L a is independently selected from the following 1), 2) or a combination thereof:

[0285] 1) one or more natural or unnatural amino acid or an oligomeric natural or unnatural amino acid having a degree of polymerization of 2-20;

[0286] 2) a chemical bond or C 1-20 alkylene, wherein the carbon chain units of said alkylene are optionally replaced by at least one substituent selected from the group consisting of -0-, -S-, -NH-, -(CO)-, C2-6 alkynyl, C 3-10 cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 arylene and 5-10 membered heteroarylene is replaced by a substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl and 3-10 membered heterocycloalkyl;

[0287] each L b and each L c is independently, when occurring, a bond or C 1-20 alkylene, wherein the carbon chain units of said alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -(CO)-, -NH-, -(C=S)-, C 6-10 arylene and 5-10 membered heteroarylene is replaced by a substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1- 10 alkyl and 3-10 membered heterocycloalkyl;

[0288] each G 1 or G 3 is independently, when occurring, selected from the group consisting of a bond or C 1-20 alkylene, wherein the carbon chain units of said alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -NH- and -(CO)-, wherein said alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10 alkyl and 3-10 membered heterocycloalkyl; preferably, each G 1 or G 3 is independently, when occurring, selected from the group consisting of a bond, optionally substituted -NH-(C 1-10 alkylene)-CO-, optionally substituted -NH-PEG-CO-, optionally substituted -NH-PEG-(C 1-10 alkylene)-CO-, optionally substituted -NH-(C 1-10 alkylene)-PEG-CO-; said substituents are selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C1-10alkyl, C 1-10 alkoxy, aminyl and sulfonyl-C 1-10Alkyl-; the PEG is -(CH2CH2O) x -or-(OCH2CH2) y -, x or y is an integer from 1 to 20;

[0289] Each G 2 or G 4 It is independently a branch unit when it appears; preferably, it is selected from one or more combinations of the following groups: 1) one or more branched natural or non-natural amino acid fragments; preferably, the branched natural or non-natural amino acid fragment has the following structure: -NH-(CR 2 R 3 )-CO-, where R 2 and R 3 are each independently selected from hydrogen, optionally substituted -(C 1-10 Alkylene)-NH-, optionally substituted-(C 1-10 Alkylene)-CO-; wherein R 2 and R 3 More preferably, the branched natural or unnatural amino acid is a glutamic acid fragment, an aspartic acid fragment, a lysine fragment; the substituted substituent is selected from hydroxyl, halogen, amino, sulfhydryl, nitro, cyano, sulfonyl, C 1- 10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Alkyl-;2)C 1-20 A straight or branched chain alkylene group, wherein the carbon chain unit of the alkylene group is optionally replaced by at least one substituent selected from -O-, -NH- and -(CO)-, wherein the alkylene group is optionally replaced by at least one substituent selected from hydroxyl, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Substitution of alkyl groups;

[0290] n1 and n2 are each independently an integer from 0 to 10;

[0291] j1, j2, k1, k2 are each independently an integer from 0 to 10;

[0292] o is an integer or non-integer greater than 0 and less than or equal to 8.

[0293] In some embodiments, preferably,

[0294] G 1 and G 3 are independently selected from a chemical bond or the following structures:

[0295] Each G2 or G 4 is independently at the occurrence a branching unit; preferably, it is selected from the group of one or more combinations of the following groups:

[0296] 1) one or more branched natural or unnatural amino acid fragments; preferably, the branched natural or unnatural amino acid fragments have the following structure: -NH-(CR 2 R 3 )-CO-, wherein R 2 and R 3 are each independently selected from the group consisting of hydrogen, optionally substituted -(C 1-10 alkylene)-NH-, optionally substituted -(C 1-10 alkylene)-CO-; wherein R 2 and R 3 are not simultaneously hydrogen; more preferably, the branched natural or unnatural amino acid is a glutamic acid fragment, an aspartic acid fragment, a lysine fragment; the substituting substituents are selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1- 10 alkyl, C 1-10 alkoxy, amido and sulfonyl-C 1-10 alkyl-; 2) C 1-20 straight chain or branched alkylene, wherein the carbon chain units of the alkylene are optionally replaced by at least one substituent selected from the group consisting of -O-, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one substituent selected from the group consisting of hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 alkyl, C 1-10 alkoxy, amido and sulfonyl-C 1-10 alkyl-;

[0297] preferably, G 2 and G 4 are each independently the following structural fragment or combinations thereof,

[0298] wherein the wavy line with * at one end is the end close to ;

[0299] In some embodiments, the albumin binding unit is a small molecule.

[0300] In some embodiments, the targeting moiety and the payload unit are coupled by an enzyme, and the enzyme used in the enzyme coupling is a ligating enzyme selected from the group consisting of Sortase enzyme, transglutaminase, formylglycine-generating enzyme, tyrosinase and asparagine ligase.

[0301] In some embodiments, under the action of the ligase, the targeting moiety and the load unit to form a covalent bond by enzyme coupling; wherein the targeting portion By L a’ The reaction forms the load unit L a .

[0302] In some embodiments, the ligase is a Sortase enzyme and the targeting moiety and L a’ Contains a Sortase enzyme donor substrate recognition sequence and an acceptor substrate sequence respectively; preferably, the donor substrate recognition sequence is LPX1TGX2, and the acceptor substrate recognition sequence is (Gly) n , wherein X1 is any natural or non-natural amino acid, X2 does not exist or is an amino acid fragment containing 1-10 amino acids, and n is an integer of 2-20.

[0303] In some embodiments, the ligase is a transglutaminase and the targeting moiety and L a’ respectively contain a transglutaminase donor substrate recognition structure and a transglutaminase acceptor substrate recognition structure; preferably, the L a’ Containing -NH2, the targeting moiety Contains glutamine; More preferably, the L a’ Contains-C 1-10 Alkylene-NH2 or lysine.

[0304] In some embodiments, the ligase is a formylglycine generating enzyme and the targeting moiety and L a’ Respectively comprising a formylglycine generating enzyme donor substrate recognition structure and a formylglycine generating enzyme acceptor substrate recognition structure; preferably, the L a’ Include The wavy line indicates the G or G of the load unit. 1 or G 2 The site of attachment, the targeting moiety It comprises a recognition sequence CX3PX4R, wherein X3 and X4 are any natural or non-natural amino acids.

[0305] In some embodiments, the ligase is tyrosinase and the targeting moiety and L a’ respectively comprise a tyrosinase donor substrate recognition structure and a tyrosinase acceptor substrate recognition structure; preferably, the L a’ Contains a bicyclo[6.1.0]nonyne structure, the targeting moiety Contains tyrosine.

[0306] In some embodiments, the ligase is a glutamine ligase, the targeting moiety and L a’ comprise a glutamine ligase donor substrate recognition structure and a glutamine ligase acceptor substrate recognition structure, respectively; preferably, the glutamine ligase is Singzyme, the L a’ comprises the amino acid fragment GI, the targeting moiety comprises the recognition sequence NX5L, wherein X5 is any natural or unnatural amino acid; or the glutamine ligase is butelase, the L a’ comprises the amino acid fragment GI, the targeting moiety comprises the recognition sequence NHV.

[0307] In some embodiments, the L a are each independently selected from the following structures:

[0308] -(Gly) n -, wherein n is an integer selected from 2-20, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably an integer from 2-10, more preferably 3;

[0309] -NH-C 1-10 alkylene-(CO)-, preferably -NH-(CH2)4-(CO)-.

[0310] In some embodiments, when n2 is 0, G 2 is absent, G 4 is selected from the following structural fragments,

[0311] when n2 is not 0, G 2 and G 4 are each the following structural fragment:

[0312] wherein the wavy line with * indicates the site of attachment to L a or G 1 or G 3 .

[0313] In some embodiments, the L b are each independently selected from the following structures:

[0314] a chemical bond;

[0315] wherein the wavy line with * indicates the site of attachment to D, and the wavy line indicates the site of attachment to G or G2 or G 4 the site of attachment;

[0316] wherein the wavy line with * indicates the site of attachment to D, and the wavy line indicates the site of attachment to G or G 2 or G 4 the site of attachment; or

[0317] wherein the wavy line with * indicates the site of attachment to D, and the wavy line indicates the site of attachment to G or G 2 or G 4 the site of attachment.

[0318] In some embodiments, the L c is a chemical bond; or,

[0319] wherein the wavy line with * indicates the site of attachment to D, and the wavy line indicates the site of attachment to G.

[0320] In some embodiments, the Q is a HSA small molecule binder; preferably, the Q are each independently selected from the following structures:

[0321] wherein,

[0322] R 1 is selected from H, C 1-6 alkyl, halogen, methoxy, trifluoromethyl; preferably, R 1 is selected from methyl or iodine;

[0323] R a1 to R a11 are each independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino, or hydroxyl.

[0324] In some embodiments, D is a chelating group chelated with a radionuclide. In some embodiments, the D, when not chelating a radionuclide, are each independently selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl- (hydroxy)amino]-4-oxobutryoyl]amino]pentyl]-N-hydroxyoxal diamide (DFO), 4,11- bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), a-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-azacyclododecane-N,N',N",N"'-1,4,7,10-tetra(methylene)phosphonic acid (DOTMP), N,N'-dipyridooxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphonate) (DPDP), diethylenetriamine N,N',N"-penta(methylene)phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra 3-hydroxy-N-methyl-2-pyridinone chelator abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2- (bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridine-bis(methylene amine tetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1-yl]methyl-hydroxy-phosphoryl]propanoic acid, and triethylenetetramine hexaacetic acid (TTHA), and N. 1 -(5-aminopentyl)-N 1 -hydroxy-N 4 -(5-(N-hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4-oxobutanamido)pentyl)butanediamide.

[0325] In some embodiments, the radionuclide is selected from any one of the radioactive cations or anions of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At, and Er.

[0326] In some embodiments, the targeting moiety is an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-epidermal growth factor receptor 2 (HER2) antibody, or an anti-Delta-like ligand 3 (DLL3) antibody; preferably, is an anti-PSMA single domain antibody.

[0327] In some embodiments, the radioconjugate is selected from:

[0328] wherein the chelator group is further chelated with a radionuclide; -GA-LPET- in the above structure is a part of the amino acid contained in the targeting moiety .

[0329] In some embodiments, the method for preparing the radioconjugate comprises the steps of: covalently linking the albumin binding unit, the linker, and the chelator group first, and then forming the conjugate with the targeting moiety via immobilized ligase, and chelating the radioconjugate with the radionuclide to obtain the radioconjugate of formula (I); or

[0330] The albumin binding unit, the linker, the chelator group are covalently linked, and then chelated with the radionuclide to form a payload unit, and the payload unit is connected with the targeting moiety through an immobilized ligase to obtain the radionuclide conjugate shown in formula (I);

[0331] wherein,

[0332] Q is an albumin binding unit;

[0333] D and D' are each independently a chelator group of a radionuclide, and D and D' are each chelated with a radionuclide;

[0334] A is a single domain antibody or a single chain antibody, or an antigen binding fragment thereof;

[0335] Ld is selected from a chemical bond or C 1-60 alkylene, wherein the alkylene is optionally interrupted by at least one substituent selected from -O-, -NH- and -(CO)-;

[0336] each L1, L2, L 1’ and L 2’ are each independently a chemical bond, a polymeric fragment of 1-10 amino acids, or one or a combination of the following divalent groups: C 1-10 alkylene, -NH- and -(CO)-, wherein the alkylene is optionally substituted with at least one substituent selected from hydroxyl, halogen, amino, nitro, cyano and C 1-10 alkyl;

[0337] m is an integer selected from 0-20;

[0338] n is an integer selected from 2-20;

[0339] z is an integer selected from 1-20.

[0340] In some embodiments, the albumin binding unit is a small molecule.

[0341] In some embodiments, the targeting moiety A is coupled to (Gly)n in formula (I) through a modification at the end of the targeting moiety A under the action of a ligase (sortase enzyme).

[0342] In some embodiments, the ligase is a Sortase enzyme; and / or

[0343] The targeting moiety A comprises a C-terminal modified antibody: the modification comprises a sequential connection of the antibody, a spacer (SP) and a ligase donor substrate recognition sequence, or a sequential connection of the antibody and the ligase donor substrate recognition sequence; and / or

[0344] said SP is selected from the group consisting of GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS; and / or

[0345] said ligase donor substrate recognition sequence is LPX1TGX2, said X1is any natural or unnatural amino acid, X2is absent or an amino acid stretch comprising 1-10 amino acids.

[0346] In some embodiments, Ldis selected from the group consisting of a chemical bond, -NH-C 1-20 alkylene-(CO)- and -NH-(PEG) i -(CO)-, said (PEG) i comprises 1-20 structural units selected from -(O-C2H4)- or -(C2H4-O)-, and optionally is attached at at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene. In some embodiments, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0347] In some embodiments, Ldis -NH-(PEG) i -(CO)-, said (PEG) i 1-20 consecutive -(O-C2H4)- or -(C2H4-O)- structural units, and optionally is attached at at least one end of said -(O-C2H4)- or -(C2H4-O)- structural units with C 1-10 alkylene;

[0348] Preferably, Ldis -NH-(PEG) i -C 1-10 alkylene-(CO)-;

[0349] More preferably, Ldis -NH-PEG4-C2H4-(CO)-;

[0350] Further preferably, Ldis -NH-(C2H4-O)4-C2H4-(CO)-.

[0351] In some embodiments, L1and L 1’ are each independently selected from the group consisting of a chemical bond, C 1-10 alkylene, -NH- and -(CO)-, or any combination thereof;

[0352] Preferably, L1is selected from -(CH2)4-NH-, -CO-NH-C2H4-NH- or -NH-;

[0353] Preferably, L1’ is selected from a chemical bond, -(CH2)4-NH-, -CO-NH-C2H4-NH-, or -NH-.

[0354] In some embodiments, L2and L 2’ are each independently selected from a chemical bond, a polymeric segment of 1-10 amino acids, -(CO)-, C 1-10 any one of alkylene, -(CO)-, and -NH-, or any combination thereof;

[0355] Preferably, L2is selected from -(CO)-, -(CH2)4-NH-, -CO-polymeric segment of 1-10 amino acids-, or -CO-Lys-.

[0356] Preferably, L 2’ is selected from a chemical bond, -(CO)-, -(CH2)4-NH-, -CO-polymeric segment of 1-10 amino acids-, or -CO-Lys-. In some embodiments, m is an integer selected from 0-10; preferably, m is 0, 1, or 2; more preferably, m is 0 or 1; and / or

[0357] n is an integer selected from 2-10, preferably, n is 2, 3, or 4; more preferably, n is 3; and / or

[0358] when Ldis -NH-(PEG) i -C 1-10 when Ldis -NH-(PEG)

[0359] z is an integer selected from 1-10, preferably, z is 1, 2, 3, or 4; more preferably, z is 1.

[0360] In some embodiments, the D and D’ are each independently selected from the group consisting of bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)- methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5- aminopentyl-(hydroxy)amino]-4-oxobutyryl]amino]pentyl]-N-hydroxyoxal diamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), a-(2- carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10- azacyclododecane-N,N',N",N"'-1,4,7,10-tetramethylene phosphonic acid (DOTMP), N,N'-dipyridooxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphonate) (DPDP), diethylenetriamine N,N',N"-pentamethylene phosphonic acid (DTMP), diethylenetriamine pentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra 3-hydroxy-N-methyl-2-pyridinone chelator abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2- (bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] Hexadecane (TE2A), 1,4,8,1 1 -Tetraazacyclododecane-1,4,8,1 1 -tetraacetic acid (TETA), Tris(hydroxypyridinone) (THP), Terpyridyl-bis(methylene amine tetraacetic acid (TMT), 1,4,7-Triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-Tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1 -yl]methyl-hydroxy-phosphoryl]propanoic acid and triethylenetetraminehexaacetic acid (TTHA).

[0361] Preferably,

[0362] D is 1,4,7,10-Tetraazacyclododecane-N,N',N",N"'-tetraacetic acid;

[0363] D' is 1,4,7,10-Tetraazacyclododecane-N,N',N",N"'-tetraacetic acid.

[0364] In some embodiments, the radionuclide is selected from any one of the radioactive cations or anions of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At and Er;

[0365] Preferably,

[0366] the radionuclide is selected from 18 F, 77 Br, 131 I, 125 I, 43 Sc, 44 Sc, 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 90 In, 111 In, 177 Lu, 94 Tc, 99 Tc, 153 Sm, 89 Sr, 223 Ra, 151 Tb, 166 Ho, 186 Re, 188 Re, 212Pb, 213 Bi, 212 Bi, 225 Ac, 227 Th, 55 Co, 57 Co, 152 Gd, 153 Gd, 157 Gd, 166 Dy, 89 Zr or 211 At.

[0367] In some embodiments, Q is selected from

[0368] wherein,

[0369] R 1 is selected from H, C 1-6 alkyl, halogen, methoxy, trifluoromethyl; preferably, R 1 is selected from methyl or iodine;

[0370] R a1 to R a11 are each independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino or hydroxyl;

[0371] Preferably, Q is selected from

[0372] In some embodiments, the targeting moiety A is an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-epidermal growth factor receptor 2 (HER2) antibody or an anti-Delta-like ligand 3 (DLL3) antibody; preferably, A is an anti-PSMA single domain antibody.

[0373] In some embodiments, the radionuclide conjugate comprises a conjugate of the following structure, wherein the chelator group further chelates a radionuclide;

[0374] wherein, individually, A is a targeting molecule, and A in GA is alanine.

[0375] In some embodiments,

[0376] The targeting moiety A comprises a HCDR1 as set forth in SEQ ID NO: 1, a HCDR2 as set forth in SEQ ID NO: 2 and a HCDR3 as set forth in SEQ ID NO: 3; or

[0377] Targeting moiety A comprises a HCDR1 as set forth in SEQ ID NO: 4, a HCDR2 as set forth in SEQ ID NO: 5, and a HCDR3 as set forth in SEQ ID NO: 6; or

[0378] Targeting moiety A comprises a HCDR1 as set forth in SEQ ID NO: 7, a HCDR2 as set forth in SEQ ID NO: 8, and a HCDR3 as set forth in SEQ ID NO: 9; or

[0379] Targeting moiety A comprises a HCDR1 as set forth in SEQ ID NO: 23, a HCDR2 as set forth in SEQ ID NO: 24, and a HCDR3 as set forth in SEQ ID NO: 25.

[0380] In some embodiments, the ligase is covalently linked to Halo, and is immobilized on a support comprising a haloalkyl linker via Halo, which is a dehalogenase or a variant thereof or a functionally active portion thereof; preferably, the ligase is a Sortase enzyme; more preferably, the ligase is Sortase A or a functional variant thereof.

[0381] In some embodiments, one end of the Sortase enzyme is covalently linked to Halo, and the other end is covalently linked to His; or

[0382] The amino terminus of the Sortase enzyme is covalently linked to Halo, and the carboxyl terminus is covalently linked to His, i.e., Halo-Sortase enzyme-His.

[0383] In some embodiments, the support comprises a chloroalkyl linker, such that the ligase is immobilized on the support via covalent interaction between the chloroalkyl linker and Halo.

[0384] In some embodiments, the fusion protein comprising a ligase and Halo comprises an amino acid sequence of SEQ ID No: 26 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity thereto.

[0385] In other embodiments, the fusion protein comprising a ligase and Halo is packed in a pre-packed column. In yet other embodiments, the ligase is covalently linked to Halo, and is immobilized on a support comprising a haloalkyl linker via Halo, and the support is packed in a pre-packed column. In some embodiments, the support is a resin microsphere. In other embodiments, the fusion protein is covalently linked to the resin microsphere.

[0386] In some embodiments, the chloroalkyl linker is generated from a chloroalkyl substrate having the following structure:

[0387] wherein u is an integer from 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, v is an integer from 0-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and w is an integer from 1-19, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0388] In some embodiments, the support has the structure:

[0389] wherein u is an integer from 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, v is an integer from 0-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and w is an integer from 1-19, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

[0390] is a resin, a bead, a membrane, a gel, a matrix, a film, a plate, a well, a tube, a slide, or a surface, preferably a resin, more preferably an agarose resin, a silicone resin, a polymethyl methacrylate resin, or a cellulose resin, most preferably a highly cross-linked agarose resin or a polymethyl methacrylate. In some embodiments, u is 3, v is 2, and w is 5. Patent application invention WO2022160156A is incorporated by reference in its entirety. Beneficial effects

[0391] The present application provides a novel radionuclide conjugate preparation method, which can realize one-step and site-specific coupling of albumin binding units and chelating groups to engineered targeting moieties (such as Fab, scFv, nanobody, and sdAb, etc.) through the catalysis of ligase to obtain corresponding radionuclide conjugates. The radionuclide conjugates prepared by the preparation method of the present application are uniform and stable in structure, have excellent in vitro stability on the basis of retaining the binding ability of the original targeting moiety to the antigen. At the same time, the coupling process is simple and easy to scale up.

[0392] Compared with the prior art, the radionuclide conjugate of the present application has a proper blood circulation half-life, while its tumor enrichment amount is higher, and its tumor retention time is longer, showing higher tumor inhibition activity; the radionuclide conjugate of the present application has stable structure, low radionuclide off-target rate, and greatly reduced toxic side effects. In particular, only an appropriate amount of radionuclide is needed to achieve the desired therapeutic effect, which can enable the patient to experience a lower total absorbed radiation dose. In another aspect, while reducing manufacturing costs, the environmental burden is also reduced.

[0393] Embodiment

[0394] The technical solutions of the present application will be further described in detail below in combination with specific embodiments.

[0395] It should be noted that the following examples are only examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the present application. Those skilled in the art can make other different forms of changes or variations on the basis of the present application, and here it is not necessary or possible to exhaust all the embodiments, and the obvious changes or changes derived therefrom are still within the protection scope of the present application. Unless otherwise specified, the instruments and reagent materials used herein are commercially available.

[0396] Example 1: Synthesis of compound LP0

[0397] 1.1 Synthesis of intermediate compound LP0a

[0398] Step 1: Swelling and condensation of resin

[0399] Weigh 2-CTC-Resin (CAS No.: 42074-68-0, 1.9 g, 2.0 mmol) into a solid-phase synthesis tube, add dichloromethane (20 mL), stir well, and swell for 30 minutes, then filter. Weigh Fmoc-Lys(Dde)-OH (CAS No.: 150629-67-7, 3.2 g) into a conical flask, and add dichloromethane (20 mL), shake until completely dissolved. Then, add diisopropyl ethylamine (0.78 g) to the conical flask and mix well. Add the solution in the conical flask to the above-mentioned solid-phase synthesis tube, stir well. After bubbling nitrogen through the bottom of the solid-phase synthesis tube for about 2 hours, add a methanol / diisopropyl ethylamine mixed solution (6 mL, v / v = 5:1) to the system to cap for 30 minutes. Then, filter the reaction system, and wash it with dichloromethane (20 mL) once, and N,N'-dimethylformamide three times, each time using 20 mL.

[0400] Step 2: Deprotection and condensation

[0401] To the solid phase synthesis tube from the previous step, add the Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4: 1, containing 1% 1-hydroxybenzotriazole (HOBt), 20 mL) and stir for 10 minutes. Filter the reaction completely. Add the same Fmoc deprotection reagent solution (20 mL) and stir for 10 minutes. Filter the reaction completely. Wash the resin with N,N'-dimethylformamide four times, 20 mL each time. Test the resin with ninhydrin, which should appear dark blue.

[0402] Weigh Fmoc-PEG4-CH2CH2COOH (CAS No. 557756-85-1, 2.9 g) and Oxyma (CAS No. 57361-81-6, 0.8 g) into a conical flask, add N,N'-dimethylformamide (20 mL) and shake until dissolved completely. Add N,N'-diisopropylcarbodiimide (DIC, 0.75 g) to the conical flask and mix well. Place the conical flask in an ice bath at 0-10 °C for 3-5 minutes to activate. Then, add the solution in the conical flask to the solid phase synthesis tube described above and mix well. Bubble nitrogen through the bottom of the solid phase synthesis tube for about 2 hours to react, and test the resin with ninhydrin until it is essentially colorless. Drain the solvent from the reaction and wash the resin with N,N'-dimethylformamide three times, 20 mL each time.

[0403] Step 3: Deprotection and condensation

[0404] To the solid phase synthesis tube from the previous step, add the Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4: 1, containing 1% 1-hydroxybenzotriazole (HOBt), 20 mL) and stir for 10 minutes. Filter the reaction completely. Add the same Fmoc deprotection reagent solution (20 mL) and stir for 10 minutes. Filter the reaction completely. Wash the resin with N,N'-dimethylformamide four times, 20 mL each time. Test the resin with ninhydrin, which should appear dark blue.

[0405] Weigh Boc-Gly-Gly-Gly-OH (CAS No.: 28320-73-2, 1.7 g) and Oxyma (CAS No.: 57361-81-6, 0.8 g) into a conical flask, add N,N'-dimethylformamide (20 mL) and shake to dissolve completely. Then add DIC (0.75 g) into the conical flask, mix well and place in 0-10 °C for activation for 3-5 minutes. Subsequently, add the solution in the conical flask into the above-mentioned solid-phase synthesis tube, mix well. Then, bubble nitrogen gas from the bottom of the solid-phase synthesis tube for about 2 hours, and use ninhydrin to detect the resin until it is basically colorless. Dry the solvent in the reaction system, and wash twice with N,N'-dimethylformamide, each time using 20 mL, and further wash three times with dichloromethane, each time using 20 mL. Dry the solvent in the reaction system, and dry the resin to a state of flowing sand.

[0406] Step 4: Cleavage and purification

[0407] Add dichloromethane (28 mL) and hexafluoroisopropanol (12 mL) into a round-bottom flask, mix well, and add the resin obtained in the above step into the above-mentioned solution, and stir for about 2 hours. Filter the reaction system, and wash the resin with a small amount of dichloromethane. After the obtained filtrate is completely concentrated under reduced pressure, it is prepared and purified by reverse phase high performance liquid chromatography (RP-HPLC), and the obtained preparation product is freeze-dried to obtain pure LP0a (1.4 g) with a yield of 85%. The LC-MS detection result is [M+H] + = 829.44.

[0408] 1.2 Synthesis of intermediate compound LP0b

[0409] Weigh (E)-4-amino-6-(((4'-amino-3,3'-dimethyl-[1,1'-biphenyl]-4-yl)diazenyl)-5- hydroxynaphthalene-1,3-disulfonic acid (EB-NH2, 0.3 g, 1.0 eq.) and LP0a (1.37 g, 3.0 eq.) into a round-bottom flask, add N,N'-dimethylformamide (12 mL) and stir to dissolve completely. Subsequently, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 0.84 g, 4.0 eq.) and diisopropylethylamine (548 μL, 6.0 eq.) into the system. Stir the reaction system at room temperature overnight, and monitor EB-NH2 using high performance liquid chromatography (HPLC) until the reaction is basically complete. Prepare and purify using reverse phase high performance liquid chromatography, and freeze-dry the obtained preparation product to obtain pure LP0b (0.36 g) with a yield of 49%. The LC-MS detection result is [M+H] + = 1352.55.

[0410] 1.3 Synthesis of intermediate compound LP0c

[0411] Step 1: Deprotection

[0412] Compound LP0b (0.36 g) was weighed into a round-bottom flask, and purified water (8 mL) was added and stirred to mix. Then, hydrazine hydrate (0.4 mL) was added to the system, and the reaction system was stirred at room temperature for 1 hour, and the reaction was monitored using HPLC until the reaction was substantially complete. Preparative purification was performed using reverse-phase high-performance liquid chromatography, and the resulting preparative product was lyophilized to obtain pure LP0b-1 (0.15 g) with a yield of 47%. LC-MS detection result was [M+H] + = 1189.6.

[0413] Step 2: Condensation

[0414] Compound LP0b-1 (0.15 g, 1.0 eq.) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester (DOTA-(COO t Bu)3, 80 mg, 1.1 eq.) were weighed into a round-bottom flask, and N,N'-dimethylformamide (6 mL) was added and stirred to dissolve completely. Subsequently, HATU (72 mg, 1.5 eq.) and diisopropylethylamine (63 μL, 3.0 eq.) were added to the system. The reaction system was stirred at room temperature overnight, and the reaction was monitored using HPLC until the reaction was substantially complete. Preparative purification was performed using reverse-phase high-performance liquid chromatography, and the resulting preparative product was lyophilized to obtain pure LP0c (0.125 g) with a yield of 60%. LC-MS detection result was [1 / 2M+H] + = 872.89.

[0415] 1.4 Synthesis of compound LP0

[0416] Compound LP0c (0.12 g) was weighed into a round-bottom flask, and a purified water / trifluoroacetic acid mixed solution (2 mL, v / v = 5:95) was added and stirred to mix. The reaction was carried out at room temperature for 2 hours, and the reaction was monitored using HPLC until the reaction was substantially complete. Preparative purification was performed using reverse-phase high-performance liquid chromatography, and the resulting preparative product was lyophilized to obtain pure LP0 (74 mg) with a yield of 73%. LC-MS detection result was [M+H] + = 1476.4.

[0417] Example 2: Synthesis of compound LP1

[0418] 2.1 Synthesis of intermediate compound LP1a

[0419] Step 1: Swelling and condensation of the resin

[0420] Weigh 2-CTC-Resin (CAS No.: 42074-68-0, 1.9 g, 2.0 mmol) into a solid phase synthesis tube, add dichloromethane (20 mL), stir to mix and swell for 30 minutes, then filter. Weigh Fmoc-Lys(Dde)-OH (CAS No.: 150629-67-7, 3.2 g) into a conical flask, add dichloromethane (20 mL), shake to dissolve completely. Then, add diisopropylethylamine (0.78 g) into the conical flask and mix well. Add the solution in the conical flask into the above solid phase synthesis tube, stir to mix. Bubble nitrogen through the bottom of the solid phase synthesis tube for about 2 hours to react, then add a methanol / diisopropylethylamine mixed solution (6 mL, v / v = 5:1) to cap for 30 minutes. Then, filter the reaction system, and wash with dichloromethane (20 mL) once, and N,N'-dimethylformamide three times, each time using 20 mL.

[0421] Step 2: Deprotection and condensation

[0422] Add a Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 20 mL) into the solid phase synthesis tube of the above step, stir to react for 10 minutes, and filter the reaction system completely. Add the same Fmoc deprotection reagent solution (20 mL) again, stir to react for 10 minutes, and filter the reaction system completely. Then, wash the resin with N,N'-dimethylformamide four times, each time using 20 mL. Use ninhydrin to detect the resin, which presents a deep blue color.

[0423] Weigh p-tolylbutyric acid (1.07 g) and Oxyma (0.8 g) into a conical flask, add N,N'-dimethylformamide (20 mL), shake to dissolve completely. Add DIC (0.75 g) into the conical flask, mix well and activate at 0-10°C for 3-5 minutes. Then, add the solution in the conical flask into the above solid phase synthesis tube, stir to mix. Then, bubble nitrogen through the bottom of the solid phase synthesis tube for about 2 hours to react, and use ninhydrin to detect the resin until it is basically colorless. Dry the solvent in the reaction system, and wash with N,N'-dimethylformamide three times, each time using 20 mL.

[0424] Step 3: Deprotection and condensation

[0425] To the solid phase synthesis tube from the previous step, add the Dde deprotection reagent solution (N,N'-dimethylformamide / hydrazine hydrate = 95:5, 20 mL) and stir for 10 minutes. Filter the reaction completely. Add the same Dde deprotection reagent solution (20 mL) and stir for 10 minutes. Filter the reaction completely. Wash the resin with N,N'-dimethylformamide (20 mL) four times. Test the resin with ninhydrin and a dark blue color is observed.

[0426] Weigh Fmoc-PEG4-CH2CH2COOH (2.9 g) and Oxyma (0.8 g) into a conical flask, add N,N'-dimethylformamide (20 mL) and shake until dissolved. Add DIC (0.75 g) to the conical flask and mix. Place the conical flask in an ice bath for 3-5 minutes to activate the reagents. Then, add the solution in the conical flask to the solid phase synthesis tube described above and mix. Bubble nitrogen gas through the reaction from the bottom of the solid phase synthesis tube for about 2 hours and test the resin with ninhydrin until the resin is essentially colorless. Filter the reaction completely and wash the resin with N,N'-dimethylformamide (20 mL) three times.

[0427] Step 4: Deprotection and Coupling

[0428] To the solid phase synthesis tube from the previous step, add the Dde deprotection reagent solution (N,N'-dimethylformamide / hydrazine hydrate = 95:5, 20 mL) and stir for 10 minutes. Filter the reaction completely. Add the same Dde deprotection reagent solution (20 mL) and stir for 10 minutes. Filter the reaction completely. Wash the resin with N,N'-dimethylformamide (20 mL) four times. Test the resin with ninhydrin and a dark blue color is observed.

[0429] Weigh Boc-Gly-Gly-Gly-OH (1.7 g) and Oxyma (0.8 g) into a conical flask, add N,N'-dimethylformamide (20 mL) and shake until dissolved. Add DIC (0.75 g) to the conical flask and mix. Place the conical flask in an ice bath for 3-5 minutes to activate the reagents. Then, add the solution in the conical flask to the solid phase synthesis tube described above and mix. Bubble nitrogen gas through the reaction from the bottom of the solid phase synthesis tube for about 2 hours and test the resin with ninhydrin until the resin is essentially colorless. Filter the reaction completely and wash the resin with N,N'-dimethylformamide (20 mL) two times and dichloromethane (20 mL) three times. Dry the resin to a crumbly consistency.

[0430] Step 5: Cleavage and Purification

[0431] To a round bottom flask was added dichloromethane (28 mL) and hexafluoroisopropanol (12 mL), mixed well, and the resin from the previous step was added to the solution and stirred for about 2 hours. The reaction was filtered and the resin was washed with a small amount of dichloromethane. The resulting filtrate was completely concentrated under reduced pressure and purified using reverse phase high performance liquid chromatography. The resulting product was lyophilized to yield LP1LP1a (1.3 g) with a yield of 79%. LC-MS results were [M+H] + = 825.37.

[0432] 2.2 Synthesis of intermediate compound LP1LP1b

[0433] To a round bottom flask was added dichloromethane (28 mL) and hexafluoroisopropanol (12 mL), mixed well, and the resin from the previous step was added to the solution and stirred for about 2 hours. The reaction was filtered and the resin was washed with a small amount of dichloromethane. The resulting filtrate was completely concentrated under reduced pressure and purified using reverse phase high performance liquid chromatography. The resulting product was lyophilized to yield LP1LP1a (1.3 g) with a yield of 79%. LC-MS results were [M+H] + = 825.37.

[0434] 2.3 Synthesis of intermediate compound LP1LP1c

[0435] To a round bottom flask was added dichloromethane (28 mL) and hexafluoroisopropanol (12 mL), mixed well, and the resin from the previous step was added to the solution and stirred for about 2 hours. The reaction was filtered and the resin was washed with a small amount of dichloromethane. The resulting filtrate was completely concentrated under reduced pressure and purified using reverse phase high performance liquid chromatography. The resulting product was lyophilized to yield LP1LP1a (1.3 g) with a yield of 79%. LC-MS results were [M+H] t Bu)3(0.26 g, 1.0 eq.) and HATU (0.26 g, 1.5 eq.) were added to the solution and stirred to dissolve. Diisopropylethylamine (305 μL, 4.0 eq.) was added to the solution and the reaction was stirred at room temperature overnight. The reaction was monitored using HPLC until it was substantially complete. The product was purified using reverse phase high performance liquid chromatography. The resulting product was lyophilized to yield LP1LP1c (0.33 g) with a yield of 51%. LC-MS results were [M+H] + = 825.37.

[0436] 2.4 Synthesis of compound LP1

[0437] Compound LP1 LP1c (0.23 g) was weighed into a round bottom flask, and a mixture of purified water / trifluoroacetic acid (2.5 mL, v / v = 5:95) was added and mixed well. The reaction was allowed to proceed at room temperature for 3 hours, and the reaction was monitored using HPLC until it was substantially complete. The product was purified using reverse phase HPLC, and the resulting product was lyophilized to give pure LP1 (115 mg) in 62% yield. LC-MS detected [M+H] = 1153.6. +

[0438] Example Three: Synthesis of compound LP2

[0439] Compound LP2 was prepared according to the procedure for synthesis of compound LP1 in Example Two, using 4-(4-iodophenyl)butyric acid instead of p-tolyl butyric acid. Pure compound LP2 was obtained (150 mg), and LC-MS detected [M+H] = 1265.44. +

[0440] Example Four: Synthesis of compound LP3 (control)

[0441] Compound LP3 was prepared according to the procedure for synthesis of compound LP1 in Example Two, using acetic acid instead of p-tolyl butyric acid. Pure compound LP3 was obtained (140 mg), and LC-MS detected [M+H] = 1035.51. +

[0442] Example Five: Synthesis of compound LP4

[0443] (1) Resin swelling and coupling of ethylenediamine:

[0444] 2-CTC-Resin (450 mg) was weighed into a solid phase synthesis tube, and dichloromethane (6 mL) was added, mixed well, and allowed to swell for 30 minutes, after which the solvent was removed by suction. Ethylenediamine (107 μL) was weighed into an EP tube, and dichloromethane (6 mL) was added, and shaken until dissolved. Diisopropylethylamine (600 μL) was then added to the conical flask, and mixed well. The solution was added to the solid phase synthesis tube, and mixed well. Nitrogen was then bubbled through the bottom of the solid phase synthesis tube for about 2 hours, after which a mixture of methanol / diisopropylethylamine (3 mL, v / v = 5:1) was added to cap the reaction for 30 minutes. The solid phase synthesis tube was then filtered by suction, and washed once with dichloromethane (10 mL) and three times with N,N'-dimethylformamide (10 mL each). ​​​

[0445] (2) Coupling of Fmoc-Lys(Dde)-OH:

[0446] Fmoc-Lys(Dde)-OH (920 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added, mixed and dissolved completely. Finally, diisopropylethylamine (600 μL) was added, mixed thoroughly, and the solution was added to the synthesis tube. Nitrogen was bubbled from the bottom of the solid-phase synthesis tube for about 2 hours, and the resin was tested with ninhydrin to be essentially colorless. The solvent was removed by suction, and N,N'-dimethylformamide was used to wash three times, 10 mL each time.

[0447] (3) Deprotection and coupling of Fmoc-PEG4-OH

[0448] The Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) was added to the solid-phase synthesis tube of the previous step, stirred for 10 minutes, and completely filtered by suction. The same Fmoc protection reagent solution (10 mL) was added, stirred for 10 minutes, and completely filtered by suction. Then, N,N'-dimethylformamide was used to wash four times, 10 mL each time. The resin was tested with ninhydrin to be deep blue.

[0449] Fmoc-PEG4-OH (585 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added, mixed and dissolved completely. Finally, diisopropylethylamine (600 μL) was added, mixed thoroughly, and the solution was added to the synthesis tube. Nitrogen was bubbled from the bottom of the solid-phase synthesis tube for about 2 hours, and the resin was tested with ninhydrin to be essentially colorless. The solvent was removed by suction, and N,N'-dimethylformamide was used to wash three times, 10 mL each time.

[0450] (4) Deprotection and coupling of Fmoc-Gly-Gly-OH

[0451] The Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) was added to the solid-phase synthesis tube of the previous step, stirred for 10 minutes, and completely filtered by suction. The same Fmoc protection reagent solution (10 mL) was added, stirred for 10 minutes, and completely filtered by suction. Then, N,N'-dimethylformamide was used to wash four times, 10 mL each time. The resin was tested with ninhydrin to be deep blue.

[0452] Fmoc-Gly-Gly-OH (567 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added, mixed and dissolved completely. Finally, diisopropylethylamine (600 μL) was added, mixed thoroughly, and the solution was added to the synthesis tube. Nitrogen was bubbled from the bottom of the synthesis tube for about 2 hours, and the resin was checked with ninhydrin to be essentially colorless. The solvent was removed by suction, and N,N'-dimethylformamide was used to wash three times, 10 mL each time.

[0453] (5) Deprotection and coupling with Boc-Gly-OH

[0454] Fmoc-deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) was added to the synthesis tube from the previous step, and stirred for 10 minutes. The resin was completely filtered by suction. The same Fmoc-deprotection reagent solution (10 mL) was added, stirred for 10 minutes, and the resin was completely filtered by suction. Subsequently, N,N'-dimethylformamide was used to wash four times, 10 mL each time. The resin was checked with ninhydrin to be deep blue.

[0455] Boc-Gly-OH (280 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added, mixed and dissolved completely. Finally, diisopropylethylamine (600 μL) was added, mixed thoroughly, and the solution was added to the synthesis tube. Nitrogen was bubbled from the bottom of the synthesis tube for about 2 hours, and the resin was checked with ninhydrin to be essentially colorless. The solvent was removed by suction, and N,N'-dimethylformamide was used to wash three times, 10 mL each time.

[0456] (6) Deprotection and coupling with ibuprofen

[0457] Dde-protection reagent solution (N,N'-dimethylformamide / hydrazine hydrate = 95:5, 10 mL) was added to the synthesis tube from the previous step, and stirred for 10 minutes. The resin was completely filtered by suction. The same Dde-protection reagent solution (10 mL) was added, stirred for 10 minutes, and the resin was completely filtered by suction. Subsequently, N,N'-dimethylformamide was used to wash four times, 10 mL each time. The resin was checked with ninhydrin to be deep blue.

[0458] Weigh Ibuprofen (330 mg) and Oxyma (432 mg) into a PE tube, add N,N'-dimethylformamide (6 mL), mix well until completely dissolved. Finally add DIC (300 μL), shake well, then add the above solution into the synthesis tube, then bubble nitrogen from the bottom of the solid-phase synthesis tube for about 2 hours, and use ninhydrin to detect that the resin is basically colorless. Dry the solvent, wash twice with N,N'-dimethylformamide, 10 mL each time, and wash three times with dichloromethane, 10 mL each time. Dry the solvent, and dry the resin to a flow state.

[0459] (7) Cleavage

[0460] Add dichloromethane (7 mL) and hexafluoroisopropanol (3 mL) into a round-bottom flask, mix well, and add the resin obtained in the previous step into the above solution, stir for about 2 hours. Filter and wash the resin with a small amount of dichloromethane. After the obtained filtrate is completely concentrated under reduced pressure, an oil-like crude peptide is obtained, which is weighed.

[0461] (8) Coupling of DOTA

[0462] Weigh DOTA (916 mg) and HOBt (212 mg) into a PE tube, add N,N'-dimethylformamide (1 mL), mix well until completely dissolved. Add the above solution into a 50 mL round-bottom flask containing the crude peptide, and add dichloromethane (5 mL), shake well, slowly add DIC (300 μL), stir overnight, then spin dry the solution to obtain the pure product by using a rotary evaporator.

[0463] (9) Deprotection and purification

[0464] Prepare 10 mL of regular cleavage solution: phenol (500 mg), Tips (1 mL), purified water (0.5 mL), and make up to 10 mL with TFA. Pour the above deprotection solution into the pure product obtained in (8), stir for 2 hours. Add ethyl ether (20 mL) to precipitate thoroughly. Then use a centrifuge at 3500 rpm for 3 min; after centrifugation, pour off the supernatant and centrifuge three times. Dry at room temperature. The obtained pure product is separated by semi-preparative liquid chromatography, and then freeze-dried to obtain compound LP4 (20 mg, purity 99.74%), with a total yield of 11%. LC-MS detection result: [M+H] + = 1182.4, consistent with the theory.

[0465] Example Six: Synthesis of Compound LP5

[0466] Refer to the synthesis method of compound LP4 in Example Five, and synthesize compound LP5 according to the route shown above. LC-MS detection result: [M+H]+ = 1277.35, consistent with theory.

[0467] Example Seven: Synthesis of Compound LP6

[0468] Compound LP6 was synthesized according to the route shown above, with reference to the synthesis of Compound LP4 in Example Five. LC-MS detection gave [M+H] + = 1120.25, consistent with theory.

[0469] Example Eight: Synthesis of Compound LP7

[0470] Compound LP7 was synthesized according to the route shown above, with reference to the synthesis of Compound LP4 in Example Five. LC-MS detection gave [1 / 2 M+H] + = 630.6, consistent with theory.

[0471] Example Nine: Synthesis of Compound LP8

[0472] Compound LP8 was synthesized according to the route shown above, with reference to the synthesis of Compound LP4 in Example Five. LC-MS detection gave [M+H] + = 1279.85, consistent with theory.

[0473] Example Ten: Synthesis of Compound LP9

[0474] Compound LP9 was synthesized according to the route shown above, with reference to the synthesis of Compound LP4 in Example Five. LC-MS detection gave [M+H] + = 1131.45, consistent with theory.

[0475] Example Eleven: Synthesis of Compound LP10

[0476] Compound LP10 was synthesized according to the route shown above, with reference to the synthesis of Compound LP4 in Example Five. LC-MS detection gave [1 / 2 M+H] + = 676.70, consistent with theory.

[0477] Example Twelve: Synthesis of Compound LP11

[0478] (1) Resin Swelling and Coupling of Ethylenediamine:

[0479] Weigh 2-CTC-Resin (450 mg) into a solid phase synthesis tube, add dichloromethane (6 mL), stir to mix and swell for 30 minutes, then remove the solvent. Weigh ethylenediamine (107 μL) into an EP tube, add dichloromethane (6 mL), shake to dissolve completely. Then, add diisopropylethylamine (600 μL) into the conical flask, mix well. Add the above solution into the solid phase synthesis tube, stir to mix. Then, bubble nitrogen from the bottom of the solid phase synthesis tube for about 2 hours, then add a mixture of methanol / diisopropylethylamine (3 mL, v / v = 5:1) to cap for 30 minutes. Then, filter and wash once with dichloromethane (10 mL), and three times with N,N'-dimethylformamide, 10 mL each time.

[0480] (2) Coupling of Fmoc-Lys(Dde)-OH:

[0481] Weigh Fmoc-Lys(Dde)-OH (920 mg), HATU (576 mg) and HOAT (208 mg) into a PE tube, add DMF (12 mL), mix and dissolve completely. Finally, add diisopropylethylamine (600 μL), shake well, then add the above solution into the synthesis tube, then bubble nitrogen from the bottom of the solid phase synthesis tube for about 2 hours, and use ninhydrin to detect that the resin is basically colorless. Remove the solvent, and wash three times with N,N'-dimethylformamide, 10 mL each time.

[0482] (3) Deprotection and coupling of Fmoc-PEG4-OH

[0483] Add a Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) into the solid phase synthesis tube of the previous step, stir for 10 minutes, and filter completely. Add the same Fmoc deprotection reagent solution (10 mL) again, stir for 10 minutes, and filter completely. Then, wash four times with N,N'-dimethylformamide, 10 mL each time. Use ninhydrin to detect the resin, which appears dark blue.

[0484] Weigh Fmoc-PEG4-OH (585 mg), HATU (576 mg) and HOAT (208 mg) into a PE tube, add DMF (12 mL), mix and dissolve completely. Finally, add diisopropylethylamine (600 μL), shake well, then add the above solution into the synthesis tube, then bubble nitrogen from the bottom of the solid phase synthesis tube for about 2 hours, and use ninhydrin to detect that the resin is basically colorless. Remove the solvent, and wash three times with N,N'-dimethylformamide, 10 mL each time.

[0485] (4) Deprotection and coupling of Fmoc-Lys(Dde)-OH

[0486] To the solid phase synthesis tube from the previous step, add Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) and stir for 10 minutes. Repeat the addition of the same Fmoc deprotection reagent solution (10 mL) and stir for 10 minutes. Wash the resin with N,N'-dimethylformamide (10 mL) four times. Test the resin with ninhydrin and a dark blue color is observed.

[0487] Weigh Fmoc-Lys(Dde)-OH (920 mg), HATU (576 mg) and HOAT (208 mg) into a PE tube, add DMF (12 mL) and mix until dissolved. Finally, add diisopropylethylamine (600 μL) and mix well. Add the solution to the solid phase synthesis tube, then bubble nitrogen through the bottom of the tube for about 2 hours. Test the resin with ninhydrin and a basic colorless solution is observed. Wash the resin with N,N'-dimethylformamide (10 mL) three times.

[0488] (5) Deprotection and coupling with Fmoc-PEG4-OH

[0489] To the solid phase synthesis tube from the previous step, add Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) and stir for 10 minutes. Repeat the addition of the same Fmoc deprotection reagent solution (10 mL) and stir for 10 minutes. Wash the resin with N,N'-dimethylformamide (10 mL) four times. Test the resin with ninhydrin and a dark blue color is observed.

[0490] Weigh Fmoc-PEG4-OH (585 mg), HATU (576 mg) and HOAT (208 mg) into a PE tube, add DMF (12 mL) and mix until dissolved. Finally, add diisopropylethylamine (600 μL) and mix well. Add the solution to the solid phase synthesis tube, then bubble nitrogen through the bottom of the tube for about 2 hours. Test the resin with ninhydrin and a basic colorless solution is observed. Wash the resin with N,N'-dimethylformamide (10 mL) three times.

[0491] (6) Deprotection and coupling with Fmoc-Gly-Gly-OH

[0492] To the solid phase synthesis tube from the previous step, Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. The same Fmoc deprotection reagent solution (10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. Subsequently, the resin was washed four times with N,N'-dimethylformamide (20 mL each). The resin was tested with ninhydrin and appeared dark blue.

[0493] Fmoc-Gly-Gly-OH (567 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added and mixed until completely dissolved. Diisopropylethylamine (600 μL) was finally added, mixed well and the solution was added to the synthesis tube. Nitrogen was then bubbled through the bottom of the solid phase synthesis tube for about 2 hours and the resin was tested with ninhydrin and appeared essentially colorless. The solvent was suctioned off and the resin was washed three times with N,N'-dimethylformamide (10 mL each).

[0494] (7) Deprotection and coupling with Boc-Gly-OH

[0495] To the solid phase synthesis tube from the previous step, Fmoc deprotection reagent solution (N,N'-dimethylformamide / piperidine = 4:1, containing 1% HOBt, 10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. The same Fmoc deprotection reagent solution (10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. Subsequently, the resin was washed four times with N,N'-dimethylformamide (10 mL each). The resin was tested with ninhydrin and appeared dark blue.

[0496] Boc-Gly-OH (280 mg), HATU (576 mg) and HOAT (208 mg) were weighed into a PE tube, DMF (12 mL) was added and mixed until completely dissolved. Diisopropylethylamine (600 μL) was finally added, mixed well and the solution was added to the synthesis tube. Nitrogen was then bubbled through the bottom of the solid phase synthesis tube for about 2 hours and the resin was tested with ninhydrin and appeared essentially colorless. The solvent was suctioned off and the resin was washed three times with N,N'-dimethylformamide (10 mL each).

[0497] (8) Deprotection and coupling with p-iodophenylbutyric acid

[0498] To the solid phase synthesis tube from the previous step, Dde deprotection reagent solution (N,N'-dimethylformamide / hydrazine hydrate = 95:5, 10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. The same Dde deprotection reagent solution (10 mL) was added and stirred for 10 minutes. The resin was washed thoroughly by suction filtration. Subsequently, the resin was washed four times with N,N'-dimethylformamide (10 mL each). The resin was tested with ninhydrin and appeared dark blue.

[0499] Weigh p-iodophenylbutyric acid (696 mg) and Oxyma (864 mg) into a PE tube, add N,N'-dimethylformamide (10 mL), mix well until completely dissolved. Finally add DIC (600 μL), after shaking well, add the above solution into the synthesis tube, then bubble nitrogen from the bottom of the solid-phase synthesis tube for about 2 hours, and use ninhydrin to detect that the resin is basically colorless. Dry the solvent, wash twice with N,N'-dimethylformamide, 10 mL each time, and wash three times with dichloromethane, 10 mL each time. Dry the solvent, and dry the resin to a flow state.

[0500] (9) Cleavage

[0501] Add dichloromethane (7 mL) and hexafluoroisopropanol (3 mL) into a round-bottom flask, mix well, and add the resin obtained in the previous step into the above solution, stir for about 2 hours. Filter and wash the resin with a small amount of dichloromethane. After completely concentrating the obtained filtrate under reduced pressure, an oil-like crude peptide is obtained, which is weighed.

[0502] (10) Coupling of DOTA

[0503] Weigh DOTA (916 mg) and HOBt (212 mg) into a PE tube, add N,N'-dimethylformamide (1 mL), mix well until completely dissolved. Add the above solution into a 50 mL round-bottom flask containing the crude peptide, and add dichloromethane (5 mL), shake well, slowly add DIC (300 μL), stir overnight, and then spin dry the solution using a rotary evaporator to obtain the pure product.

[0504] (11) Deprotection and purification

[0505] Prepare 10 mL of regular cleavage solution: phenol (500 mg), Tips (1 mL), purified water (0.5 mL), and dilute to 10 mL with TFA. Pour the above deprotection solution into the pure product obtained in (8), stir for 2 hours. Add ethyl ether (20 mL) to precipitate completely. Then use a centrifuge at 3500 rpm for 3 min; after centrifugation, pour off the supernatant and centrifuge three times. Dry at room temperature. The obtained pure product is separated using semi-preparative liquid chromatography, and then freeze-dried to obtain LP11 (20 mg, purity 96.46%). LC-MS detection result: [1 / 2M+H] + = 957.25, consistent with the theory.

[0506] Example Thirteen: Synthesis of Compound LP12

[0507] Compound LP12 was synthesized according to the route shown above, referring to the synthesis method of compound LP4 in Example 5. LC-MS detection was consistent with the theory.

[0508] Example Fourteen: Synthesis of compound LP13

[0509] Compound LP13 was synthesized according to the route shown above, referring to the synthesis method of compound LP4 in Example 5. LC-MS detection result was [1 / 2M+H] + = 816.70, consistent with the theory.

[0510] Example Fifteen: Synthesis of compound LP14

[0511] Compound LP14 was synthesized according to the route shown above, referring to the synthesis method of compound LP4 in Example 5. LC-MS detection result was [1 / 2M+H] + = 665.50, consistent with the theory.

[0512] Example Sixteen: Synthesis of compound H0274

[0513] Positive control radionuclide conjugate precursor H0274 was synthesized according to the method in reference Mol. Pharmaceutics 2018, 15, 934-946. LC-MS detection result was [M+H] + = 1330.95, consistent with the theory.

[0514] Example Seventeen: Preparation of monoclonal single-domain antibody specifically targeting human prostate-specific membrane antigen (PSMA)

[0515] 17.1 Vector construction and expression

[0516] Monoclonal single-domain antibodies with high affinity and specifically targeting human PSMA were selected, and their sequences are shown in the following table.

[0517] Note: Antibodies Ab60 and Ab61 are unmodified VHH; Antibody Ab62 is modified VHH: its C-terminus is connected with connecting sequence (GA), sortase enzyme donor substrate recognition sequence (LPETGG, SEQ ID NO: 18) and purification sequence (HHHHHH, SEQ ID NO: 20) in turn, Ab62 antibody is cut off sequence GGHHHHHH (SEQ ID NO: 19) and connected with glycine in formula (I) under the action of sortase enzyme; Antibody Ab63 is modified VHH: its C-terminus is connected with connecting sequence (GGGGSGGGGS, SEQ ID NO: 15) and sortase enzyme donor substrate recognition sequence (LPETGG, SEQ ID NO: 18) in turn, Ab63 antibody is cut off sequence GG and connected with glycine in formula (I) under the action of sortase enzyme.

[0518] To generate expression vectors encoding anti-human PSMA single domain antibodies, the nucleic acid sequence of the single domain antibody was cloned into pCDNA 3.4 vector; transformed into E. coli competent cells by ligation, and single colonies were picked for sequencing confirmation. Positive clones were cultured and amplified for plasmid extraction to obtain antibody eukaryotic expression plasmid, which was transformed into Chinese hamster ovary cells (CHO cells) that had been adapted for suspension growth by electroporation. After electroporation, the cells in the electroporation tube were evenly divided into 100 ml of culture medium in a shake flask, and incubated for 40 min. After incubation, the shake flask was incubated at 37°C, 120 rpm, 8% CO2, and the antibody was harvested.

[0519] 17.2 Antibody purification

[0520] The antibody was purified by Ni affinity chromatography column. First, the chromatography column was equilibrated using 20 ml of 1x PBS at a flow rate of 1 ml / min. After loading, the column was washed with 20 ml of 1x PBS, 5 mM imidazole (pH 8.0) at a flow rate of 1 ml / min. Then the sample was eluted using 150 mM imidazole (pH 8.0) at a flow rate of 1 ml / min, and collected in separate tubes. The high concentration of protein was transferred to a dialysis bag and placed in a beaker containing 50 mM Tris + 150 mM NaCl, pH 8.0 for dialysis.

[0521] Example Eighteen: Preparation of conjugate Ab62-LP1

[0522] 18.1 Experimental principle

[0523] The engineered small antibody fragments (such as Fab, scFv, nanobody, sdAb and affibody, etc.) are specifically recognized and cleaved by the immobilized Sortase enzyme recognition site (e.g., LPETGG) on the engineered small antibody fragments, and are site-specifically coupled to the structure of the chelating group connected with the radionuclide to form the corresponding conjugate.

[0524] 18.2 Preparation of immobilized Sortase

[0525] After incubation of Halo-Sortase and Chloro resin (see WO2022160156A) at room temperature for 10 min-24 h, the elution is performed with 20 mM Tris-HCl, 150 mM NaCl, pH=6.0-10.0 buffer. The eluted immobilized Sortase is subjected to activity detection, and after passing the detection, the immobilized ligase (Sortase enzyme) resin is washed with 20 mM Tris-HCl, 150 mM NaCl and stored at 4°C for later use. The Halo-Sortase contains the following amino acid sequence:

[0526] 18.3 Coupling and purification

[0527] The single-domain antibody Ab62 is treated by ultrafiltration, dialysis or desalting column method, and the buffer solution thereof is replaced with 50 mM Tris-HCl, 150 mM NaCl, pH=5.0-8.0 solution.

[0528] The single-domain antibody Ab62 is mixed with the compound LP1 at an appropriate molar ratio (1:1 to 1:100), added to a certain amount of immobilized Sortase enzyme, and a certain amount of buffer solution and CaCl2 solution is added and mixed uniformly. The above system is coupled at 4-40°C for 0.5-20 hours. After the reaction is completed, centrifugation is performed and the supernatant is taken, and an appropriate amount of EDTA solution is added to the supernatant, and incubated at room temperature for 0.1-5 hours. Subsequently, purification, ultrafiltration or dialysis is performed to remove unreacted small molecules. The purified conjugate Ab62-LP1 is stored in acetic acid buffer solution at an appropriate pH value at 4°C or -80°C for later use.

[0529] 18.4 Detection of the number of chelating groups (DAR value) in the conjugate Ab62-LP1

[0530] The number of structures of chelating groups connected with radionuclides (DAR value) coupled on the single-domain antibody in the conjugate Ab62-LP1 is detected by HIC-HPLC, and the specific method is as follows:

[0531] Column: Proteomix HIC Butyl-NP5, 4.6*100mm, 5μm, Non-Porous column (Vendor: Sartomer, PN: 431NP5-4610);

[0532] Mobile phase A: 1.5M Ammonium Sulfate + 20mM Phosphate Buffer Saline, pH 7.0;

[0533] Mobile phase B: 20mM Phosphate Buffer Saline, pH 7.0 mixed with Isopropanol, 7:3 by volume;

[0534] Flow rate: 0.8mL / min;

[0535] Detection wavelength: 280nm;

[0536] Elution gradient: 0-8min, B phase from 10% to 100%;

[0537] The detection result is shown in Figure 1. According to the peak area, it can be calculated that one single-domain antibody Ab62 is coupled with about 0.79 radionuclide-chelated groups on average, i.e. the DAR of the conjugate Ab62-LP1 is 0.79.

[0538] 18.5 Purity detection of conjugate Ab62-LP1

[0539] The purity of conjugate Ab62-LP1 was detected by SEC-HPLC, and the specific method was as follows:

[0540] Column: TSKgel G3000SWXL 7.8mm I.D.*30cm, 5μm column (Vendor: TOSOH, PN: 0008541);

[0541] Mobile phase: 2xPBS mixed with acetonitrile, 90:10 by volume;

[0542] Flow rate: 1.0mL / min;

[0543] Detection wavelength: 280nm

[0544] The detection result is shown in Figure 2. According to the peak area, it can be calculated that the purity of conjugate Ab62-LP1 is close to 100%.

[0545] Example Nineteen: Preparation of conjugate Ab62-LP0, conjugate Ab62-LP2, conjugate Ab62-LP3 and conjugate Ab63-LP1

[0546] Reference to the preparation method of Example Eighteen, the compound LP1 is replaced by the compound LP0, the compound LP2 and the compound LP3 to prepare the conjugate Ab62-LP0, the conjugate Ab62-LP2, the conjugate Ab62-LP3, respectively; the single-domain antibody Ab62 is replaced by the single-domain antibody Ab63 to prepare the conjugate Ab63-LP1. Further, the obtained conjugates are respectively purified and DAR test is carried out, and the results are shown in the following table:

[0547] Example Twenty: Affinity detection of conjugates (SPR method)

[0548] The affinity of the single-domain antibody Ab62 and its conjugates (Ab62-LP1, Ab62-LP2 and Ab62-LP3) to human PSMA was measured on a Biacore T200 molecular interaction instrument.

[0549] First, a 1×HBS-EP solution was prepared as the flow buffer of the whole detection system. The human PSMA protein was diluted to 15 μg / mL in a 10 mM sodium acetate solution at pH 4.5, and was coupled to the 2-4 channels of a Series S CM5 biochip using the amino coupling reagent EDC-NHS, about 2500 RU. Subsequently, quenching was performed in a 1M ethanolamine solution at pH 8.5. The 1st channel was left untreated as a blank subtraction channel. The single-domain antibody and conjugates to be tested were diluted to 0.625 nM with a 10 nM highest concentration, two-fold gradient, and the affinity of the samples at different concentrations to the human PSMA coupled Series S CM5 biochip was measured in turn using the single-cycle mode according to the analyte concentration from low to high. Among them, the analysis temperature was 25°C, the data acquisition frequency was 10 Hz, the flow rate was 30 μL / min, and the binding and dissociation times were 120 s and 600 s, respectively. After completing data acquisition, the sensorgram traces of each sample were blank-subtracted using the Biacore T200 Evaluation Software 3.2.1 software, and the blank-subtracted sensorgram traces were used for kinetic analysis of 1:1 binding model fitting, and finally the affinity strength of the single-domain antibody and the conjugate to the human PSMA antigen binding was calculated. The results are shown in the following table:

[0550] According to the results in the above table, the affinities of the conjugate Ab62-LP1, the conjugate Ab62-LP2 and the conjugate Ab62-LP3 have no obvious difference from Ab62, indicating that the conjugation of different payloads-linkers does not affect the affinity of the single-domain antibody.

[0551] Example Twenty-One: Affinity detection of conjugates (FACS method)

[0552] Using PSMA + The PC-3 tumor cell line was used to determine the affinity of the conjugates Ab62-LP1, Ab62-LP2, Ab62-LP3 and the single-domain antibody Ab62 to PSMA positive tumor cells using the conventional FACS method, and the results are shown in Figure 3. It can be seen that conjugation of different payloads-linkers does not affect the affinity of the single-domain antibody to the tumor cells.

[0553] Example XXII: Endocytosis activity detection

[0554] Using PSMA + The PC-3 tumor cell line was used to determine the endocytosis activity of the conjugates Ab62-LP1, Ab62-LP2, Ab62-LP3 and the single-domain antibody Ab62 to PSMA positive tumor cells using the conventional endocytosis activity detection method, and the results are shown in Figure 4. It can be seen that conjugation of different payloads-linkers does not affect the endocytosis of the single-domain antibody by PSMA positive tumor cells.

[0555] Example XXIII: Radionuclide conjugates 68 Preparation of Ga-Ab62-LP1

[0556] The conjugate Ab62-LP1 (about 288 μg) was diluted in about 200 μL of 1 M NaOAc buffer (pH about 4.5), and [Ga]Ga solution (5 mCi, about 1 mL) was added to the system. 68 Ga]Ga 3+ Then it was incubated at 20-50 °C for 10-120 minutes. The reaction solution was purified by SEC using a PD-10 desalting column and eluted with 0.01 M sterile PBS buffer (pH about 7.4), and the desired fractions were collected. The specific activity was about 0.01 mCi / μg, and the chemical purity and radiochemical purity were detected using radio-HPLC, and the results are shown in Figures 5 and 6, respectively.

[0557] Example XXIV: Radionuclide conjugates 177 Preparation of Lu-Ab62-LP1

[0558] Preparation I: The conjugate Ab62-LP1 (about 200-1000 μg) was diluted in about 200-500 μL of 0.5 M NH4OAc buffer (pH about 4.0), and [Lu]Lu solution (5 mCi, about 1 mL) was added to the system. 177 Lu]Lu 3+Solution (5-250 mCi). Then incubate at 20-50 °C for 10-120 min. SEC purification of the reaction solution was performed using PD-10 desalting column and eluted with 0.01 M sterile PBS buffer (pH about 7.4), and collect the desired fractions. The specific activity was about 10-150 mCi / mg, and the chemical purity and radiochemical purity were detected by radio-HPLC, and the results were shown in FIG. 7 and FIG. 8, respectively.

[0559] Preparation two: Refer to the method of preparation two of example twenty-four, the conjugate Ab62-LP1 was replaced by the conjugate Ab62-LP2, the conjugate Ab62-LP3 and PSMA-617 (CAS: 1702967-37-0) respectively to prepare, and the conjugate 177 Lu] Lu 3+ Solution (20-25 mCi), then incubate at 20-50 °C for 10-120 min. SEC purification of the reaction solution was performed using PD-10 desalting column and eluted with 0.01 M sterile PBS buffer (pH about 7.4), and collect the desired fractions to obtain the conjugate 177 Lu-Ab62-LP1, and add an appropriate amount of NaVc solution (100 mg / mL) to the solution. The specific activity was about 0.04 mCi / μg, and the chemical purity and radiochemical purity were detected by radio-HPLC, and the results were shown in FIG. 9 and FIG. 10, respectively.

[0560] Example twenty-five: radionuclide conjugates 177 Lu-Ab62-LP2, 177 Lu-Ab62-LP3 and 177 Lu-PSMA-617

[0561] Preparation one: Refer to the method of preparation one of example twenty-four, the conjugate Ab62-LP1 was replaced by the conjugate Ab62-LP2, the conjugate Ab62-LP3 and PSMA-617 (CAS: 1702967-37-0) respectively to prepare, and the conjugate 177 Lu-Ab62-LP2, 177 Lu-Ab62-LP3 and 177 Lu-PSMA-617, and after purification, the chemical purity and radiochemical purity were detected by radio-HPLC, which could meet the subsequent test requirements.

[0562] Preparation two: Refer to the method of preparation two of example twenty-four, the conjugate Ab62-LP1 was replaced by the conjugate Ab62-LP2, the conjugate Ab62-LP3 and PSMA-617 (CAS: 1702967-37-0) respectively to prepare, and the conjugate 177 Lu-Ab62-LP2, 177 ​​​​Lu-Ab62-LP3 and 177 Lu-PSMA-617, the chemical purity and radiochemical purity were both satisfied the requirement of following test after purification by radio-HPLC.

[0563] Example twenty-six: radionuclide conjugates 177 Lu-Ab62-LP1, 177 Lu-Ab62-LP2, 177 Lu-Ab62-LP3 and 177 Serum stability test of Lu-PSMA-617

[0564] Respectively, 20-50 μL radionuclide conjugates prepared by the method of Example twenty-four preparation two and Example twenty-five preparation two 177 Lu-Ab62-LP1, 177 Lu-Ab62-LP2, 177 Lu-Ab62-LP3 and 177 Lu-PSMA-617 and an equal volume of mouse serum were mixed and incubated in a 37℃ constant temperature incubator. The samples were taken at 0h, 8h, 24h for radio thin layer scanning analysis to detect the radiochemical purity of the system.

[0565] The results showed that the radionuclide conjugates 177 The radiochemical purity of Lu-PSMA-617 at 0h was 99.2%, the radiochemical purity at 8h was slightly reduced to 99.1%, but the radiochemical purity at 24h dropped to 97.0%, while the radionuclide conjugates 177 Lu-Ab62-LP1, 177 Lu-Ab62-LP2 and 177 Lu-Ab62-LP3 had the same radiochemical purity at 0h, 8h and 24h in serum, all of which were 100%. Compared with the radionuclide conjugates 177 Lu-PSMA-617, the radionuclide conjugates 177 Lu-Ab62-LP1, 177 Lu-Ab62-LP2 and 177 Lu-Ab62-LP3 showed higher stability.

[0566] Example twenty-seven: different 177 Lu-labeled radionuclide conjugates in PSMA + Evaluation of in vivo tumor inhibition activity in LNCaP tumor-bearing mouse model

[0567] Respectively, 20-50 μL radionuclide conjugates prepared by the method of Example twenty-four preparation two and Example twenty-five preparation two 177Lu-Ab62-LP1, 177 Lu-Ab62-LP2, 177 Lu-Ab62-LP3 (control 1) and radionuclide conjugates 177 Lu-PSMA-617 (control 2) were injected into PSMA + LNCaP tumor-bearing mice, and then the changes in tumor volume and body weight of the mice were recorded at different time points, as shown in the following table.

[0568] It can be seen that the radionuclide conjugates 177 Lu-Ab62-LP2 showed the highest tumor inhibition activity, while the radionuclide conjugates 177 Lu-Ab62-LP1 also had higher activity than the radionuclide conjugates 177 Lu-Ab62-LP3 (control 1) and radionuclide conjugates 177 Lu-PSMA-617 (control 2).

[0569] The body weight of the mice then tended to recover, and it can be seen that the RDCs of the present application were safe and well tolerated.

[0570] Example Twenty-Eight: Preparation of radionuclide conjugates 64 Cu-Ab62-LP2

[0571] The conjugate Ab62-LP2 (about 100 μg) was diluted in about 200 μL of 1M NaOAc buffer (pH about 4.5), and about 100 μL of [Cu]Cu 64 Cu]Cu 2+ solution (6 mCi, about 300 μL) was added to the system, and then incubated at 20-50°C for 10-120 minutes. The reaction solution was purified by SEC using a PD-10 desalting column, and eluted with 0.01M sterile PBS buffer (pH about 7.4), and the desired fraction was collected to obtain the radionuclide conjugate 64 Cu-Ab62-LP2. The specific activity was about 0.19 mCi / μg, and the chemical purity and radiochemical purity were detected by radio-HPLC, and the results are shown in Figures 11 and 12, respectively.

[0572] Example Twenty-Nine: Preparation of radionuclide conjugates 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP3 and 64 Cu-PSMA-617

[0573] Referring to the method of Example 28, the conjugate Ab62-LP2 was replaced by the conjugate Ab62-LP1, the conjugate Ab62-LP3 and PSMA-617 (CAS: 1702967-37-0) to prepare the radionuclide conjugates. 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP3 and 64 After purification, the chemical purity and radioactive purity of Cu-PSMA-617 were both approximately 100% as determined by radio-HPLC.

[0574] Example 30: Radionuclide Conjugate 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2, 64 Cu-Ab62-LP3 and 64 Study on the stability of Cu-PSMA-617 in mouse serum

[0575] A certain amount of radionuclide conjugate 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2, 64 Cu-Ab62-LP3 and conjugates 64 Cu-PSMA-617 was mixed with mouse serum and incubated. Samples were taken at 0h, 8h and 24h and the radiochemical purity of the samples was detected by Radio-iTLC. The results are shown in Figure 13. 64 Cu-PSMA-617, radionuclide conjugate 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2 and 64 Cu-Ab62-LP3 exhibited higher stability and almost no degradation occurred.

[0576] Example 31: Radionuclide Conjugate 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2, 64 Cu-Ab62-LP3 and 64 Cu-PSMA-617 uptake and endocytosis in tumor cells

[0577] PSMA + LNCaP and PSMA - PC-3 tumor cells were seeded into multi-well plates and allowed to adhere and grow overnight. The cells were washed with PBS, and a certain amount of culture medium was added to each well. Then, radionuclide conjugates were added to each well. 64 Cu-Ab62-LP1,64 Cu-Ab62-LP2, 64 Cu-Ab62-LP3 and 64 Cu-PSMA-617, and each sample was plated in duplicate. Then, it was diluted with physiological saline and incubated in the well plate for 4 hours. In order to detect the uptake activity of tumor cells for radionuclide conjugates, the cells were washed three times with ice-cold PBS, and NaOH lysis solution was added to the cell sample and measured in a γ counter. In order to detect the endocytosis of each conjugate by tumor cells, the cells were first washed with ice-cold PBS, then incubated in acidic buffer for 10 minutes, and then washed with ice-cold PBS, and NaOH lysis solution was added to the cell sample and measured in a γ counter. The results are shown in Figure 14, and it can be seen that the three single-domain antibody radionuclide conjugates 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2 and 64 Cu-Ab62-LP3 on PSMA + The activity of uptake and endocytosis in LNCaP tumor cells 64 Cu-PSMA-617 is comparable to and is hardly affected by PSMA - Uptake by PC-3 tumor cells.

[0578] Example 32: Different 64 Cu-labeled radionuclide conjugates in PSMA + Positron Emission Tomography (PET) Imaging Study in LNCaP Tumor-Bearing Mouse Model

[0579] Radionuclide conjugates 64 Cu-Ab62-LP1, 64 Cu-Ab62-LP2, 64 Cu-Ab62-LP3 (control 3) and radionuclide conjugates 64 Cu-PSMA-617 (control 4) was injected into PSMA + In LNCaP tumor-bearing mice, PET / CT was then used to scan the tumor-bearing mice at different time points to collect corresponding images and the presence of tumor and key organ tissues. 64 The results are shown in the following table.

[0580] It can be seen that compared with 64 Cu-Ab62-LP3 (control 3) and conjugates 64 Cu-PSMA-617 (control 4), 64 Cu-Ab62-LP1 and 64 Cu-Ab62-LP2 in tumors 64 Cu enrichment (AUC1-48h ) higher, and longer residence time. Also, the ratio of Cu-enrichment was significantly higher at 24h and 48h compared to the other three radionuclide conjugates, 64 Cu-Ab62-LP2 in tumor vs. blood and key normal tissues 64 The ratio of Cu-enrichment was significantly higher at 24h and 48h compared to the other three radionuclide conjugates.

[0581] Example Thirty-Three: Preparation of a monoclonal single-domain antibody specifically targeting human epidermal growth factor receptor 2 (HER2)

[0582] 33.1 Vector construction and expression

[0583] A monoclonal single-domain antibody with high affinity and specificity to human HER2 was selected, and its sequence is shown in the following table.

[0584] To generate the expression vector encoding the anti-human HER2 single-domain antibody, the nucleic acid sequence of the single-domain antibody was cloned into the pCDNA 3.4 vector; transformed into E. coli competent cells by ligation, and single colonies were picked for sequencing confirmation. Positive clones were cultured for plasmid extraction, and the antibody eukaryotic expression plasmid was obtained. The plasmid was transformed into Chinese hamster ovary cells (CHO cells) that had been adapted for suspension growth by electroporation. After electroporation, the cells in the electroporation tube were evenly divided into 100 ml of culture medium in a flask, and incubated for 40 min. After incubation, the flask was placed in a 37°C, 120 rpm, 8% CO2 incubator for culture, and the antibody was harvested.

[0585] 33.2 Antibody purification

[0586] The antibody was purified by Ni affinity chromatography column. First, the column was equilibrated with 20 ml of 1x PBS at a flow rate of 1 ml / min. After loading, the column was washed with 20 ml of 1x PBS, 5 mM imidazole (pH 8.0) at a flow rate of 1 ml / min. Then the sample was eluted with 150 mM imidazole (pH 8.0) at a flow rate of 1 ml / min, and collected in separate tubes. The high-concentration protein was transferred to a dialysis bag and placed in a beaker containing 50 mM Tris + 150 mM NaCl, pH 8.0 for dialysis.

[0587] Example Thirty-Four: Preparation of Conjugate Ab5-LP10

[0588] According to the preparation method of Reference Example Eighteen, the compound LP1 was replaced by compound LP10, and the single-domain antibody Ab62 was replaced by single-domain antibody Ab5 to prepare the conjugate Ab5-LP10. The resulting conjugate was further purified and tested for DAR, and the measured DAR value and purity analysis (monomer rate) results are shown in the following table:

[0589] Example Thirty-Five: Affinity detection (SPR method)

[0590] According to the method of Reference Example Twenty, the human PSMA protein was replaced by human HER2 protein, and the single-domain antibody Ab62 was replaced by single-domain antibody Ab5, and the affinity strength was tested. The results are shown in the following table:

[0591] Example Thirty-Six: Radionuclide conjugate 64 Preparation of Cu-Ab5-LP10

[0592] According to the preparation method of Reference Example Twenty-Eight, the conjugate Ab62-LP2 was replaced by the conjugate Ab5-LP10 to prepare the radionuclide conjugate 64 Cu-Ab5-LP10, after purification, the chemical purity and radioactivity purity were detected by radio-HPLC, and both met the subsequent test requirements.

[0593] Example Thirty-Seven: 64 PET imaging study of Cu-labeled radionuclide conjugate in HER2 high expression tumor-bearing mouse model SKOV-3

[0594] The radionuclide conjugate 64 Cu-Ab5-LP10 was injected into HER2 high expression SKOV-3 tumor-bearing mice, and then PET / CT was used to scan the tumor-bearing mice at different time points, and the corresponding images and the amount of Cu in the tumor and key organ tissues 64 were collected. The results are shown in the following table.

[0595] It can be seen that, similar to 64 Cu-Ab62-LP1 and 64 Cu-Ab62-LP2 in Example Thirty-Two, the radionuclide conjugate 64 Cu-Ab5-LP10 has a high 64 Cu enrichment amount (AUC 1-72h ) in the tumor, and also has a longer retention time.

[0596] Example Thirty-Eight: Preparation of targeting DLL3 conjugates Ab12-LP10 and Ab9-LP5

[0597] Reference to the preparation method of Example Eighteen, replace compound LP1 with compound LP10 and compound LP5, replace single-domain antibody Ab62 with anti-DLL3 single-domain antibody Ab12 and scFv fragment Ab9, respectively, to prepare conjugates Ab12-LP10 and Ab9-LP5. Further purify the obtained conjugates and test the DAR, and the results of purity analysis (monomer rate) are shown in the following table; wherein Ab9 is scFv and Ab12 is VHH.

[0598] Example Thirty-Nine: Radionuclide Conjugate 64 Cu-Ab12-LP10 and 64 Preparation of Cu-Ab9-LP5

[0599] Reference to the preparation method of Example Twenty-Eight, replace conjugate Ab62-LP2 with target conjugates Ab12-LP10 and Ab9-LP5, to prepare radionuclide conjugates 64 Cu-Ab12-LP10 and 64 Cu-Ab9-LP5, after purification, the chemical purity and radioactivity purity detected by radio-HPLC meet the subsequent test requirements.

[0600] Example Forty: 64 PET Imaging Study of Cu-Labeled Radionuclide Conjugate in DLL3-Expressing Tumor-Bearing Mouse Model H82

[0601] Inject radionuclide conjugate 64 Cu-Ab12-LP10 into DLL3-expressing H82 tumor-bearing mice, then scan the tumor-bearing mice at different time points using PET / CT, collect the corresponding images, and collect the amount of 64 Cu in tumor and key organ tissues, and calculate the ratio of 64 Cu in tumor and blood and muscle, and the results are shown in the following table:

[0602] It can be seen that, 64 Cu-Ab12-LP10 can better enrich the tumor site.

Claims

1. A method for preparing a radionuclide conjugate, characterized in that: The radionuclide conjugate comprises a covalently linked targeting portion and a loading unit, wherein the loading unit comprises an albumin binding unit, a linker, a radionuclide and a chelating group; the method comprises the following steps: the targeting portion and the portion comprising the linker are covalently linked by catalysis of an immobilized ligase.

2. The preparation method according to claim 1, comprising the steps of: The albumin binding unit, the linker, and the chelator group are first covalently linked, and then a conjugate is formed with the targeting portion via an immobilized ligase, and the conjugate is chelated with the radionuclide to obtain a radionuclide conjugate; or The albumin binding unit, the linker, and the chelator group are covalently linked and then chelated with the radionuclide to form a loading unit. The loading unit is connected to the targeting part through an immobilized ligase to obtain a radionuclide conjugate.

3. The preparation method according to claim 1, comprising the steps of: The intermediate compound I forms a conjugate with the targeting moiety through an immobilized ligase, wherein the intermediate compound I comprises a covalently linked albumin binding unit, a linker, and a chelating agent group; The conjugate is chelated with the radionuclide to obtain a radionuclide conjugate; or The loading unit and the targeting part are connected by an immobilized ligase to obtain a radionuclide conjugate.

4. The preparation method according to claim 1 or 2, characterized in that The ligase comprises Sortase enzyme, transglutaminase, formylglycine generating enzyme, tyrosinase or asparagine ligase; preferably Sortase A enzyme or a functional variant thereof.

5. The preparation method according to claim 4, wherein The ligase is covalently linked to Halo and immobilized on a support containing a haloalkyl linker via Halo. The Halo is a dehalogenase or a variant thereof or a truncated functionally active portion thereof.

6. The preparation method according to claim 5, wherein The fusion protein comprising the ligase and Halo comprises the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having at least 85% sequence identity thereto.

7. The preparation method according to claim 6, wherein The support is resin microspheres, and the fusion protein covalently linked to the resin microspheres is filled in a prepacked column.

8. The preparation method according to claim 3, wherein The coupling reaction catalyzed by the ligase requires the addition of a calcium ion solution; preferably, the calcium ion solution is a CaCl2 solution.

9. The preparation method according to claim 3, wherein After the ligase-catalyzed coupling reaction is completed, EDTA is added for incubation.

10. The preparation method according to any one of claims 1 to 9, characterized in that The albumin binding unit is a small molecule.

11. The preparation method according to claim 10, characterized in that The targeting moiety is selected from a ligand, a polypeptide, an antibody or an antigen-binding fragment thereof that specifically binds to a target; preferably an antibody or an antigen-binding fragment thereof; more preferably a single domain antibody or a single chain antibody.

12. A method for preparing a radionuclide conjugate, characterized in that: The method comprises the following steps: firstly covalently linking the albumin binding unit, the linker, and the chelator group, then forming a conjugate with the targeting portion through an immobilized ligase, and then chelating the conjugate with the radionuclide to obtain a radionuclide conjugate represented by formula (III); or The albumin binding unit, the linker, and the chelator group are covalently linked, and then chelated with the radionuclide to form a loading unit, and the loading unit is linked to the targeting portion via an immobilized ligase to obtain a radionuclide conjugate of formula (III); or The intermediate compound I forms a conjugate with the targeting portion through an immobilized ligase, wherein the intermediate compound I comprises a covalently linked albumin binding unit, a linker, and a chelating agent group; the conjugate is chelated with a radionuclide to obtain a radionuclide conjugate represented by formula (III); or The loading unit and the targeting portion are connected by an immobilized ligase to obtain a radionuclide conjugate represented by formula (III); in, is the targeting portion, and the rest is the loading unit, wherein the targeting portion Forming a covalent bond with the load unit by enzyme coupling; Each Q is independently an albumin binding unit; Each D is independently a chelating group chelated with a radionuclide; L a To connect the targeting moiety and G coupling unit, each L a Each independently selected from the following 1), 2) or a combination thereof: 1) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids with a degree of polymerization of 2-20; 2) Chemical bond or C 1-20 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one selected from -O-, -S-, -NH-, -(CO)-, C 2-6 Alkynyl, C 3-10 Cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 The alkylene, alkynyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are optionally replaced by at least one selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino, sulfonyl-C 1-10 Alkyl and 3-10 membered heterocycloalkyl substituents; Each L b and each L c When they appear, they are independently chemical bonds or C 1-20 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one selected from -O-, -(CO)-, -NH-, -(C=S)-, C 6-10 alkylene and 5-10 membered heteroarylene, wherein the alkylene, arylene and heteroarylene are optionally replaced by at least one selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1- 10 Substitution of alkyl groups; G is a branch portion having a branching function, directly or indirectly connected to Q and D; wherein each G is independently selected from the following 3), 4) or a combination thereof: 3) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids with a degree of polymerization of 2-20; 4) Chemical bond or C 1-60 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one substituent selected from -O-, -NH- and -(CO)-, wherein the alkylene is optionally replaced by at least one substituent selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Substitution of alkyl groups; j is an integer selected from 1-30; k is an integer selected from 1-20; o is an integer or non-integer greater than 0 and less than 20.

13. The preparation method according to claim 12, wherein The radionuclide conjugate comprises the following structure: in, is the targeting portion, and the rest is the loading unit, wherein the targeting portion Forming a covalent bond with the load unit by enzyme coupling; Each Q is independently an albumin binding unit; Each D is independently a chelating group chelated with a radionuclide; L a To connect the targeting moiety and G coupling unit, each L a Each independently selected from the following 1), 2) or a combination thereof: 1) one or more natural or unnatural amino acids or oligomeric natural or unnatural amino acids with a degree of polymerization of 2-20; 2) Chemical bond or C 1-20 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one selected from -O-, -S-, -NH-, -(CO)-, C 2-6 Alkynyl, C 3-10 Cycloalkylene, 3-10 membered heterocycloalkylene, C 6-10 The alkylene, alkynyl, cycloalkylene, heterocycloalkylene, arylene and heteroarylene groups are optionally replaced by at least one selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino, sulfonyl-C 1-10 Alkyl and 3-10 membered heterocycloalkyl substituents; Each L b and each L c When they appear, they are independently chemical bonds or C 1-20 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one selected from -O-, -(CO)-, -NH-, -(C=S)-, C 6-10 alkylene and 5-10 membered heteroarylene, wherein the alkylene, arylene and heteroarylene are optionally replaced by at least one selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1- 10 Substitution of alkyl groups; Each G 1 or G 3 When present, independently selected from a chemical bond or C 1-20 Alkylene, wherein the carbon chain unit of the alkylene is optionally replaced by at least one substituent selected from -O-, -NH- and -(CO)-, wherein the alkylene is optionally replaced by at least one substituent selected from hydroxy, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 The substituents of the alkyl group are substituted; preferably, each G 1 or G 3 independently selected from a chemical bond, an optionally substituted -NH-(C 1-10 Alkylene)-CO-, optionally substituted-NH-PEG-CO-, optionally substituted-NH-PEG-(C 1-10 Alkylene)-CO-, optionally substituted-NH-(C 1-10 Alkylene)-PEG-CO-; the substituted substituent is selected from hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Alkyl-; the PEG is -(CH2CH2O) x -or-(OCH2CH2) y -, x or y is an integer from 1 to 20; Each G 2 or G 4 It is independently a branch unit when it appears; preferably, it is selected from one or more combinations of the following groups: 1) one or more branched natural or non-natural amino acid fragments; preferably, the branched natural or non-natural amino acid fragment has the following structure: -NH-(CR 2 R 3 )-CO-, where R 2 and R 3 are each independently selected from hydrogen, optionally substituted -(C 1-10 Alkylene)-NH-, optionally substituted-(C 1-10 Alkylene)-CO-; wherein R 2 and R 3 are not hydrogen at the same time; more preferably, the branched natural or unnatural amino acid is a glutamic acid fragment, an aspartic acid fragment, a lysine fragment; the substituted substituent is selected from hydroxyl, halogen, amino, thiol, nitro, cyano, sulfonyl, C 1- 10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Alkyl-;2)C 1-20 A straight or branched chain alkylene group, wherein the carbon chain unit of the alkylene group is optionally replaced by at least one substituent selected from -O-, -NH- and -(CO)-, wherein the alkylene group is optionally replaced by at least one substituent selected from hydroxyl, halogen, amino, mercapto, nitro, cyano, sulfonyl, C 1-10 Alkyl, C 1-10 Alkoxy, amino and sulfonyl-C 1-10 Substitution of alkyl groups; n1 and n2 are each independently an integer from 0 to 10; j1, j2, k1, k2 are each independently an integer from 0 to 10; o is an integer or non-integer greater than 0 and less than or equal to 8.

14. The preparation method according to claim 12, wherein The albumin binding unit is a small molecule.

15. The preparation method according to claim 12, wherein The targeting moiety The loading unit is coupled with the loading unit through an enzyme, and the enzyme used in the enzyme coupling is a ligase, and the ligase is selected from sortase, transglutaminase, formylglycine generating enzyme, tyrosinase and asparagine ligase.

16. The preparation method according to claim 12, wherein Under the action of ligase, the targeting moiety and the load unit to form a covalent bond by enzyme coupling; wherein the targeting portion By L a’ The reaction forms the load unit L a ; The ligase is Sortase enzyme, and the targeting moiety and L a’ Contains a Sortase enzyme donor substrate recognition sequence and an acceptor substrate sequence respectively; preferably, the donor substrate recognition sequence is LPX1TGX2, and the acceptor substrate recognition sequence is (Gly) n , wherein X1 is any natural or non-natural amino acid, X2 does not exist or is an amino acid fragment containing 1-10 amino acids, and n is an integer of 2-20; or The ligase is transglutaminase, and the targeting moiety and L a’ respectively contain a transglutaminase donor substrate recognition structure and a transglutaminase acceptor substrate recognition structure; preferably, the L a’ Containing -NH2, the targeting moiety Contains glutamine; More preferably, the L a’ Contains-C 1-10 Alkylene-NH2 or lysine; or The ligase is a formylglycine generating enzyme, and the targeting moiety and L a’ Respectively comprising a formylglycine generating enzyme donor substrate recognition structure and a formylglycine generating enzyme acceptor substrate recognition structure; preferably, the L a’ Include The wavy line indicates the G or G of the load unit. 1 or G 2 The site of attachment, the targeting moiety Containing the recognition sequence CX3PX4R, wherein X3 and X4 are any natural or unnatural amino acids; or The ligase is tyrosinase, and the targeting moiety and L a’ respectively comprise a tyrosinase donor substrate recognition structure and a tyrosinase acceptor substrate recognition structure; preferably, the L a’ Contains a bicyclo[6.1.0]nonyne structure, the targeting moiety contains tyrosine; or The ligase is asparagine ligase, and the targeting moiety and L a’ They respectively comprise an asparagine ligase donor substrate recognition structure and an asparagine ligase acceptor substrate recognition structure; preferably, the asparagine ligase is Singzyme, the L a’ Contains amino acid fragment GI, the targeting portion Contains a recognition sequence NX5L, wherein X5 is any natural or unnatural amino acid; or, the asparagine ligase is butelase, and the L a’ Contains amino acid fragment GI, the targeting portion Contains the recognition sequence NHV.

17. The preparation method according to claim 12, wherein The Q is a small molecule binder for HSA; preferably, the Q is independently selected from the following structures: in, R 1 Selected from H, C 1-6 Alkyl, halogen, methoxy, trifluoromethyl; preferably, R 1 selected from methyl or iodine; R a1 to R a11 are each independently selected from hydrogen, C 1-6 Alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino or hydroxy.

18. The preparation method according to claim 12, wherein When the D is not chelated with a radionuclide, each D is independently selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxysuccinamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo, [6.6.2]hexadecane (D O2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-azacyclododecane-N,N',N",N"'-1,4,7,10-tetra(methylene)phosphonic acid (DOTMP), N,N'-dipyridyloxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphate) (DPDP), diethylenetriamine N,N',N"-penta(methylene)phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethyl diamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazino-N-methylpyridine-3-carboxamide (HYNIC), tetrakis 3-hydroxy-N-methyl-2-pyridone chelating agent abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1- methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane triacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridone) (THP), terpyridine-bis(methyleneaminetetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-N,N',N",N''-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1-yl]methyl-hydroxy-phosphoryl]propionic acid and triethylenetetraaminehexaacetic acid (TTHA) and N. 1 -(5-aminopentyl)-N 1 -Hydroxy-N 4 -(5-(N-hydroxy-4-((5-(N-hydroxyacetamido)pentyl)amino)-4-oxobutanamido)pentyl)succinamide.

19. The preparation method according to claim 12, wherein The radionuclide is selected from any radioactive cation or anion of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At and Er.

20. The preparation method according to claim 12, wherein The targeting moiety These are anti-prostate-specific membrane antigen (PSMA) antibodies, anti-epidermal growth factor receptor 2 (HER2) antibodies, or anti-Delta-like ligand 3 (DLL3) antibodies.

21. A method for preparing a radionuclide conjugate, characterized in that: The method comprises the following steps: firstly covalently linking the albumin binding unit, the linker, and the chelator group, then forming a conjugate with the targeting portion through an immobilized ligase, and then chelating the conjugate with the radionuclide to obtain the radionuclide conjugate represented by formula (I); or The albumin binding unit, the linker, and the chelator group are covalently linked, and then chelated with the radionuclide to form a loading unit, and the loading unit is connected to the targeting portion via an immobilized ligase to obtain a radionuclide conjugate represented by formula (I); in, Q is the albumin binding unit; D and D' are each independently a chelating group for a radionuclide, and both D and D' are chelated with a radionuclide; A is a single-domain antibody or single-chain antibody, or an antigen-binding fragment thereof; Ld is selected from chemical bonds or C 1-60 an alkylene group, wherein the alkylene group is optionally interrupted by at least one substituent selected from the group consisting of -O-, -NH-, and -(CO)-; Each L1, L2, L 1’ and L 2’ Each independently represents a chemical bond, an amino acid fragment with a degree of polymerization of 1-10, or is selected from one or a combination of the following divalent groups: C 1-10 Alkylene, -NH- and -(CO)-, wherein the alkylene is optionally substituted by at least one selected from hydroxy, halogen, amino, nitro, cyano and C 1-10 Substitution of alkyl groups; m is an integer selected from 0-20; n is an integer selected from 2 to 20; z is an integer selected from 1-20.

22. The preparation method according to claim 21, wherein The albumin binding unit is a small molecule.

23. The preparation method according to claim 21, wherein The A terminus of the antibody is modified and coupled with (Gly)n in formula (I) under the action of Sortase enzyme.

24. The preparation method according to any one of claims 21 to 23, characterized in that The ligase is a Sortase enzyme; and / or A. An antibody comprising a C-terminal modification: the modification comprises sequentially linking an antibody, a spacer (SP), and a ligase donor substrate recognition sequence, or sequentially linking an antibody and a ligase donor substrate recognition sequence; and / or The SP is selected from GA, GGGGS, GGGGSGGGGS and GGGGSGGGGSGGGGS; and / or The ligase donor substrate recognition sequence is LPX1TGX2, wherein X1 is any natural or non-natural amino acid, and X2 does not exist or is an amino acid fragment containing 1-10 amino acids.

25. The preparation method according to claim 24, characterized in that Ld is selected from chemical bonds, -NH-C 1-20 Alkylene-(CO)- and -NH-(PEG) i -(CO)-, the (PEG) i The invention comprises 1-20 structural units selected from -(O-C2H4)- or -(C2H4-O)-, and optionally at least one end of the -(O-C2H4)- or -(C2H4-O)- structural unit is connected to C 1-10 Alkylene.

26. The preparation method according to claim 25, characterized in that Ld is -NH-(PEG) i -(CO)-, the (PEG) i It is 1-20 consecutive -(O-C2H4)- or -(C2H4-O)- structural units, and optionally at least one end of the -(O-C2H4)- or -(C2H4-O)- structural unit is connected to C 1-10 alkylene; Preferably, Ld is -NH-(PEG) i -C 1-10 Alkylene-(CO)-; More preferably, Ld is -NH-PEG4-C2H4-(CO)-; More preferably, Ld is -NH-(C2H4-O)4-C2H4-(CO)-.

27. The preparation method according to claim 24, wherein L1 and L 1’ Each independently selected from a chemical bond, C 1-10 any one of alkylene, -NH- and -(CO)- or any combination thereof; Preferably, L1 is selected from -(CH2)4-NH-, -CO-NH-C2H4-NH- or -NH-; Preferably, L 1’ Selected from a chemical bond, -(CH2)4-NH-, -CO-NH-C2H4-NH- or -NH-.

28. The preparation method according to claim 24, wherein L2 and L 2’ Each independently selected from a chemical bond, an amino acid fragment with a degree of polymerization of 1-10, -(CO)-, C 1-10 Any one of alkylene and -NH- or any combination thereof; Preferably, L2 is selected from -(CO)-, -(CH2)4-NH-, -CO-amino acid fragment with a degree of polymerization of 1-10-, or -CO-Lys-; Preferably, L 2’ Selected from chemical bonds, -(CO)-, -(CH2)4-NH-, -CO-, amino acid fragments with a degree of polymerization of 1-10, or -CO-Lys-.

29. The preparation method according to claim 24, wherein m is an integer selected from 0-10, preferably, m is 0, 1 or 2; more preferably, it is 0 or 1; n is an integer selected from 2-10, preferably, n is 2, 3 or 4; more preferably, n is 3; and / or When Ld is -NH-(PEG) i -C 1-10 When alkylene-(CO)-, i is an integer selected from 1-12, preferably, i is 2, 3, 4, 5 or 6; more preferably, i is 4; and / or z is an integer selected from 1-10, preferably, z is 1, 2, 3 or 4; more preferably, z is 1.

30. The preparation method according to claim 24, wherein Said D and D' are each independently selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA), N'-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxysuccinamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA), α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTAGA), 1,4,7,10-azacyclododecane-N,N',N",N"'-1,4,7,10-tetra(methylene)phosphonic acid (DOTMP), N,N'-dipyridyloxyethylenediamine-N,N'-diacetic acid-5,5"-bis(phosphate) (DPDP), diethylenetriamine N,N',N"-penta(methylene)phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), Ethylenediamine-N,N'-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-N,N,N",N"-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N"-diacetic acid (HBED), hydroxyethylethylenediaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazino-N-methylpyridine-3-carboxamide (HYNIC), tetrakis 3-hydroxy-N-methyl-2-pyridone chelating agent abbreviated as Me-3,2-HOPO (4-((4-(3-(bis(2-(3-hydroxy-1 -methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclononane triacetic acid (NOTA), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2] hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridone) (THP), terpyridine-bis(methyleneaminetetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-N,N',N",N"'-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazacyclononan-1-yl]methyl-hydroxy-phosphoryl]propionic acid and triethylenetetraaminehexaacetic acid (TTHA).

31. The preparation method according to claim 24, wherein The radionuclide is selected from any radioactive cation or anion of F, Br, I, Sc, Cu, Ga, Y, In, Lu, Tc, Sm, Sr, Ra, Tb, Ho, Re, Pb, Bi, Ac, Th, Co, Gd, Dy, Zr, At and Er.

32. The preparation method according to claim 24, wherein The Q is selected from in, R 1 Selected from H, C 1-6 Alkyl, halogen, methoxy, trifluoromethyl; preferably, R 1 selected from methyl or iodine; R a1 to R a11 are each independently selected from hydrogen, C 1-6 Alkyl, C 1-6 alkoxy, halogen, cyano, nitro, amino or hydroxy; Preferably, Q is selected from 33. The preparation method according to claim 24, wherein A is an anti-prostate-specific membrane antigen (PSMA) antibody, an anti-epidermal growth factor receptor 2 (HER2) antibody, or an anti-Delta-like ligand 3 (DLL3) antibody.

34. The preparation method according to any one of claims 1 to 33, characterized in that The ligase is covalently linked to Halo and immobilized on a support containing a haloalkyl linker via Halo, wherein the Halo is a dehalogenase or a variant thereof or a truncated functionally active portion thereof; Preferably, the ligase is a Sortase enzyme; more preferably, the ligase is Sortase A or a functional variant thereof.

35. The preparation method according to claim 34, wherein One end of the Sortase enzyme is covalently linked to Halo, and the other end is covalently linked to His; or The amino terminus of the Sortase enzyme is covalently linked to Halo, and the carboxyl terminus is covalently linked to His, ie, Halo-Sortase enzyme-His.

36. The preparation method according to claim 34, wherein The support comprises a chloroalkyl linker, such that the ligase is immobilized on the support via a covalent interaction between the chloroalkyl linker and Halo.

37. The preparation method according to claim 36, wherein The chloroalkyl linker is generated from a chloroalkyl substrate having the following structure: Wherein, u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19.

38. The preparation method according to claim 36, characterized in that The support has the following structure: Wherein u is an integer from 1 to 20, v is an integer from 0 to 20, and w is an integer from 1 to 19; It is a resin, bead, membrane, gel, matrix, film, plate, well, tube, slide or surface, preferably a resin, more preferably agarose resin, silicone resin, polymethyl methacrylate resin or cellulose resin, most preferably highly cross-linked agarose resin or polymethyl methacrylate.

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