Radionuclide-protein conjugate and use thereof

Abstract

Provided is a radionuclide-protein conjugate comprising a fusion protein. The fusion protein is formed by fusing at least one receptor-binding fragment with an Fc domain derived from human immunoglobulin G (IgG), wherein sites of modification comprise L234, L235, and H435 in the Fc domain, and may further comprise P329 and / or D265, and mutation combinations, for example, may comprise L234A, L235A, and H435Q. The provided fusion protein exhibits low-level uptake in tissues such as blood, kidney, and liver following radionuclide chelation, can be rapidly cleared therefrom, and achieves high-level uptake and sustained retention in tumor tissue, thereby greatly improving radiopharmaceutical therapy safety and efficacy.

Description

A radioactive protein-coupled drug and its application

[0001] This application claims priority to Chinese Patent Application No. 202510379379.X, filed on March 28, 2025, entitled “A Radioactive Protein-Conjugated Drug and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure pertains to the field of biomedicine, specifically relating to a radioactive protein-coupled drug and its applications. Background Technology

[0003] Radionuclide drug conjugates (RDCs) are a new type of precision oncology drug developed based on radionuclide-targeting ligand molecular conjugation technology. By utilizing tumor antigen-specific molecular carriers, radionuclides are precisely delivered to tumor cells, achieving the dual purpose of diagnosis and treatment. The basic structure of a RDC includes several key components: an antibody, peptide or small molecule mediating targeting, a linker arm, a chelate, and a radioisotope.

[0004] Radionuclide-conjugated drugs combine the advantages of precise targeting and potent killing. Specific monoclonal antibodies carrying a radionuclide payload target cancer cells, concentrating energy release within a range several times the cell diameter. This kills cancer cells while minimizing damage to surrounding normal tissue cells, resulting in good efficacy, few adverse reactions, and prolonged survival. They are crucial for the treatment of advanced tumors and offer unique advantages in early tumor diagnosis, staging, and treatment evaluation.

[0005] Radionuclide conjugates also have some common problems, such as low targeting and specificity, high uptake of radionuclides by non-target tissues, and low target-to-non-target ratio.

[0006] Patent application CN202280030415.6 describes a combined mutant developed by mutating the Fc fragment of an antibody. This combined mutant can achieve high-dose absorption in tumor tissue while ensuring low-dose absorption in the liver and blood. However, this mutant still has indicators that urgently need improvement, such as low biodistribution in tumor tissue, which may affect the drug's efficacy. Therefore, further research is necessary to improve the distribution of radionuclide-conjugated drugs in tumor tissue. Summary of the Invention

[0007] To address one of the aforementioned problems to some extent, this disclosure presents a radioactive protein-coupled drug and its application.

[0008] On one hand, this disclosure discloses a fusion protein comprising at least one receptor-binding fragment and an IgG-derived Fc domain fused to the receptor-binding fragment; for example, the fusion protein is formed by fusing at least one receptor-binding fragment with an IgG-derived Fc domain; the Fc domain is mutated at the following positions: L234, L235, and H435.

[0009] In some embodiments, the Fc domain described in this disclosure may also be mutated at the following locations: P329 and / or D265.

[0010] In some embodiments, the IgG described in this disclosure includes human IgG1, IgG2, or IgG4 subtypes.

[0011] In some embodiments, the IgG described in this disclosure is human IgG1.

[0012] In some embodiments, the original amino acid at the mutation site of the Fc domain described in this disclosure can be mutated to any one of Gly, Ala, Val, Leu, Ile, Phe, Pro, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Trp, or Met.

[0013] In some embodiments, the amino acid sequence prior to the mutation of the Fc domain described in this disclosure is shown in SEQ ID NO.32.

[0014] In some embodiments, the Fc domain mutation sites described in this disclosure include at least one of L234A, L235A, D265A, P329G, and / or H435Q.

[0015] In some specific embodiments of this disclosure, the amino acid sequence after the Fc domain mutation is shown below:

[0016] In some embodiments, the combination of Fc domain mutation sites described in this disclosure includes:

[0017] (1) L234A, L235A and H435Q;

[0018] (2) L234A, L235A, P329G and H435Q;

[0019] (3) L234A, L235A, D265A and H435Q.

[0020] In certain specific embodiments of this disclosure, the amino acid sequence of combination (1) is shown in SEQ ID NO.21; the amino acid sequence of combination (2) is shown in SEQ ID NO.22; and the amino acid sequence of combination (3) is shown in SEQ ID NO.23.

[0021] Specifically, the receptor-binding fragment is connected to the Fc domain via a hinge region.

[0022] In some specific embodiments of this disclosure, the amino acid sequence of the hinge region is as shown in SEQ ID NO.35.

[0023]

[0024] Specifically, the receptor-binding fragment includes: a ligand naturally present in the body or a truncated form thereof, a single-domain heavy chain antibody (VHH), a single-chain variable fragment (scFv), or a bispecific antibody-binding fragment.

[0025] In some embodiments, the receptor-binding fragment described in this disclosure is a single-domain heavy chain antibody.

[0026] In certain specific embodiments of this disclosure, the amino acid sequence comprising the receptor-binding fragment, Fc domain, and hinge region is shown below:

[0027] On the other hand, this disclosure discloses a nucleic acid that encodes the aforementioned fusion protein.

[0028] Furthermore, this disclosure provides a vector comprising the aforementioned nucleic acid.

[0029] On the other hand, this disclosure discloses a host cell, which includes the aforementioned nucleic acid or vector.

[0030] Specifically, the host cell is a prokaryotic host cell or a eukaryotic host cell. The prokaryotic host cell includes, but is not limited to, one or more of the following: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella, and Streptomyces. The eukaryotic host cell includes, but is not limited to, one or more of the following: Saccharomyces cerevisiae, Pichia pastoris, Chinese hamster oocytes (CHO), monkey kidney cells (COS), juvenile hamster kidney cells (BHK), mouse embryonic fibroblasts (NIH3T3), or mouse myeloma cells (SP2 / 0 cells).

[0031] On the other hand, this disclosure discloses the application of the aforementioned fusion proteins, nucleic acids, vectors, and host cells in the preparation of radioprotein-coupled drugs.

[0032] In another aspect, this disclosure provides a radioprotein-coupled drug comprising the aforementioned fusion protein, and a small molecule cytotoxic drug or a compound chelating a radionuclide conjugated to the aforementioned fusion protein; for example, the radioprotein-coupled drug uses the aforementioned fusion protein as a carrier to conjugate a small molecule cytotoxic drug or a compound capable of chelating a radionuclide.

[0033] On another front, this disclosure discloses a protein-conjugated drug, wherein the protein-conjugated drug comprises the fusion protein described in this disclosure, and a small molecule cytotoxic drug or a compound chelated with a radionuclide conjugated to the fusion protein; for example, the protein-conjugated drug uses the fusion protein described in this disclosure as a carrier to conjugate a small molecule cytotoxic drug or a compound chelated with a radionuclide.

[0034] Specifically, the radionuclides include, but are not limited to, those mentioned above. 111 In、 223 Ra、 67 Ga、 68 Ga、 44 Sc、 90 Y、 177 Lu、 225 Ac、 212 Bi、 213 Bi、 212 Pb, 227 Th、 64 Cu、 67 Cu、 89 Zr.

[0035] Specifically, the pathway by which the fusion protein is coupled to the compound that chelates the radionuclide includes:

[0036] A: Coupling is achieved through a connector and a chelating ring;

[0037] B: Coupling via free or reduced thiol groups;

[0038] C: Coupling via exposed amino groups.

[0039] In another aspect, this disclosure provides a pharmaceutical composition comprising the above-described fusion protein, nucleic acid, vector, or host cell.

[0040] Specifically, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0041] More specifically, the excipients include, but are not limited to, at least one of the following: ethanol, gentic acid, vitamin C, vitamin E, resveratrol, quercetin, glutathione, and / or lipoic acid.

[0042] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, including but not limited to at least one of: binders, fillers, lubricants, preservatives, antioxidants, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0043] On the other hand, this disclosure discloses the use of the aforementioned fusion proteins, nucleic acids, vectors, host cells, radioactive protein-conjugated drugs, or drug compositions in the preparation of products for the diagnosis and / or treatment of tumors.

[0044] Specifically, the tumors include, but are not limited to: squamous cell papilloma, adenoma, fibroma, lipoma, rhabdomyosarcoma, lymphangioma, chondroma, glioma, ganglioneuroma, meningioma, hydatidiform mole, squamous cell carcinoma, adenocarcinoma, fibrosarcoma, liposarcoma, angiosarcoma, malignant mesenchymal tumor, malignant lymphoma, leukemia, invasive hydatidiform mole, malignant melanoma, lymphoma, neuroendocrine tumors and sarcomas, leiomyomas, hemangiomas, gastric stromal tumors, gastric sarcomas, gastric cancer, colorectal polyps, colon cancer, and rectal cancer. Focal nodular hyperplasia of the liver, hepatic hemangioma, hepatic cyst, hepatic adenoma, cirrhosis, liver cancer, sebaceous cyst, sebaceous nevus, syringoma, keloid, fibrosarcoma, basal cell carcinoma, squamous cell carcinoma, eczematous carcinoma, schwannoma, meningioma, glioma, congenital tumors (epidermoid cyst, dermoid cyst, teratoma), cavernous hemangioma, hemangioblastoma, metastatic tumor, granuloma, hematologic malignancies, multiple myeloma, nervous system tumors, urinary system tumors, head and neck malignancies.

[0045] In another aspect, this disclosure discloses a treatment method comprising administering the aforementioned fusion protein, nucleic acid, vector, host cell, radioactive protein-conjugated drug, or drug composition to a subject.

[0046] The method for treating tumors according to this disclosure includes administering, simultaneously or sequentially, a therapeutically effective amount of: (a) a first active ingredient, which is a radioactive protein-conjugated drug or pharmaceutical composition according to this disclosure; and (b) a second active ingredient, which is at least one antitumor therapeutic agent selected from antibody-conjugated drugs and peptide-conjugated drugs. The first active ingredient may be administered before or after the administration of the second active ingredient.

[0047] Compared with the prior art, this disclosure has the following advantages:

[0048] 1. The radionuclide-labeled mutants provided in this disclosure can be rapidly cleared from the blood and liver of normal mice, and the absorbed dose in the above organs at different time points remains at a very low level.

[0049] 2. The novel mutant provided in this disclosure can achieve long-term, high-dose uptake at the tumor site while maintaining low uptake in normal tissues, enabling the drug to achieve better tumor treatment effects without damaging normal organs.

[0050] 3. Compared to unmodified molecules and molecular structures reported in other patents, the fusion protein structure in this disclosure exhibits significant differences in both conformation and sequence, resulting in radiopharmaceutical uptake at tumor sites and in normal tissues that are distinctly different from previously reported mutants. This unique and innovative structure provides a new therapeutic strategy for the field of radiopharmaceuticals. Attached Figure Description

[0051] Figure 1 shows a SPECT / CT image of αTrop2-Fc v5 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0052] Figure 2 shows SPECT / CT images of αTrop2-Fc v6 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0053] Figure 3 shows SPECT / CT images of αTrop2-Fc v7 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0054] Figure 4 shows SPECT / CT images of αTrop2-Fc v8 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0055] Figure 5 shows SPECT / CT images of αTrop2-Fc v9 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0056] Figure 6 shows SPECT / CT images of αTrop2-Fc v10 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0057] Figure 7 shows SPECT / CT images of αTrop2-Fc v11 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0058] Figure 8 shows SPECT / CT images of αTrop2-Fc v12 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0059] Figure 9 shows SPECT / CT images of αTrop2-Fc v13 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0060] Figure 10 shows SPECT / CT images of αTrop2-Fc v14 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0061] Figure 11 shows SPECT / CT images of αTrop2-Fc v15 molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0062] Figure 12 shows SPECT / CT images of αTrop2-ABD Fc molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0063] Figure 13 is 89 The results of radioactive uptake measurements of Zr-labeled molecules in tumor tissue are shown in the white dashed elliptical line, which represents the tumor tissue.

[0064] Figure 14 shows the deconvolution map of αTrop2-Fc v5.

[0065] Figure 15 shows the deconvolution map of αTrop2-ABD Fc.

[0066] Figure 16 shows SPECT / CT images of αTrop2-Fc v15-Mal-DOTA molecules in tumor-bearing mice, with the white elliptical dashed line representing the tumor tissue.

[0067] Figure 17 shows 177 Tissue distribution of Lu-αTrop2-Fc v5 in tumor-bearing mice.

[0068] Figure 18 shows 177 Antitumor efficacy of Lu-αTrop2-Fc v5 in tumor-bearing mice.

[0069] Figure 19 shows 177 Effect of Lu-αTrop2-Fc v5 on tumor volume in tumor-bearing mice.

[0070] Figure 20 is 177 SPECT / CT image of Lu-αDLL3-Fc v5 molecule in tumor-bearing mice, with the white elliptical dashed line indicating the tumor tissue.

[0071] Figure 21 shows 177 Tissue distribution of Lu-αDLL3-Fc v5 in tumor-bearing mice.

[0072] Figure 22 shows 177 Antitumor efficacy of Lu-αDLL3-Fc v5 in tumor-bearing mice. Detailed Implementation

[0073] The present disclosure will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present disclosure, but only to illustrate the present disclosure. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0074] Terminology Definition

[0075] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0076] The term “and / or”, such as “X and / or Y”, is understood to mean “X and Y” or “X or Y”, and should be understood to provide explicit support for both meanings or either meaning.

[0077] The amino acid three-letter codes or single-letter codes used in this disclosure are as described in J. biol. chem, 243, p3558 (1968, IUPAC--IUB Committee).

[0078] The peptide sequences described herein are written according to usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomers of amino acids are known, they are the L-forms of the amino acids represented unless otherwise explicitly stated.

[0079] The "antibody" described in this disclosure refers to immunoglobulins, which have specific molecular structures. Due to differences in the composition and sequence of amino acids in the constant region of the heavy chain of immunoglobulins, they exhibit varying antigenicity. Based on this characteristic, immunoglobulins can be classified into five categories, also known as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ chain, δ chain, γ chain, α chain, and ε chain, respectively.

[0080] Within the same category of Ig, based on the composition of its hinge region amino acids and the differences in the number and position of heavy chain disulfide bonds, it can be further subdivided into different subclasses. For example, IgG can be further divided into IgG1, IgG2, IgG3, and IgG4.

[0081] Light chains are classified as κ chains or λ chains based on the differences in their constant regions. In the five classes of Ig mentioned above, each class contains both κ and λ chains.

[0082] In the molecular structure of an antibody, the sequence of the approximately 110 amino acids near the N-terminus of the heavy and light chains exhibits considerable variability; this region is defined as the variable region, or Fv region. The remaining amino acid sequence near the C-terminus, on the other hand, exhibits relative stability and is considered the constant region.

[0083] The variable region contains three hypervariable regions, known as HVRs, and four relatively conserved backbone regions, known as FRs. Among them, the three hypervariable regions play a decisive role in the formation of antibody specificity and are therefore also called complementarity-determining regions, or CDRs for short.

[0084] The term "Fc" refers to a fragment in an antibody molecule that neither interferes with antigen binding nor hinders crystallization. By analyzing the three-dimensional structure of the Fc, the interaction pattern between the two constant domains CH2 and CH3 of each heavy chain (HC) can be clearly understood. Proteins containing Fc fragments, such as common antibodies and Fc fusion proteins, can specifically bind to cell surface receptors, such as FcγRs and FcRn, thereby enabling the protein to exert a series of specific biological effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and a relatively long half-life.

[0085] Fc receptors can bind to various Fcγ receptors, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRII can be further subdivided into three subclasses: FcγRIIa (CD32a), FcγRIIb (CD32b), and FcγRIIc (CD32c). FcγRIII can be further subdivided into two subclasses: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Additionally, Fc can bind to proteins in the complement system, such as C1q, and may eventually bind to the cell membrane. These Fc receptors are expressed on various normal immune cells. If antibody-drug conjugates (ADCs) bind to these receptors via the antibody's Fc receptors, it may lead to drug damage to normal tissues.

[0086] The division of the Fc region amino acid coding in this disclosure is based on the EU coding rules for Fc coding established by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5). th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0087] In this case, the term "half-life" specifically refers to the "half-life of a drug," which is the time required for the plasma drug concentration to decrease by half. It is an important parameter reflecting the rate at which a drug is eliminated from the body. The process of drug clearance from the blood mainly depends on the body's metabolic (primarily liver) and excretion (primarily kidney) functions. Half-life is an important indicator of the rate at which a drug is cleared from the blood. Long-half-lived drugs are eliminated slowly from the body. If used continuously for a long period, the drug is prone to accumulate in the body. Long-half-lived radiopharmaceuticals may cause liver damage such as hepatocyte necrosis and inflammatory reactions, leading to drug-induced hepatitis. Some long-half-lived drugs may interfere with the normal secretion and excretion of bile, leading to bile stasis in the liver. Bile stasis can further cause damage to intrahepatic bile ducts, and in severe cases, can lead to liver fibrosis. Long-half-lived radiopharmaceuticals also have a significant impact on bone marrow damage. These drugs act on bone marrow hematopoietic stem cells for a long time, damaging the DNA structure of bone marrow cells, preventing hematopoietic stem cells from dividing normally, and resulting in a decrease in the production of white blood cells, red blood cells, and platelets. Subjects may experience leukopenia, which makes them more susceptible to infection; leukopenia can lead to anemia, causing symptoms such as fatigue, dizziness, and palpitations; and thrombocytopenia can increase the risk of bleeding.

[0088] The term "single-domain antibody," also known as "nanobody," "VHH," or "heavy chain antibody (HCAb)," refers to antibodies found in camels or small sharks that lack the CH1 domain required for light chain pairing. Therefore, they have only two heavy chains, each with a variable antigen-binding (VHH) domain. Due to their small size (only 15 kDa) and low serum persistence or rapid renal clearance, they are disadvantageous in diagnostic screening and therapeutic applications because the glomerular filtration threshold is approximately 60 kDa.

[0089] The term "mutant" refers to a new molecule with a sequence different from the original polypeptide or protein, formed by insertion, deletion, or substitution at the gene level or amino acid level. The new molecule has a substantially homologous amino acid sequence to the original molecule, and these amino acid sequences can have enhanced, similar, or diminished functions or properties due to the deletion, insertion, and / or substitution of one or more different amino acids. In this disclosure, "mutant" refers to a molecule that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid identity with the original sequence. The identity of two amino acid sequences or two nucleic acid sequences can be determined by visual inspection and / or mathematical calculation, or more readily by comparing sequence information using known computer programs for sequence comparison (e.g., Clustal package version 1.83). Variants may contain sequences with at least one conserved amino acid substitution, meaning that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conserved substitutions include substitution of one aliphatic residue for another aliphatic residue, such as substitution of Ile, Val, Leu, or Ala for each other, or substitution of one polar residue for another, such as substitution between Lys and Arg; substitution between Glu and Asp; or substitution between Gln and Asn. Other such conserved substitutions, such as substitution of entire regions with similar hydrophobic characteristics, are well known (Kyte, Jack & Doolittle, Russell. (1982). "A Simple Method for Displaying the Hydropathic Character of a Protein." J. Mol. Biol. 157. 105-132.).

[0090] Table 1 shows exemplary amino acid substitutions.

[0091] Table 1. Exemplary amino acid substitutions

[0092] Amino acids can be classified according to common side chain characteristics as follows: (1) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (2) Hydrophobic: Met, Ala, Val, Leu, Ile; (3) Basic: His, Lys, Arg; (4) Acidic: Asp, Glu; (5) Residues that affect chain orientation: Gly, Pro; and (6) Aromatic ring: Trp, Tyr, Phe.

[0093] "Fusion proteins" are novel proteins with multiple functions produced by linking the coding sequences of two or more different genes together using genetic engineering techniques, allowing them to be expressed within the same reading frame. Examples include the fusion of cytokines with antibody Fc, the fusion of antibody-binding fragments with antibody Fc, and the fusion of the extracellular domain of cell surface receptors with antibody Fc.

[0094] "Antibody-binding fragment" refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be, for example, Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and their utility can be screened in the same manner as that of the whole antibody. Antigen-binding fragments can be generated by recombinant DNA technology, enzymatic or chemical cleavage of the whole immunoglobulin, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0095] A "host cell" is a cell capable of receiving and expressing a foreign gene (target gene) within itself to produce the desired protein. For example, *Escherichia coli* is one of the most commonly used prokaryotic host cells; Chinese hamster ovary (CHO) cells are currently the most widely used mammalian cell for producing protein drugs. CHO cells can correctly fold proteins and perform complex glycosylation modifications, making the protein drugs they produce structurally and functionally closer to natural proteins in the human body; insect cells such as Sf9 and Sf21 cells can use the baculovirus expression vector system (BEVS) to express foreign genes.

[0096] "Vectors" include nucleic acid vectors, such as DNA vectors, RNA vectors, and viruses. The expression vectors described herein may contain polynucleotide sequences and additional sequence elements, such as those for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. These sequence elements may include 5' and 3' untranslated regions and polyadenylation signal sites to guide the efficient transcription of genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors; suitable markers include genes encoding antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, and norsinolate).

[0097] The term “affinity” here specifically refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its pair (e.g., an antigen). Unless otherwise specified, “binding affinity” as used herein refers to internal binding affinity, which reflects the 1:1 interaction strength between the two members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its pair Y can typically be quantified by the dissociation constant (KD). This affinity can be measured and evaluated using standard methods known in the art, including those mentioned herein.

[0098] "Couplet" refers to the combination or connection of two or more objects. When referring to chemical or biological compounds, coupling can refer to the covalent connection between two or more chemical or biological compounds.

[0099] "Protein-drug conjugates" are a special type of drug that typically involves chemically or biologically conjugating a pharmacologically active ingredient (such as cytokines, protein toxins, cytotoxic compounds, radionuclides, nucleotides, etc.) to a target molecule. The target molecule can be a monoclonal antibody, antibody fragment, recombinant protein, etc.

[0100] These conjugated drugs combine the properties of pharmacodynamic components and targeting molecules, aiming to improve drug efficacy, reduce toxic side effects, and achieve more specific targeted therapy. For example, cytotoxic compounds can include maytansine, camptothecin and its derivatives, auristatin toxins, maytansine thio derivatives, docetaxel, gemcitabine, and docetaxel. Radionuclide-conjugated protein drugs are a class of drugs that combine a radionuclide with a targeted protein drug through a specific chemical linker. This combination allows the drug to utilize the targeting properties of the protein drug to precisely deliver the radionuclide to diseased cells or tissues, playing a diagnostic or therapeutic role.

[0101] The term "chelation" refers to the process by which radioactive metal ions are bound to a chelating agent through coordinate bonds to form a stable complex with a specific structure. Chelating agents are typically organic compounds containing multiple coordinating atoms. These coordinating atoms are generally atoms with lone pairs of electrons, such as nitrogen (N), oxygen (O), and sulfur (S). For example, ethylenediaminetetraacetic acid (EDTA) is a classic chelating agent; its molecular structure contains four carboxyl groups (-COOH) and two amino groups (-NH2), in which the oxygen and nitrogen atoms can act as coordinating atoms. Other common chelating agents include desferrioxamine (DFO), diethylenetriaminepentaacetic acid (DTPA) and its derivatives, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives, 1-nitro-2,3,4,6,7,8-hexaazatricyclo[3.3.1.13,7]decane (NOTA) and its derivatives, 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) and its derivatives, and 3,6,9,15-tetraazabicyclo[9.3.1]pentadecan-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and its derivatives.

[0102] When radioactive metal ions are present, the coordinating atoms in the chelating agent molecule form coordinate bonds with the metal ions. Different chelating agents can alter the physical and chemical properties of radiopharmaceuticals. For example, the hydrophilicity or hydrophobicity of a chelating agent affects the distribution of radiopharmaceuticals in the body. Hydrophilic chelating agents can facilitate the dispersion and transport of radiopharmaceuticals in aqueous environments such as blood, while hydrophobic chelating agents may guide the distribution of radiopharmaceuticals more towards adipose tissue, etc. Furthermore, the size, charge, and other properties of the chelated radiopharmaceutical are also altered depending on the chelating agent, and these properties have a significant impact on processes such as drug penetration of physiological barriers (e.g., the blood-brain barrier) and cellular uptake.

[0103] Chelating agents can be linked to molecules with targeting functions (such as antibodies, peptides, and small molecule ligands) to form targeted radiopharmaceuticals. For example, by linking an antibody that recognizes specific receptors on the surface of tumor cells with a chelating agent that chelates radioactive metal ions, such a radiopharmaceutical can specifically bind to tumor cells in vivo, enabling precise imaging or treatment of tumors. This targeted chelation strategy can reduce the damage of radiopharmaceuticals to normal tissues, improving the therapeutic efficacy and safety of the drug.

[0104] The term "radiopharmaceutical" refers to a radionuclide preparation or its labeled compound used for clinical diagnosis or treatment. These drugs contain radionuclides that emit radiation, such as alpha rays, beta rays, or gamma rays. Examples of such radionuclides are as follows: 18 F,11 C 14 C 13 N、 32 P, 111 In、 113m In、 66 Ga、 67 Ga、 68 Ga、 51 Cr 52 Fe、 52 Mn, 51 Mn, 123 I, 124 I, 125 I, 131 I, 90 Y、 149 Tb, 152 Tb, 155 Tb, 161 Tb, 177 Lu、 211 At、 225 Ac、 227 Ac、 212 Pb, 223 Ra、 226 Th、 227 Th、 86 Y、 90 Y、 89 Zr、 89 Sr、 99m Tc, 99 Mo、 94m Tc, 61 Cu、 62 Cu、 64 Cu、 67 Cu、 89 Sr、 212 Bi、 213 Bi et al.

[0105] The term "radiochemical purity" refers to the percentage of radionuclides existing in a specific chemical form in a radioactive sample. This value reflects the proportion of radionuclides existing in a specific chemical form in the total number of radionuclides.

[0106] The term "uptake" refers to the process by which a radionuclide-conjugated drug (RCD) is absorbed and incorporated into target cells or tissues through a series of physiological processes. This is similar to the process by which cells take in nutrients, but in this context, it refers to RCDs. For example, when an RCD is injected into the body, it needs to reach diseased cells (such as tumor cells) and enter these cells through a specific mechanism; this process of entering the cell can be called uptake.

[0107] The term "distribution" refers to the process by which a drug, after being absorbed into the bloodstream, is transported to various tissues, organs, and body fluids through various physiological mechanisms. It is a crucial step in the dynamic changes of drugs within the body and is closely related to their efficacy and safety. Drugs are more easily reached and distributed in tissues and organs with high blood flow. For example, organs with large blood flows, such as the heart, liver, and kidneys, typically experience faster drug distribution in these areas. Drugs have different affinities for target sites in different tissues, leading to selective accumulation of drugs in certain tissues.

[0108] The terms "subject" or "object" refer to a primate (e.g., a human), dog, rabbit, guinea pig, pig, rat, or mouse undergoing diagnosis or treatment for a specific disease. In some embodiments, the subject is a primate. In other embodiments, the subject or "object" is a human.

[0109] The term "%ID / g" refers to the percentage of the injected dose of radiopharmaceutical per gram of tissue. By measuring the %ID / g values ​​at different time points, in different tissues or organs, we can understand the dynamic changes of the drug in the body, providing important information for clinical diagnosis and treatment.

[0110] The term "tumor" refers to a mass formed by the abnormal proliferation of cells in the body. This abnormal proliferation can be benign or malignant, and is usually determined based on the growth rate and invasiveness of the tumor cells. These tumors are classified into the following types: squamous cell papilloma, adenoma, fibroma, lipoma, rhabdomyosarcoma, lymphangioma, chondroma, glioma, ganglioneuroma, meningioma, hydatidiform mole, squamous cell carcinoma, adenocarcinoma, fibrosarcoma, liposarcoma, angiosarcoma, malignant stromal tumor, malignant lymphoma, leukemia, invasive hydatidiform mole, malignant melanoma, lymphoma, neuroendocrine tumors and sarcomas, leiomyomas, hemangiomas, gastric stromal tumors, gastric sarcomas, gastric cancer, colorectal polyps, colon cancer, rectal cancer. Cancer, focal nodular hyperplasia of the liver, hepatic hemangioma, hepatic cyst, hepatic adenoma, cirrhosis, liver cancer, sebaceous cyst, sebaceous nevus, syringoma, keloid, fibrosarcoma, basal cell carcinoma, squamous cell carcinoma, eczematous carcinoma, schwannoma, meningioma, glioma, congenital tumors (epidermoid cyst, dermoid cyst, teratoma), cavernous hemangioma, hemangioblastoma, metastatic tumor, granuloma, hematologic malignancies, multiple myeloma, nervous system tumors, urinary system tumors, head and neck malignancies, etc.

[0111] The term “treatment” means to reduce or improve a disease, disorder, or condition (i.e., to slow or stop the development or progression of a disease, disorder, or condition or at least one of its clinical symptoms); or to reduce or improve at least one physical parameter or biomarker associated with the disease, disorder, or condition, including those physical parameters or biomarkers that the patient may not be able to identify.

[0112] The effective therapeutic dose described in this disclosure depends on the species, weight, age and individual condition of the subject, the disorder or disease being treated or its severity.

[0113] In one embodiment, the fusion protein of this disclosure is present in a pharmaceutical formulation as a single dose. Those skilled in the art can readily determine a suitable therapeutically effective dose. A suitable dose of the fusion protein disclosed in this disclosure can be calculated based on the subject's weight; for example, a suitable dose range may be about 0.1 to about 5 mg / kg, such as about 0.5 to about 5 mg / kg. In one embodiment, the fusion protein is administered at a dose of 0.1 to 5 mg / kg every 2 to 6 weeks. For effective treatment of diseases such as breast cancer, lung cancer, colorectal cancer, or pancreatic cancer in humans, a suitable dose range may be about 0.1 to about 300 mg, such as about 0.1 to about 300 mg, such as about 300 mg, such as about 0.1 to about 100 mg, or about 0.1 to about 50 mg, or about 0.1 to about 20 mg, or about 0.1 to about 10 mg. The fusion protein provided in this disclosure, in doses of 1 to about 15 mg, or for example 1 to about 30 mg, or 1 to about 60 mg, can be administered via a parenteral route, such as subcutaneous, intravenous, or intratumoral injection. If necessary, the physician may choose appropriate intervals for repeated administration.

[0114] In one embodiment, the radioprotein-conjugated drug of this disclosure is present in a single-dose form in a pharmaceutical formulation. Those skilled in the art can readily determine an appropriate therapeutically effective dose of the radioprotein-conjugated drug. An appropriate dose can be calculated based on the subject's weight; for example, an appropriate dose range may be about 0.01 to about 4 mCi / kg, such as about 0.1 to about 4 mCi / kg. In one embodiment, the radioprotein-conjugated drug is administered at a dose of 0.1 to 4 mCi / kg every 2 to 6 weeks. For the effective diagnosis or treatment of diseases such as breast cancer, lung cancer, colorectal cancer, or pancreatic cancer in humans, an appropriate dose range may be about 1 to about 200 millicuries, such as about 0.1 to about 200 millicuries, such as about 200 millicuries, such as about 0.1 to about 120 millicuries, or about 0.1 to about 80 millicuries, or about 0.1 to about 60 millicuries, or about 0.1 to about 40 millicuries. The radioactive protein-coupled drug disclosed herein, in doses of 1 to about 60 millicuries, or for example 1 to about 80 millicuries, or 1 to about 120 millicuries, can be administered via parenteral route, such as subcutaneous, intravenous, or intratumoral injection. If necessary, the drug can be repeated at appropriate intervals at the physician's discretion.

[0115] The term "diagnosis" includes tumor imaging examinations, such as using imaging techniques to determine the location, size, and shape of lesions, assess the effectiveness of disease treatment, or determine the metastasis of tumors.

[0116] TROP2, short for Trophoblast cell surface antigen-2, is a glycoprotein encoded by the TACSTD2 gene that participates in transmembrane calcium signaling. TROP2 is overexpressed in various malignant tumors, such as cervical cancer, urothelial carcinoma, papillary thyroid carcinoma, breast cancer, squamous cell carcinoma of the lung, endometrial cancer, prostate cancer, colon cancer, lung adenocarcinoma, ovarian epithelial cancer, gastric cancer, and pancreatic cancer, and is associated with poor prognosis and increased risk of metastasis. TROP2 is expressed at low levels in normal tissues.

[0117] DLL3 (Delta-like ligand 3) is highly expressed in a variety of neuroendocrine tumors (such as small cell lung cancer, neuroendocrine prostate cancer, and gastrointestinal neuroendocrine carcinoma), but its expression is extremely low in normal tissues.

[0118] Example 1: Preparation of antibody samples

[0119] 1.1 Construction of antibody expression vector

[0120] The antibody sequence (SEQ ID NO. 36) specifically targeting human Trop2 was optimized for amino acid codons according to the human host cell expression system. A nucleotide sequence encoding a protein expression-related signal peptide was added, and the gene was synthesized. The synthesized target gene was then cloned into the pTT5 (ampicillin-resistant) vector via 5'EcoRI and 3'HindIII restriction enzyme digestion. After construction, the vector was transformed. Clones were selected for sequencing, and cells with correct sequencing results were cultured, plasmids were extracted, and their concentrations were determined.

[0121] The antibody proteins in Table 2 were expressed and purified for further experimental testing, and mutants affecting antibody half-life were studied.

[0122] Table 2. Names, sequences, and Fc mutation information of the expressed antibodies used in half-life studies.

[0123] 1.2 Cell Culture and Antibody Expression

[0124] Cell transfection and antibody expression were performed using the extracted plasmids as follows:

[0125] (1) Measure cell density. Viability should be greater than 95%. Adjust the density of HEK293 cells (purchased from ATCC, catalog number CRL--1573.3) to 3×10⁻⁶ using preheated HEK293 medium. 6 Cells / mL, gently shake and dispense cells, ensuring the cell volume in the shake flask does not exceed 1 / 3 of the flask's capacity, and place on a shaker for later use.

[0126] (2) Calculate the volume of the transfection buffer opti-MEM based on the transfected cell volume. This volume is 1 / 10 of the transfection system. Calculate the amount of the transfection reagent PEI (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-K2014), which is 9 μg / mL for transfecting cells. Calculate the total amount of transfected DNA, which is 1 μg / mL for transfecting cells.

[0127] The specific transfection procedure is as follows:

[0128] Take one 50mL centrifuge tube, add 10% Opti-MEM (purchased from Beijing Zhong Sheng Aobang Biotechnology Co., Ltd., catalog number 03.18001A) to the transfection system, add plasmid, mix well, filter, and let stand for 5 min. Add PEI to the DNA suspension, mix gently (invert gently 2-3 times), and let stand for 15-20 min. Then gently add the complex to the aliquoted cells, gently shaking the flask while adding. Incubate the transfected cells in a shaker at 37℃. Add 293mg of serum-free feed medium on days 1, 3, and 5 after transfection. Purify the protein after 7 days of cell culture.

[0129] 1.3 Antibody Purification

[0130] (1) Sample preparation

[0131] The collected cell supernatant was centrifuged using a benchtop centrifuge at 4000g for 30 minutes. The cell supernatant was then collected and filtered through a 0.45μM filter membrane.

[0132] (2) Antibody purification

[0133] The cell culture supernatant after centrifugation was purified using the protein G affinity purification method (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number P2017) to harvest monoclonal antibodies. The operation steps are as follows.

[0134] 1) Ligation system: Select a protein G column of appropriate specifications based on the expression level and ligate it to the purification system;

[0135] 2) Water balance: Rinse the 3CV with ultrapure water to replace the 25% ethanol preservation solution;

[0136] 3) Equilibration column: Equilibrate the column with 5CV equilibration buffer (50mM Tris, 100mM NaCl, pH 8.0);

[0137] 4) Sample loading: Adjust the flow rate to a suitable level for sample loading;

[0138] 5) Washing: Elute the column with equilibration buffer (50mM Tris, 100mM NaCl, pH 8.0) for 10 CV, at the same flow rate as the loading flow rate;

[0139] 6) Elution: Elute and collect antibodies using elution buffer (100mM Glycine, 10mM NaCl, pH 3.0);

[0140] 7) Neutralization: Add 2M Tris, pH 8.0 to neutralize and elute the antibody;

[0141] 8) Desalting: Prepare a PD10 desalting column and wash the column with 6CV PBS 7.4. Add 3mL of elution buffer from step (7), and then add 3mL of PBS 7.4 to elute and obtain the antibody.

[0142] Example 2: Effect of different Fc mutants on molecular half-life

[0143] 2.1 Affinity test of different Fc mutants for FcRn binding

[0144] The equilibrium dissociation constant (KD) of the antibody sample described above bound to human FcRn protein was determined using biological membrane optical interferometry (BLI). The BLI affinity assay procedure is briefly described below:

[0145] (1) Immobilized ligands

[0146] Dilute antibody samples to 5 μg / mL using running buffer PBS, fix with a ProA sensor for 300 s; dilute ligand samples to 5 μg / mL using running buffer, fix with a His1K sensor for 180 s.

[0147] (2) Preparation and detection of analytes

[0148] Human FcRn protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number CT009-H08H) was diluted to 200 nM using running buffer. Binding time: 120 s; dissociation time: 180 s. Antibody samples were also diluted to 50 nM using running buffer, and then serially diluted 2-fold. Binding time: 120 s; dissociation time: 180 s.

[0149] 3) Analysis of experimental results

[0150] The results were analyzed using Data Analysis 12.0 software to obtain the binding rate, dissociation rate, and affinity constant, as shown in Table 3.

[0151] Table 3. Results of the determination of the affinity of different Fc mutants for FcRn. Note: "Non-binding" means that no clear binding effect was detected when using the BLI method for multi-concentration affinity testing, and the relevant parameters KD(M), kon(1 / Ms), and kdis(1 / s) could not be obtained.

[0152] Affinity assays of different Fc variants with FcRn showed that the H435Q mutation introduced into the Fc region, or combinations of mutations such as H435A, H435Q, H310A, and H310Q, can completely eliminate the binding effect of Fc to FcRn. This suggests that αTrop2-Fc v1, αTrop2-Fc v2, αTrop2-Fc v3, and αTrop2-Fc v4 may be cleared more rapidly in vivo compared to αTrop2-WT Fc.

[0153] 2.2 Animal in vivo test of the effect of different Fc mutants on antibody half-life

[0154] Healthy adult SD rats (weighing 200-300 grams) were selected and randomly divided into 5 groups of 3 rats each, according to the experimental design. Each group was injected with one of the 5 antibody samples listed in Table 2 at 5 mg / kg via tail vein.

[0155] Blood samples were collected at different time points after antibody injection: 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 168 hours post-injection. ELISA testing was performed on the collected samples. The antibody concentration data over time were fitted using an exponential decay model. The half-life was calculated based on the fitted parameters to assess antibody metabolism in rats (see Table 4).

[0156] Table 4. Results of half-life determination of naked antibodies against different Fc mutants in rats.

[0157] 2.3 Antibodies 177 Lu-labeled and marker-based half-life testing in animal blood

[0158] Antibody conjugation: Dissolve 4 mg of antibody in 0.1 M NaHCO3 (pH 9.0) to a final concentration of 4 mg / mL. Add 10 equivalents of DOTA-NHS-ester (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-128890) and react overnight at room temperature. Centrifuge with PBS to remove unreacted DOTA-NHS-ester small molecule compounds.

[0159] Chelation of radionuclides: Open the metal bath reactor and preheat to 40°C. Dilute the conjugated antibody with PBS solution to 5 mg / mL to obtain the precursor solution. Take 37 MBq (1 mCi). 177 LuCl3 solution (50 pmol) was added, followed by 4 equivalents of the precursor solution, and then the volume was increased to 50 μL with sodium vitamin C buffer. The mixture was reacted at 40 °C and 200 rpm for 2 h to obtain the desired product. 177 Lu-labeled antibody molecules exist in solution form.

[0160] Thin-layer chromatography (TLC) was performed using 1% sodium citrate solution as the developing solvent. If the purity was greater than 95%, no further processing was required. If the purity was less than 95%, further purification was necessary: ​​centrifugation using an ultrafiltration centrifuge tube was performed to remove uncoordinated components. 177 Lu. Determine the radioactivity of the chelated and purified drug.

[0161] 177Half-life test of Lu-antibody molecules in animal blood: Healthy adult SD rats (weighing 200-300 grams) were selected and randomly divided into 5 groups of 3 rats each, according to the experimental design. Each group was injected with 300 μCi of one of the chelated samples mentioned above via tail vein. Blood samples were collected at different time points after antibody injection: 0.5 hours, 1 hour, 17 hours, 24 hours, 48 ​​hours, and 72 hours post-injection. Gamma counter analysis was performed, and the activity of the radiopharmaceutical in the blood at different time points was calculated based on the gamma counter results. The activity of different Fc mutants was then calculated. 177 The half-life of Lu-antibodies in animal blood is shown in Table 5.

[0162] Table 5 is different. 177 Half-life determination results of Lu-labeled Fc mutant in rats

[0163] The results above show that, after... 177 The Lu-labeled antibody showed a basically unchanged or reduced half-life in rat blood. The mutant with a reduced half-life also means that the radioactive material can be cleared from the body more quickly.

[0164] These Fc mutation points are important for 177 The effects of Lu labeling on the half-life of antibodies are different, and it is uncertain whether radionuclide-labeled antibodies prepared based on this will affect their uptake in animals.

[0165] Example 3: Effects of different Fc mutants on the binding of the molecule to FcγRs and C1q.

[0166] The Fc fragment can bind to cells expressing FcγRs in vivo, and it can also bind to proteins in the complement system such as C1q. This may ultimately lead to antibody molecules binding to normal cells and tissues through the Fc-mediated action. If labeled with a radionuclide, it will inevitably cause stronger toxic side effects on normal tissues. Therefore, it is necessary to remove or partially remove these effects by modifying the amino acids on the Fc fragment. In this example, we also used the Fc mutant from patent application number CN202280030415.6 to construct the antibody αTrop2-ABD Fc for comparative studies. The information is summarized below. The antibodies in Table 6 were expressed and purified according to the method in Example 1 for further affinity determination.

[0167] Table 6. Names, sequences, and Fc mutation information of expression antibodies used in FcγRs and C1q binding studies.

[0168] Using αTrop2-WT Fc as a reference, the degree of change in binding affinity between different mutants and different FcγR proteins and C1q complement proteins was compared. Among them, the mutant combinations of αTrop2-Fc v8, αTrop2-Fc v9, αTrop2-Fc v10 and αTrop2-Fc v11 antibodies affecting the binding affinity of FcγRs and C1q were duplicated with some other antibody Fc region mutation positions. Therefore, the affinity test of antibody Fc region affecting the binding affinity of αTrop2-Fc v8, αTrop2-Fc v9, αTrop2-Fc v10 and αTrop2-Fc v11 for binding affinity of FcγRs and C1q was not performed here. The test results are shown in Table 7.

[0169] Table 7 shows the percentage change in affinity for FcγRs and C1q among different mutants compared to αTrop2-WT Fc. Note: "(↓)" indicates the percentage decrease in affinity compared to αTrop2-WT Fc. For example, "396.3(↓)" means that the antibody with the L234A and L235A mutation combination in the Fc region has a 396.3% decrease in affinity for the CD64 receptor protein compared to αTrop2-WT Fc. "No binding" means that the corresponding antibody mutant cannot be detected to bind significantly to the receptor protein using this experimental method, or that the corresponding antibody mutant has completely lost its ability to bind to the receptor.

[0170] Based on the experimental results, the following inferences can be drawn:

[0171] (1) Antibody mutants that have completely lost their ability to bind to different receptors may have lower uptake in normal tissues after being labeled with radionuclides. Specifically, compared with αTrop2-WT Fc molecules without any mutations and antibody variants that still have the ability to bind to CD64, such as αTrop2-Fc v5, αTrop2-Fc v9, and αTrop2-Fc v13, these antibody variants that have removed Fc and FcγRs and C1q may have lower radioactive uptake in normal tissues (e.g., liver), while radioactive uptake in tumor tissues may be higher or remain unchanged.

[0172] (2) Among these antibody variants that remove Fc and FcγRs, C1q, under the condition of containing the same combination of mutation sites affecting half-life, the uptake of these mutants in normal and / or tumor tissues in animals after labeling with radionuclides may be the same. For example, αTrop2-Fc v6, αTrop2-Fc v7 and control molecule αTrop2-ABD Fc, αTrop2-Fc v8 and αTrop2-Fc v10, αTrop2-Fc v11 and αTrop2-Fc v12, αTrop2-Fc v14 and αTrop2-Fc v15 have the same uptake in animal tissues after labeling with radionuclides.

[0173] Next, we will conduct experimental verification of the above hypothesis through specific and extensive experiments.

[0174] Example 4: Imaging test of antibody molecules containing different mutant combinations in tumor-bearing mice.

[0175] 4.1 Antibody conjugation and labeling

[0176] The antibody molecules in Table 6 were conjugated and 177 Lu labeling, preparation 177 Lu-antibody radiopharmaceuticals are used for in vivo imaging studies in tumor-bearing mice.

[0177] Amide bond coupling procedure: Take a 1.5 mL EP tube, add 0.1 M NaHCO3, pH 8-10 solution, and 2 mg of antibody to make the reaction concentration 2 mg / mL. Add 10 times the molar concentration of NHS-DOTA (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-128890), shake at room temperature for 24 hours, and then replace with phosphate buffer to remove unreacted NHS-DOTA.

[0178] 4.2 Establishment of animal tumor models

[0179] Experimental balb / c nude mice were subcutaneously inoculated with 5 × 10⁸ cells in the right axilla. 6 A certain number of MDA-MB-468 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 mL / cell). Tumor growth was observed periodically until the tumors reached an average volume of 180-220 mm². 3 (approximately 200mm) 3 The mice were randomly grouped and given medication based on tumor size and body weight.

[0180] 4.3 Single-photon emission computed tomography (SPECT / CT) and data processing

[0181] Will 177Lu-antibody was diluted with physiological saline to 7.4 MBq (200 μCi) / 200 μL, and 7.4 MBq (200 μCi) of the drug was administered to each tumor-bearing mouse via tail vein injection. Mice were continuously anesthetized with isoflurane at 24, 48, 72, 96, and 120 hours post-administration, placed prone on an examination table, and scanned using single-photon emission computed tomography (SPECT) (model: IVIS spectrum, manufacturer: MILabs). Acquisition methods included static 10-minute SPECT and medium-resolution whole-body computed tomography. During the scans, animal weight, injection dose, injection time, and residual dose were recorded according to the log, as well as the time for measuring the injection dose and the time for measuring the residual dose. Data were reconstructed after scanning, and PMOD software was used to analyze and delineate tumor, liver, and kidney tissues, quantify drug distribution, save images and data, and perform further statistical analysis.

[0182] The radioactive uptake of tumor, liver, and kidney tissues at different time points was analyzed using PMOD software. The experimental results are shown in Table 8.

[0183] Table 8. Uptake (%ID / g) of different radiolabeled antibody mutants in tumors, liver, and kidneys at different time points.

[0184] The results above show that the uptake of radiopharmaceuticals in tumor tissue varies among these different Fc mutants. Among the 11 mutants from αTrop2-Fc v5 to αTrop2-Fc v15, αTrop2-Fc v5, αTrop2-Fc v6, and αTrop2-Fc v7 showed superior or comparable uptake of radiopharmaceuticals in tumor tissue at different time points. In particular, αTrop2-Fc v5 showed the best tumor uptake at all tested time points and was superior to the control molecule αTrop2-ABD Fc. This is completely contrary to the prediction in Example 3, which is an unexpected result. Furthermore, in addition to the low level of tumor uptake, some molecules in the mutants αTrop2-Fc v8 to αTrop2-Fc v15 also exhibited prolonged and high uptake in normal liver tissue, such as αTrop2-Fc v8, αTrop2-Fc v11, αTrop2-Fc v12, αTrop2-Fc v13, αTrop2-Fc v14, and αTrop2-Fc v15, which is completely contrary to our speculation in Example 3.

[0185] In summary, not all mutants that reduce or completely eliminate binding to FcγRs, C1q, and FcRn exhibit ideal uptake in tumors and the liver. Among the tested series of Fc mutation combinations, the combinations of L234A, L235A, H435Q, L234A, L235A, P329G, H435Q, and L234A, L235A, D265A, H435Q showed excellent characteristics of high uptake of radionuclide-labeled antibody molecules in mouse tumor tissues and low uptake in liver tissues. Among them, the L234A, L235A, H435Q mutation combination showed a greater advantage in high uptake in tumors than the control mutant αTrop2-ABD Fc.

[0186] Figures 1 to 12 illustrate these results more intuitively, with tumor tissue circled by white elliptical dashed lines.

[0187] 4.4 αTrop2-Fc v5 and αTrop2-ABD Fc 89 Zr labeling and positron emission tomography (PET-CT) testing

[0188] To compare the differences in tumor uptake using different radionuclide labels αTrop2-Fc v5 and αTrop2-ABD Fc, we used 89 Zr was used for antibody labeling and imaging. The chelating agent used was p-SCN-Bn-deferoxamine (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-134797), conjugated with and 89 The specific steps of the Zr labeling method are as follows: Take 4 mg of antibody, dissolve it in 0.1 M NaHCO3 solution, pH 8.5-9.5, add 5 equivalents of p-SCN-Bn-deferoxamine chelating agent, react overnight at room temperature, and then concentrate the antibody using an ultrafiltration tube. Take 2 mg of the concentrated antibody and mix it with 90 MBq of... 89 Mix Zr, adjust pH to 7.2, and incubate at room temperature for 60 minutes. Use EDTA with unbound Zr. 89 The Zr reaction was incubated at room temperature for 15 minutes. Purification was then performed using gel filtration on a disposable PD10 column.

[0189] The labeled molecules were injected via tail vein into a mouse model bearing MDA-MB-468 tumors. Micro-PET / CT (Super Nova, manufacturer: Ping Sheng Medical Technology (Kunshan) Co., Ltd.) scans were performed to measure the %ID / cc of tumor lesions. Small animal positron emission tomography (PET) scans were performed at 24, 48, 72, 96, 120, and 144 hours to analyze the radioactive uptake of tumor tissue at multiple time points, as shown in Figure 13. Comparison revealed that…89 Zr-αTrop2-Fc v5 molecules showed superior radioactive uptake by tumor tissue at all time points compared to [other methods]. 89 The Zr-αTrop2-ABD Fc molecule further demonstrates the superiority of the L234A, L235A, and H435Q mutant combination.

[0190] Example 5: Test of Thiol Coupling

[0191] The following simplified method was used for DOTA chelating and thiol-coupling of αTrop2-ABD Fc and αTrop2-Fc v5: First, the disulfide bonds of the antibody were reduced for 1 hour at 37°C using 10 equivalents of tris(2-carboxyethyl)phosphine (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number: HY-W011500). Then, 4 equivalents of the chelating agent Maleimide-DOTA (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number: HY-133540) dissolved in DMSO were added to the solution. After incubation at 37°C for 12 hours, the chelating agent bound to the thiol group of the antibody. The coupled sample was then purified using PBS in ultrafiltration centrifuge tubes.

[0192] The products αTrop2-ABD Fc-Mal-DOTA and αTrop2-Fc v5-Mal-DOTA, which are conjugated from αTrop2-ABD Fc and αTrop2-Fc v5, were tested by LC-MS according to the following steps:

[0193] 1) Sample processing

[0194] After desalting a 2 mg / mL sample, incubate it with PNGase F (catalog number: P0704, manufacturer: New England Biotechnology Co., Ltd.) at 37℃ for 1 hour to remove sugar. After incubation, transfer the sample to a vial for analysis.

[0195] 2) Liquid chromatography-mass spectrometry (LC-MS)

[0196] The test sample was separated using an ultra-high performance liquid chromatography (UHPLC) system (Vanquish UPLC, Thermo Fisher Scientific). Phase A was a 0.1% formic acid-water solution, and Phase B was a 0.1% formic acid-acetonitrile solution. The test sample was loaded using an autosampler and then passed through a chromatographic column (ACQUITY UPLC BEH C4, 2.1*50mm, 1.7μm). Gradient separation was performed using a Waters spectrometer (model: Q Exactive HF-X, brand: Thermo Fisher Scientific). The analysis time was 10 min, the detection mode was positive ion, and the precursor ion scan range was 500–4000 m / z. The experimental results are shown in Figures 14-15 and Table 9.

[0197] Table 9 Summary of mass spectrometry results after thiol coupling of the two molecules

[0198] The results above clearly show that, when using thiol coupling, the Fc v5 mutant is more efficient and produces a more homogeneous product than the positive control ABD Fc mutant.

[0199] Example 6: Single-photon emission computed tomography (SPECT / CT) results of αTrop2-Fc v5 with thiol coupling in tumor-bearing mice.

[0200] The αTrop2-Fc v5-Mal-DOTA molecule from Example 5 was subjected to... 177 Lu-labeled mice bearing MDA-MB-468 tumors were injected via the tail vein. SPECT / CT scans were performed at different time points, and PMOD software was used to delineate the tumors, liver, and kidneys. The imaging results are shown in Figure 16.

[0201] Table 10 shows the uptake results of three different tissues at different time points after PMOD software criterion processing:

[0202] Table 10 PMOD treatment results (%ID / g) of αTrop2-Fc v5 via thiol coupling in tumor-bearing mice.

[0203] The results showed that molecules constructed using thiol coupling underwent [a process / process]. 177 Even after Lu radiolabeling, high uptake values ​​can still be achieved in tumor tissue over a long period of time.

[0204] Example 7: Distribution test of αTrop2-Fc v5 in tumor-bearing mice

[0205] Referring to the antibody-chelating agent conjugation scheme in Example 6, the DOTA-NHS (purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number: HY-128890) chelating agent corresponding antibody αTrop2-Fc v5 was conjugated and purified. After completion, it was used... 177Lu nuclide-labeled mice were administered the drug at a dose of 50 μCi per mouse. The mice were euthanized at 1 hour, 4 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours and 120 hours after administration. Different tissue samples were collected for weight measurement and gamma particle counting. The proportion of drug dose per unit mass organ at the corresponding time point to the total injected dose (%ID / g) was calculated. Three mice were selected at each time point.

[0206] The results are shown in Figure 17. 177 Lu-labeled αTrop2-Fc v5 drug accumulated in tumor tissues over time after injection, reaching a maximum distribution value of 43.35% ID / g at 72 hours. In other normal organs, the drug concentration decreased over time after injection.

[0207] Example 8: Antitumor efficacy test of αTrop2-Fc v5 in tumor-bearing mice

[0208] MDA-MB-468 cells were inoculated into Balb / c Nude mice, and tumors grew to 200–300 mm in size. 3 Mice were divided into groups of 8 at intervals. DOTA-NHS conjugation was performed on αTrop2-Fc v5 and... 177 After Lu labeling, mice were administered the drug via tail vein at three different activity doses: 0.45 mCi / mouse, 0.90 mCi / mouse, and 1.35 mCi / mouse. Control groups were administered with PBS buffer and unconjugated αTrop2-Fc v5 naked antibody (100 μg / mouse). Tumor volume was measured periodically after administration. Seventeen days later, mice were euthanized, and tumor samples were harvested and photographed.

[0209] Figures 18 and 19 show the antitumor drug efficacy results, indicating that αTrop2-Fc v5, after... 177 Lu-labeled drugs showed excellent efficacy in inhibiting tumor growth.

[0210] Example 9: Tissue distribution test of αDLL3-Fc v5 in tumor-bearing mice

[0211] 9.1 Antibody labeling and establishment of tumor-bearing mouse model

[0212] After initial screening, a nanobody specifically binding to the DLL3 protein (underlined sequence) was obtained. This nanobody was then fused with Fc v5 for expression, preparing αDLL3-Fc v5 for tissue distribution and antitumor efficacy testing in tumor-bearing mice. The αDLL3-Fc v5 sequence information is as follows:

[0213] Table 11 αDLL3-Fc v5 antibody sequence information

[0214] The method described in Example 5 is used to perform DOTA-Mal coupling on αDLL3-Fc v5, and the tag is... 177 Following the administration of Lu radionuclide to tumor-bearing mice, the labeled drug was named... 177 Lu-αDLL3-Fc v5. The information regarding the establishment of the tumor-bearing mouse model is as follows:

[0215] Balb / c nude mice were subcutaneously inoculated with 5 × 10⁸ cells under the right axilla. 6 A certain number of NCI-H82 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 mL / cell). Tumor growth was observed periodically until the tumor reached an average volume of 180-220 mm². 3 (approximately 200mm) 3 The mice were randomly grouped and given medication based on tumor size and body weight.

[0216] 9.2 SPECT / CT Scanning and Data Processing

[0217] Will 177 Lu-αDLL3-Fc v5 was diluted with physiological saline to 7.4 MBq (200 uCi) / 200 μL, and 7.4 MBq (200 μCi) of the drug was administered to each tumor-bearing mouse via tail vein injection. Mice were continuously anesthetized with isoflurane at 1, 24, 48, 72, 96, and 120 hours after administration, placed prone on an examination table, and underwent SPECT / CT scans. Acquisition methods included static 10-minute SPECT scans and medium-resolution whole-body CT scans. During the scans, animal weight, injection dose, injection time, and residual dose were recorded according to the record sheet, as well as the time for measuring the injection dose and the time for measuring the residual dose. Reconstructed data were generated after the scans, and the imaging results are shown in Figure 20, showing the results over time. 177 Lu-αDLL3-Fc v5 accumulates in tumor tissue and is retained for a long time.

[0218] 9.3 177 Tissue distribution of Lu-αDLL3-Fc v5 in tumor-bearing mice

[0219] Mice were anesthetized and euthanized at different time points after drug administration. Tissues were dissected and analyzed for radioactive gamma particle counting. The biodistribution ratio of the drug in different tissues was then calculated. The results of the in vitro tissue distribution are shown in Figure 21. 177 Lu-αDLL3-Fc v5 achieved high numerical distribution in tumor tissue over a long period, reaching a maximum of 31.10% ID / g at 72 hours, while remaining at lower levels in other normal tissues.

[0220] 9.4177 Antitumor effect test of Lu-αDLL3-Fc v5 in animals

[0221] Determined using xenograft mouse models 177 In vivo antitumor efficacy of Lu-αDLL3-Fc v5. 5×10 6 A number of NCI-H82 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 mL / cell). Tumor growth was observed periodically until the tumor reached an average volume of 110 mm². 3 Mice were divided into groups of 8 each. After grouping, they were administered medication as shown in Table 12. Tumors were observed and measured twice weekly, and mouse body weight was measured twice weekly until the end of the study. Tumor volume was defined as TV = 0.5a × b. 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor.

[0222] Table 12 Mouse grouping and administration information

[0223] On day 10 after administration, the tumor inhibition rate of different treatment groups was calculated based on the measured tumor volume. The formula was TGI = (1-T / C) × 100%, where T is the average tumor volume of different treatment groups and C is the average tumor volume of the PBS control group. The results are shown in Table 13.

[0224] Table 13 177 Inhibition rate of Lu-αDLL3-Fc v5 on tumor growth (%)

[0225] 177 The antitumor efficacy experiment of Lu-αDLL3-Fc v5 in tumor-bearing mice showed that it had a significant tumor growth inhibition effect in both medium and high dose groups, and the effect was dose-dependent. The high dose group showed a strong antitumor efficacy (see Figure 22).

[0226] The above examples demonstrate that the Fc v5 mutant exhibits excellent tumor and normal tissue distribution and anti-tumor efficacy. Those skilled in the art can conclude from the above studies that Fc v6 and Fc v7 will also have similar excellent technical effects.

[0227] Finally, it should be noted that the above content is only used to illustrate the technical solution of this disclosure, and is not intended to limit the scope of protection of this disclosure. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of this disclosure do not depart from the substance and scope of the technical solution of this disclosure.

Claims

1. A fusion protein, wherein, The fusion protein comprises at least one receptor-binding fragment and an IgG-derived Fc domain fused to the receptor-binding fragment. For example, the fusion protein is formed by fusing at least one receptor-binding fragment with an IgG-derived Fc domain; The Fc domain is mutated at the following positions: L234, L235, and H435.

2. The fusion protein according to claim 1, wherein, The Fc domain is also mutated at the following locations: P329 and / or D265.

3. The fusion protein according to claim 1 or 2, wherein, The IgG is selected from human IgG1, IgG2 and IgG4 subtypes.

4. The fusion protein according to any one of claims 1 to 3, wherein, The original amino acid at the mutation site of the Fc domain is mutated to any one of Gly, Ala, Val, Leu, Ile, Phe, Pro, Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Trp, or Met.

5. The fusion protein according to any one of claims 1 to 4, wherein, The amino acid sequence before the Fc domain mutation is shown in SEQ ID NO.

32.

6. The fusion protein according to any one of claims 1 to 5, wherein, The combination of Fc domain mutation sites is selected from: (1) L234A, L235A and H435Q; (2) L234A, L235A, P329G, and H435Q; and (3) L234A, L235A, D265A and H435Q.

7. The fusion protein according to claim 6, wherein, The amino acid sequence of the fusion protein comprising combination (1) is shown in SEQ ID NO.21; the amino acid sequence of the fusion protein comprising combination (2) is shown in SEQ ID NO.22; and / or the amino acid sequence of the fusion protein comprising combination (3) is shown in SEQ ID NO.

23.

8. The fusion protein according to any one of claims 1 to 7, wherein, The receptor-binding fragment is connected to the Fc domain via a hinge region.

9. The fusion protein according to claim 8, wherein, The amino acid sequence of the hinge region is shown in SEQ ID NO.

35.

10. The fusion protein according to any one of claims 1 to 9, wherein, The receptor-binding fragment is selected from the group consisting of ligands naturally present in the body or their truncated forms, single-domain heavy chain antibodies, single-chain variable fragments, and bispecific antibody-binding fragments.

11. The fusion protein according to claim 10, wherein, The receptor-binding fragment is a single-domain heavy chain antibody.

12. A nucleic acid, wherein, The nucleic acid encodes the fusion protein according to any one of claims 1 to 11.

13. A carrier, wherein, The vector comprises the nucleic acid as described in claim 12.

14. A host cell, wherein, The host cell comprises the nucleic acid of claim 12 or the vector of claim 13.

15. The use of the fusion protein according to any one of claims 1 to 11, the nucleic acid according to claim 12, the vector according to claim 13, or the host cell according to claim 14 in the preparation of a radioprotein-conjugated drug.

16. A radioactive protein-coupled drug, wherein, The radioactive protein-coupled drug comprises the fusion protein according to any one of claims 1 to 11, and a small molecule cytotoxic drug or a compound chelated with a radionuclide conjugated to the fusion protein. For example, the radioactive protein-coupled drug uses the fusion protein described in any one of claims 1 to 11 as a carrier to conjugate a small molecule cytotoxic drug or a compound chelated with a radionuclide.

17. The radioprotein-conjugated drug according to claim 16, wherein, The radionuclides include, but are not limited to, those mentioned above. 111 In、 223 Ra、 67 Ga、 68 Ga、 44 Sc、 90 Y、 177 Lu、 225 Ac、 212 Bi、 213 Bi、 212 Pb, 227 Th、 64 Cu、 67 Cu、 89 Zr.

18. The radioprotein-conjugated drug according to claim 16, wherein, The pathway by which the fusion protein is coupled to the compound chelated with the radionuclide is selected from: A: Coupling is achieved through a connector and a chelating ring; B: Coupling via free or reduced thiol groups; and C: Coupling via exposed amino groups.

19. A pharmaceutical composition, wherein, The pharmaceutical composition comprises: the fusion protein of any one of claims 1 to 11, the nucleic acid of claim 12, the vector of claim 13, the host cell of claim 14, and / or the radioprotein-conjugated drug of any one of claims 16 to 18.

20. The use of the fusion protein of any one of claims 1 to 11, the nucleic acid of claim 12, the vector of claim 13, the host cell of claim 14, the radioactive protein-conjugated drug of any one of claims 16 to 18, or the pharmaceutical composition of claim 19 in the preparation of products for the diagnosis and / or treatment of tumors.

21. A method for diagnosing and / or treating tumors, wherein, This includes administering to a subject in need the fusion protein of any one of claims 1 to 11, the nucleic acid of claim 12, the vector of claim 13, the host cell of claim 14, the radioactive protein-coupled drug of any one of claims 16 to 18, or the pharmaceutical composition of claim 19.

22. The method of treating tumors according to claim 21, further comprising administering an effective amount of another antitumor therapeutic agent to a subject in need; optionally, the other antitumor therapeutic agent is an antibody-drug conjugate and / or a peptide-drug conjugate.

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