Radioactive protein coupling medicine and application thereof

Abstract

The invention provides a radioactive protein coupling medicine and application thereof, and belongs to the field of biological medicine. The invention provides a novel radioactive protein coupling drug, the novel radioactive protein is formed by fusing at least one receptor binding fragment and an Fc structural domain derived from human immunoglobulin G (IgG), mutation modification sites comprise L234, L235 and H435 of the Fc structural domain, and can also comprise P329 and / or D265, and the preferable mutation combination is L234A, L235A and H435Q. After being chelated with radionuclides, the novel fusion protein provided by the invention can be taken in tissues such as blood, kidneys, livers and the like at a low dose and can be rapidly removed, and can be taken in tumor tissues at a high dose for a long time, so that the safety and effectiveness of radiopharmaceutical treatment are greatly improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a radioactive protein-coupled drug and an application thereof. Background Art

[0002] Radiopharmaceuticals are increasingly being used in the treatment of different types of tumors as a safe and effective treatment method. Drugs that preferentially bind to cancer cells or accumulate through physiological mechanisms are usually used to reach the whole body or local lesions, and then the lesions are irradiated by emitting rays to kill the cancer cells. In addition, these radionuclides can emit imageable photons, which can non-invasively observe the biological distribution of therapeutic drugs.

[0003] Radionuclide drug conjugates (RDCs) are an emerging type of precision tumor diagnosis and treatment drug developed based on radionuclide-targeting ligand molecule conjugation technology. They achieve the dual purpose of diagnosis and treatment by using tumor antigen-specific molecular carriers to accurately deliver radionuclides to tumor cells. The basic structure of radionuclide conjugates includes several key components: antibodies, peptides or small molecules that mediate targeting, linkers, chelates, and radioisotopes. The design of the linker of radionuclide conjugates is crucial. Unlike traditional drugs, during the course of action, the linker of radionuclide conjugates does not need to break when it comes into contact with tumor cells, which makes radionuclide conjugates more stable and safe in the body, while also reducing toxicity to normal tissues.

[0004] Radionuclide-conjugated drugs combine the advantages of precise targeting and potent killing. Specific monoclonal antibodies carrying radionuclide payloads are used to target cancer cells, and the energy is concentrated and released within a range of several times the cell diameter, killing cancer cells while limiting the damage to normal tissue cells near tumor cells to the greatest extent. They have good efficacy, few adverse reactions, and can prolong patient survival. They are crucial for the treatment of advanced tumors and have unique advantages in early diagnosis, staging, and treatment evaluation of tumors.

[0005] However, there are still many problems that limit its transformation and promotion, such as low targeting and specificity, high uptake of radionuclides by non-target tissues, and low target-to-non-target ratio; the types of medical radionuclides available are limited, and there are strict requirements on the radionuclide radiation energy, penetration, half-life, etc.; in particular, the biocompatibility and in vivo stability of RDCs need to be further investigated.

[0006] Gary A. Ulaner et al. used the full-length monoclonal antibody Pertuzumab to 89Human experiments after Zr labeling revealed that the molecule was too large in molecular weight (about 150 kDa) and had a long half-life, resulting in long-term, high-dose absorption in tissues such as the liver, heart, spleen, and lungs.

[0007] Large molecular weight proteins (>60 kDa) are usually not filtered by the kidneys and therefore are not excreted in the urine. They are mainly metabolized by the liver because the liver is the main organ that processes these large molecules. When these large molecular weight proteins are prepared into radiopharmaceuticals, they will be highly accumulated in the liver and circulate in the blood for a long time, which may cause the following potential side effects and safety issues.

[0008] 1. Due to the high accumulation of radioactive drugs in the liver, direct damage to liver cells may occur. These injuries may be caused by the direct radiation effect of radioactive drugs or by toxic metabolites produced during the metabolism of drugs in the liver.

[0009] 2. Due to the large molecular weight of these macromolecular proteins, they exist in the blood circulation for a long time. This may lead to the long-term accumulation of drugs in the blood, increasing the potential toxicity to blood cells and bone marrow cells. Blood toxicity may manifest as leukopenia, thrombocytopenia, etc., while bone marrow damage may lead to decreased hematopoietic function and even develop into serious complications such as bone marrow suppression.

[0010] Patent application number 202280030415.6 discovered a combined mutant by mutation modification of the antibody Fc fragment, which can achieve high-dose absorption in tumor tissue while ensuring low-dose absorption in the liver and blood. However, the provided mutant still has indicators that need to be improved, such as low biodistribution in tumor tissue, which may affect the efficacy of the drug. Therefore, it is necessary to study better molecular mutants to further improve the distribution of radioactive drugs in tumor tissue. Summary of the invention

[0011] In order to solve the above problems, the present invention provides a radioactive protein-conjugated drug and its application.

[0012] In one aspect, the present invention provides a fusion protein, wherein the fusion protein is formed by fusion of at least one receptor binding fragment and an Fc domain derived from IgG; The Fc domain was mutated at the following positions: L234, L235 and H435.

[0013] Specifically, the Fc domain may also be mutated at the following positions: P329 and / or D265.

[0014] Specifically, the IgG includes human IgG1, IgG2 or IgG4 subtype.

[0015] Preferably, the IgG is human IgG1.

[0016] Specifically, the original amino acid at the mutation site of the Fc domain 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.

[0017] More specifically, the amino acid sequence of the Fc domain before mutation is shown in SEQ ID NO:32.

[0018] SEQ ID NO:32:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0019] Preferably, the Fc domain mutation sites include: at least one of L234A, L235A, D265A, P329G and / or H435Q.

[0020] In certain specific embodiments of the present invention, the amino acid sequence of the Fc domain after mutation is as follows: SEQ ID NO:17:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:18: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:19: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:20: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:21: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:22: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:23: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:24: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:25: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:26: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:27: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:28: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:29:APEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCQVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLPG; SEQ ID NO:30: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:31: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:34: APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG.

[0021] Further preferably, the combination of the Fc domain mutation sites includes: (1) L234A, L235A and H435Q; (2) L234A, L235A, P329G and H435Q; (3) L234A, L235A, D265A and H435Q.

[0022] In certain specific embodiments of the present invention, the amino acid sequence of the combination (1) is shown as SEQ ID NO:21; the amino acid sequence of the combination (2) is shown as SEQ ID NO:22; and the amino acid sequence of the combination (3) is shown as SEQ ID NO:23.

[0023] Specifically, the receptor binding fragment and the Fc domain are connected via a hinge region.

[0024] In certain specific embodiments of the present invention, the hinge region amino acid sequence is shown in SEQ ID NO:35.

[0025] SEQ ID NO:35:EPKSSCTHTCPPCP.

[0026] 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.

[0027] Preferably, the receptor binding fragment is a single domain heavy chain antibody.

[0028] In certain specific embodiments of the present invention, the amino acid sequence comprising the receptor binding fragment, the Fc domain and the hinge region is as follows: SEQ ID NO:1:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:2:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:3:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:4:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPPCAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:5: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO: 6: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:7: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:8: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:9: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:10:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:11:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:12:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:13: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG; SEQ ID NO:14:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPPCAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVAV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:15: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG; SEQ ID NO:16:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO:33:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG.

[0029] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned fusion protein.

[0030] In still another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid.

[0031] In yet another aspect, the present invention provides a host cell comprising the above-mentioned nucleic acid or vector.

[0032] 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 Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella and Streptomyces; the eukaryotic host cell includes but is not limited to one or more of Saccharomyces cerevisiae, Pichia pastoris, Chinese hamster ovary cells (CHO), monkey kidney cells (COS), baby hamster kidney cells (BHK), mouse embryonic fibroblasts (NIH3T3) or mouse myeloma cells (SP2 / 0 cells).

[0033] In yet another aspect, the present invention provides use of the above-mentioned fusion protein, nucleic acid, vector and host cell in the preparation of radioactive protein-conjugated drugs.

[0034] In another aspect, the present invention provides a radioactive protein-conjugated drug, wherein the radioactive protein-conjugated drug uses the above-mentioned fusion protein as a carrier and is conjugated with a small molecule cytotoxic drug or a compound that can chelate a radionuclide.

[0035] Specifically, the radionuclides include but are not limited to 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.

[0036] Specifically, the method for coupling the fusion protein with the compound chelating radionuclides includes: A: coupling with the chelating ring through a linker; B: coupling through free or reduced sulfhydryl groups; C: Coupling occurs via exposed amino groups.

[0037] In yet another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein, nucleic acid, vector or host cell.

[0038] Specifically, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0039] More specifically, the auxiliary materials include but are not limited to: at least one of ethanol, gentisic acid, vitamin C, vitamin E, resveratrol, quercetin, glutathione and / or lipoic acid.

[0040] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, which includes but is not limited to at least one of a binder, a filler, a lubricant, a preservative, an antioxidant, a cosolvent, an emulsifier, a solubilizer or an osmotic pressure regulator.

[0041] In yet another aspect, the present invention provides the use of the above-mentioned fusion protein, nucleic acid, vector, host cell, radioactive protein-conjugated drug or pharmaceutical composition in the preparation of products for diagnosing and / or treating tumors.

[0042] Specifically, the tumor includes but is not limited to: squamous cell papilloma, adenoma, fibroma, lipoma, rhabdomyoma, lymphangioma, chondroma, glioma, ganglioneuroma, meningioma, hydatidiform mole, squamous cell tumor, adenocarcinoma, fibrosarcoma, liposarcoma, angiosarcoma, malignant stromal mesenchymal tumor, malignant lymphoma, leukemia, invasive hydatidiform mole, malignant melanoma, lymphoma, neuroendocrine tumor and sarcoma, leiomyoma, hemangioma, gastric stromal tumor, gastric sarcoma, gastric cancer, colorectal polyp, colon cancer, rectal cancer , focal nodular hyperplasia of the liver, hepatic hemangioma, liver cyst, hepatic adenoma, cirrhosis, liver cancer, sebaceous cyst, sebaceous nevus, syringoma, keloid, fibrosarcoma, basal cell carcinoma, squamous cell carcinoma, eczematoid carcinoma, neurothecoma, meningioma, glioma, congenital tumors (epidermoid cyst, dermoid cyst, teratoma), cavernous hemangioma, angioreticuloma, metastasis, granuloma, hematological tumors, multiple myeloma, nervous system tumors, urinary system tumors, head and neck malignant tumors.

[0043] In yet another aspect, the present invention provides a treatment method, comprising administering to a subject a drug comprising the above-mentioned fusion protein, nucleic acid, vector, host cell, radioactive protein-conjugated drug or pharmaceutical composition.

[0044] Compared with the prior art, the present invention has the following advantages: 1. The radionuclide-labeled mutant provided by the present invention 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.

[0045] 2. The novel mutant provided by the present invention can achieve long-term, high-dose uptake in the tumor site while maintaining low uptake in normal tissues, enabling the drug to achieve a better tumor treatment effect without damaging normal organs.

[0046] 3. Compared with the unmodified molecules and the molecular structures reported in other patents, the fusion protein structure in the present invention shows significant differences in configuration and sequence, and the radioactive drug uptake in tumor sites and normal tissues is completely different from the results of the mutants that have been reported. This unique innovative structure provides a new treatment strategy in the field of radioactive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The single photon emission tomography results of αTrop2-Fc v5 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0048] Figure 2 The single photon emission tomography results of αTrop2-Fc v6 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0049] Figure 3 The single photon emission tomography results of αTrop2-Fc v7 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0050] Figure 4 The single photon emission tomography results of αTrop2-Fc v8 molecules in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0051] Figure 5 The single photon emission tomography results of αTrop2-Fc v9 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0052] Figure 6 The single photon emission tomography results of αTrop2-Fc v10 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0053] Figure 7 The single photon emission tomography results of αTrop2-Fc v11 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0054] Figure 8 The single photon emission tomography results of αTrop2-Fc v12 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0055] Fig. 9 The single photon emission tomography results of αTrop2-Fc v13 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0056] Fig.10The single photon emission tomography results of αTrop2-Fc v14 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0057] Fig.11 The single photon emission tomography results of αTrop2-Fc v15 molecule in tumor-bearing mice, the white oval dotted line is the circled tumor tissue.

[0058] Fig.12 Single photon emission tomography results of αTrop2-ABD Fc molecule in tumor-bearing mice, and the white oval dotted line is the circled tumor tissue.

[0059] Fig.13 for 89 The results of the radioactive uptake measurement of Zr-labeled molecules in tumor tissues. The white oval dotted line is the circled tumor tissue.

[0060] Fig.14 This is the deconvoluted image of αTrop2-Fc v5.

[0061] Fig.15 This is the deconvoluted image of αTrop2-ABD Fc.

[0062] Fig.16 The single photon emission tomography results of αTrop2-Fc v15-Mal-DOTA molecule in tumor-bearing mice, and the white oval dotted line is the circled tumor tissue. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0064] Definition of terms In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0065] The term "and / or", such as "X and / or Y", is understood to mean "X and Y" or "X or Y", and should be construed to provide explicit support for both meanings or either meaning.

[0066] The three-letter or one-letter codes for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968, IUPAC-IUB Committee).

[0067] The peptide sequences described herein are written according to the usual convention, wherein the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, unless otherwise specifically indicated, it is the L form of the amino acid represented.

[0068] The "antibody" described in the present invention refers to an immunoglobulin, which has a specific molecular structure. Due to the differences in the amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain, it has different antigenicity. Based on this characteristic, immunoglobulins can be divided into five categories, also known as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain.

[0069] For Ig of the same category, it can be further divided into different subclasses based on the composition of the amino acids in the hinge region and the number and location of the disulfide bonds in the heavy chain. For example, IgG can be further divided into IgG1, IgG2, IgG3, and IgG4.

[0070] Light chains are classified as either kappa chains or lambda chains based on differences in their constant regions. In each of the five types of Ig, there are two types: kappa chains or lambda chains.

[0071] In the molecular structure of antibodies, the sequences of the approximately 110 amino acids near the N-terminus of the heavy and light chains show great variability, and this area is defined as the variable region, or Fv region; while the remaining amino acid sequences near the C-terminus show relative stability, and this part belongs to the constant region.

[0072] The variable region contains three hypervariable regions, namely HVR, and four relatively conservative framework regions, namely FR. Among them, these three hypervariable regions play a decisive role in the formation of antibody specificity, so they are also called complementary determining regions, abbreviated as CDR.

[0073] The term "Fc" refers to a fragment in an antibody molecule that does not affect antigen binding and has the property of being easy to crystallize. By analyzing the three-dimensional structure of Fc, we can clearly understand the interaction pattern between the two constant domains CH2 and CH3 of each heavy chain (HC). 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 causing the protein to exert a series of specific biological effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and achieve a longer half-life and other characteristics.

[0074] Fc can bind to a variety of different FcγRs such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRII can be further divided into three subclasses: FcγRIIa (CD32a), FcγRIIb (CD32b) and FcγRIIc (CD32c), and FcγRIII can be further divided into two subclasses: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). In addition, Fc can also bind to proteins on the complement system such as C1q and may eventually bind to the cell membrane. These Fc receptors are expressed on different normal immune cells. If antibody-conjugated nuclide drugs bind to these receptors through the Fc of the antibody, it may cause damage to normal tissues.

[0075] The division of the amino acid coding of the Fc region of the present invention is based on the definition of 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).

[0076] The term "half-life" in this case specifically refers to "half-life of the drug", which refers to the time required for the plasma drug concentration to drop by half. It is an important parameter reflecting the rate of drug elimination in the body. The clearance process of drugs in the blood mainly depends on the body's metabolism (mainly the liver) and excretion (mainly the kidney) functions. Half-life is an important indicator to measure the rate of drug clearance from the blood. Long half-life drugs are slowly eliminated from the body. If used continuously for a long time, the drug is likely to accumulate in the body. Long half-life nuclear drugs may cause liver damage such as liver cell necrosis and inflammatory response, causing drug-induced hepatitis. Some long half-life drugs may interfere with the normal secretion and excretion of bile, causing bile to accumulate in the liver. Bile stasis will further cause damage to the intrahepatic bile duct, and in severe cases, it can lead to liver fibrosis. Long half-life radioactive drugs also have obvious damage to the bone marrow. These drugs act on bone marrow hematopoietic stem cells in the body for a long time, destroying the DNA structure of bone marrow cells, making hematopoietic stem cells unable to divide normally, resulting in reduced production of white blood cells, red blood cells and platelets. Patients may experience leukopenia, which can easily lead to infection; a decrease in red blood cells can lead to anemia, with symptoms such as fatigue, dizziness, and palpitations; and a decrease in platelets can increase the risk of bleeding.

[0077] The term "single domain antibody", also known as "nanoantibody", "VHH" or "heavy chain antibody (HCAb)", refers to: found in camelids or small sharks, characterized by the lack of the CH1 domain required for light chain pairing. Therefore, they have only two heavy chains, each with a variable antigen binding (VHH) domain, and their small size (only 15kDa) leads to low serum persistence or rapid renal clearance due to the threshold of glomerular filtration of about 60kDa, so they have disadvantages in diagnostic screening and therapeutic applications.

[0078] The term "mutant" refers to a new molecule different from the original polypeptide or protein sequence formed by a method of insertion, deletion or replacement at the gene level or amino acid level, the new molecule has a substantially homologous amino acid sequence with the original molecule, and these amino acid sequences can cause the new molecule to have a function or characteristic that is improved, similar or reduced with the original molecule because of one or more different amino acid deletions, insertions and or substitutions. "Mutant" in the present invention refers to a molecule with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% consistency with the original sequence at the amino acid level. The identity of two amino acid sequences or two nucleic acid sequences can be determined by visual inspection and / or mathematical calculation, or by using a known computer program for sequence comparison (e.g., Clustal package version 1.83) to compare sequence information to more easily determine. Variants can include a sequence of amino acids with at least one conservative substitution, which means that a given amino acid residue is replaced by a residue with similar physiological and chemical properties. Examples of conservative substitutions include substitutions of one aliphatic residue to another aliphatic residue, such as substitutions of Ile, Val, Leu or Ala to each other, or substitutions of one polar residue to another, such as substitutions between Lys and Arg; substitutions between Glu and Asp; or substitutions between Gln and Asn. Other such conservative substitutions, such as substitutions 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.).

[0079] Table 1 lists exemplary amino acid substitutions.

[0080] Table 1 Exemplary amino acid substitutions

[0081] Amino acids can be classified according to common side chain properties 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 affecting chain orientation: Gly, Pro; and (6) aromatic rings: Trp, Tyr, Phe.

[0082] "Fusion protein" refers to a new type of protein with multiple functions produced by connecting the coding sequences of two or more different genes together through genetic engineering technology so that they are expressed in the same reading frame. For example, the fusion of cytokines and antibody Fc, the fusion of antibody binding fragments and antibody Fc, the fusion of the extracellular domain of cell surface receptors and antibody Fc, etc.

[0083] "Antibody binding fragment" refers to one or more parts 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 a fragment 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 the effectiveness of these fragments can be screened in the same manner as complete antibodies. Antigen binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of complete immunoglobulins, or in some cases, by chemical peptide synthesis procedures known in the art.

[0084] "Host cells" refer to cells that can accept foreign genes (target genes) and express them in themselves to produce the desired proteins. 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 cells for producing protein drugs. CHO cells can correctly fold and perform complex glycosylation modifications on proteins, so that the protein drugs they produce are closer to natural proteins in the human body in structure and function; insect cells such as Sf9 and Sf21 cells can use the Baculovirus Expression Vector System (BEVS) to express foreign genes.

[0085] "Vector" includes nucleic acid vectors, such as DNA vectors, RNA vectors, viruses, etc. The expression vectors described herein may contain polynucleotide sequences and other sequence elements, such as for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells, which 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 including genes encoding antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin and nourseothricin).

[0086] The term "affinity" refers specifically to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its counterpart (e.g., an antigen). Unless otherwise specified, "binding affinity" referred to herein refers to internal binding affinity, which reflects the 1:1 interaction strength between the two members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its counterpart Y can usually be quantified by the dissociation constant (KD). This affinity can be measured and evaluated by standard methods known in the art (including the methods mentioned herein).

[0087] "Conjugation" refers to the joining or attachment of two or more objects together. When referring to chemical or biological compounds, conjugation can refer to a covalent link between two or more chemical or biological compounds.

[0088] "Protein-conjugated drugs" are a special type of drug, which usually involves coupling the pharmacological components (such as cytokines, protein toxins, cytotoxic compounds, radionuclides, nucleotides, etc.) with targeting molecules through chemical or biological methods to form conjugated drugs. The targeting molecules can be monoclonal antibodies, antibody fragments, recombinant proteins, etc.

[0089] This conjugated drug combines the characteristics of the pharmacodynamic component and the targeting molecule, aiming to improve the efficacy of the drug, reduce toxic side effects, and achieve more specific target therapy. For example, the types of cytotoxic compounds may include maytansine, camptothecin and its derivatives, auristatin toxins, maytansine thio derivatives, docetaxel, gemcitabine, docetaxel, etc. Protein drugs conjugated with radionuclides are a class of drugs that combine radionuclides with targeted protein drugs through specific chemical linkers. This combination enables the drug to utilize the targeting properties of protein drugs to accurately deliver radionuclides to diseased cells or tissues to play a diagnostic or therapeutic role.

[0090] The term "chelation" refers to the process of combining radioactive metal ions with chelating agents through coordination bonds to form stable complexes with specific structures. Chelating agents are usually organic compounds containing multiple coordinating atoms. These coordinating atoms are generally atoms with lone pairs of electrons, such as nitrogen (N), oxygen (O), sulfur (S), etc. For example, ethylenediaminetetraacetic acid (EDTA) is a classic chelating agent. Its molecular structure has four carboxyl groups (-COOH) and two amino groups (-NH2). The oxygen atoms and nitrogen atoms in these groups can serve 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, 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) and its derivatives. When radioactive metal ions are present, the coordinating atoms in the chelator molecules will form coordination bonds with the metal ions. Different chelators can change the physical and chemical properties of radiopharmaceuticals. For example, the hydrophilicity or hydrophobicity of the chelator will affect the distribution of radiopharmaceuticals in the body. Hydrophilic chelators can make radiopharmaceuticals more easily dispersed and transported in aqueous environments such as blood, while hydrophobic chelators may guide radiopharmaceuticals to be more distributed in adipose tissue, etc. In addition, the size, charge and other properties of the chelated radiopharmaceuticals will also change due to different chelators. These properties have an important impact on the process of drug penetration of physiological barriers (such as the blood-brain barrier) and cell uptake.

[0091] Chelators can be linked to molecules with targeting functions (such as antibodies, peptides, small molecule ligands, etc.) to form targeted radiopharmaceuticals. For example, antibodies that can recognize specific receptors on the surface of tumor cells are linked to chelators that chelate radioactive metal ions. Such radiopharmaceuticals can specifically bind to tumor cells in vivo to achieve accurate imaging or treatment of tumors. This targeted chelation strategy can reduce the damage of radiopharmaceuticals to normal tissues and improve the therapeutic effect and safety of drugs.

[0092] The term "radiopharmaceutical" refers to radionuclide preparations or their labeled compounds used for clinical diagnosis or treatment. These drugs contain radionuclides that can emit radiation, such as alpha rays, beta rays or gamma rays. Examples of these 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.

[0093] The term "radiochemical purity", referred to as radiochemical purity, refers to the percentage of radionuclides in a radioactive sample that exist in a certain chemical form. This value reflects the proportion of radionuclides in a specific chemical form in the total radionuclides.

[0094] The term "uptake" refers to the process by which radionuclide-conjugated drugs are absorbed and incorporated into target cells or tissues through a series of physiological processes. This is similar to the process by which cells take up nutrients, but here it is for radionuclide-conjugated drugs. For example, when a radionuclide-conjugated drug is injected into the body, it needs to reach diseased cells (such as tumor cells) and enter these cells by a certain mechanism. This process of entering the cell can be called uptake.

[0095] The term "distribution" refers to the process by which drugs are transported to various tissues, organs, and body fluids of the body through various physiological mechanisms after being absorbed into the blood circulation. It is an important part of the dynamic changes of drugs in the body and is closely related to the efficacy and safety of drugs. Drugs are more likely to reach and distribute in tissues and organs with abundant blood flow. For example, organs such as the heart, liver, and kidneys have large blood flows, and the distribution speed of drugs in these parts is usually faster. Drugs have different affinities for targets on different tissues, which can lead to selective enrichment of drugs in certain tissues.

[0096] The term "subject" refers to an organism, such as a human, who is receiving treatment for a particular disease or condition as described herein.

[0097] The term "%ID / g" refers to the percentage of the injected dose of the radioactive drug per gram of tissue. By measuring the %ID / g value at different time points, in different tissues or organs, we can understand the dynamic changes of the drug in the body and provide important information for clinical diagnosis and treatment.

[0098] Example 1 Preparation of antibody samples 1.1 Construction of antibody expression vector After the antibody sequence (SEQ ID NO: 36) that specifically binds to the human Trop2 target was optimized for amino acid codons according to the human host cell expression system, the nucleotide sequence encoding the protein expression-related signal peptide was added and gene synthesis was performed. Then, the synthesized target gene was cloned into the vector pTT5 (ampicillin resistance) through 5'EcoRI and 3'HindIII restriction digestion, and transformation was performed after the construction was completed. The clones were selected for sequencing, and the bacteria with correct sequencing were selected for culture, plasmids were extracted, and the concentration was measured.

[0099] The antibody proteins in Table 2 were expressed and purified for further experimental testing to study mutants that affect the half-life of the antibodies.

[0100] Table 2 Names, sequences and Fc mutation information of expressed antibodies used for half-life studies

[0101] 1.2 Cell culture and antibody expression The extracted plasmids were transfected into cells and the antibodies were expressed as follows: (1) Determine the cell density. The viability should be greater than 95%. Use pre-warmed HEK293 culture medium to adjust the density of HEK293 cells (purchased from ATCC, catalog number CRL-1573.3) to 3×10 6 cells / mL, shake gently and dispense the cells, making sure that the volume of cells in the flask does not exceed 1 / 3 of the flask specification, and place in a shaker for later use.

[0102] (2) Calculate the volume of the transfection buffer Opti-MEM based on the volume of the transfected cells. This volume is 1 / 10 of the transfection system. Calculate the amount of the transfection reagent PEI (purchased from Shanghai Haoyuan Biotechnology Co., Ltd., catalog number HY-K2014). The ratio is 9 μg / mL transfected cells. Calculate the total amount of transfected DNA. The ratio is 1 μg / mL transfected cells.

[0103] The specific transfection process is as follows: Take a 50mL centrifuge tube, add 10% opti-MEM (purchased from Beijing Zhongsheng Aobang Biotechnology Co., Ltd., item number 03.18001A) of the transfection system, add the plasmid, mix well, filter, let stand for 5 minutes, add PEI to the DNA suspension, gently mix (gently invert 2-3 times), and let stand for 15-20 minutes. Then gently add the complex to the packaged cells, and gently shake the shaker while adding; the transfected cells are placed in a 37℃ shaker for culture, and 293 serum-free feed solution is added on the 1st, 3rd and 5th days after transfection. Protein purification is performed after 7 days of cell culture.

[0104] 1.3 Antibody purification (1) Sample preparation The collected cell supernatant was centrifuged using a desktop centrifuge at 4000 g for 30 min, and the cell supernatant after centrifugation was collected; the cell supernatant after centrifugation was filtered using a 0.45 μM filter membrane.

[0105] (2) Antibody purification The cell culture supernatant after centrifugation was purified using protein G (purchased from Shanghai Biotime Biotechnology Co., Ltd., catalog number P2017) affinity purification method to harvest monoclonal antibodies. The operation steps are as follows.

[0106] 1) Connection system: Select a protein G column of appropriate specifications according to the expression level and connect it to the purification system; 2) Water balance: Wash with ultrapure water for 3 CV and replace the 25% ethanol storage solution; 3) Equilibrate the column: Equilibrate the column with 5CV of equilibration buffer (50mM Tris, 100mM NaCl, pH 8.0); 4) Sample loading: adjust the appropriate flow rate for sample loading; 5) Elution: Elute the column with equilibration buffer (50 mM Tris, 100 mM NaCl, pH 8.0) for 10 CV at the same flow rate as the loading flow rate; 6) Elution: Elution buffer (100mM Glycine, 10mM NaCl, pH3.0) is used to elute and collect antibodies; 7) Neutralization: Add 2M Tris, pH 8.0 to neutralize the eluted antibody; 8) Desalting: Prepare a PD10 desalting column and wash the column with 6 CV of PBS 7.4. Add 3 mL of the eluent from step (7), and then add 3 mL of PBS 7.4 to elute the antibody.

[0107] Example 2 Testing the effects of different Fc mutants on molecular half-life 2.1 Testing of the binding affinity of different Fc mutants to FcRn The equilibrium dissociation constant (KD) of the above antibody samples of the present invention binding to human FcRn protein was determined by biofilm light interferometry (BLI) assay. The BLI affinity assay steps are briefly as follows: (1) Immobilization of ligand The antibody sample was diluted to 5 µg / mL using running buffer PBS and immobilized on the ProA sensor for 300 s; the ligand sample was diluted to 5 µg / mL using running buffer and immobilized on the His1K sensor for 180 s.

[0108] (2) Analyte preparation and detection Human FcRn protein (purchased from Beijing Sino Biological Technology Co., Ltd., Cat. No. CT009-H08H) was diluted to 200 nM using running buffer. Binding time was 120 s, and dissociation time was 180 s. Antibody samples were diluted to 50 nM using running buffer, and then diluted 2-fold in sequence. Binding time was 120 s, and dissociation time was 180 s.

[0109] 3) Experimental results analysis The results were analyzed using Data Analysis 12.0 software to obtain the association rate, dissociation rate and affinity constant (see Table 3).

[0110] Table 3 Determination results of affinity of different Fc mutants for FcRn

[0111] Note: "No binding" means that no clear binding effect was detected in the multi-concentration affinity test using the BLI method, and the related parameters of KD (M), kon (1 / Ms), and kdis (1 / s) could not be obtained.

[0112] The results of affinity determination of different Fc variants with FcRn show that the H435Q mutation or the combination of H435A, H435Q and H310A, H310Q mutations introduced into the Fc region can completely eliminate the binding effect of Fc with FcRn. This indicates that αTrop2-Fc v1, αTrop2-Fc v2, αTrop2-Fc v3, αTrop2-Fc v4 may be cleared faster in vivo than αTrop2-WT Fc.

[0113] 2.2 In vivo animal testing of the effects of different Fc mutants on antibody half-life Healthy adult SD rats (weighing 200-300 g) were selected and randomly divided into 5 groups according to the experimental design, with 3 rats in each group. Each group was injected with 5 mg / kg of one of the 5 antibody samples in Table 2 through the tail vein.

[0114] Blood samples were collected at different time points after the injection of the antibody, and the time points were selected to collect blood and perform ELISA tests at 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours and 168 hours after the injection. The data of the measured antibody concentration changing with time were fitted, and the commonly used fitting model was the exponential decay model. The half-life was calculated based on the parameters obtained by fitting, so as to evaluate the metabolism of the antibody in rats, as shown in Table 4.

[0115] Table 4 Results of half-life determination of naked antibodies with different Fc mutants in rats

[0116] 2.3 Antibodies 177 Lu labeling and half-life testing of labeled substances in animal blood Antibody coupling: 4 mg of antibody was dissolved in 0.1 M NaHCO3 (pH 9.0) to a final concentration of 4 mg / mL. After adding 10 times the equivalent of DOTA-NHS-ester (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., catalog number HY-128890), the reaction was allowed to proceed overnight at room temperature, and PBS was used for centrifugal replacement to remove the unreacted DOTA-NHS-ester small molecule compound.

[0117] Chelation of radionuclides: Open the metal bath reactor and preheat to 40°C. Dilute the coupled antibody to 5 mg / mL with PBS solution to obtain the precursor solution. Take 37 MBq (1 mCi) 177 LuCl3 solution (50 pmol), then add 4 times the equivalent of precursor solution, make up the sodium vitamin C buffer to 50 μL, react at 40°C, 200 rpm for 2 h, and obtain 177Lu-labeled antibody molecules exist in solution.

[0118] Use 1% sodium citrate solution as the developing solvent for TLC purity test. If the purity is greater than 95%, no subsequent treatment is required. If the purity is less than 95%, subsequent purification is required: centrifuge with an ultrafiltration centrifuge tube to remove unliganded 177 Lu. Determine the radioactivity of the chelated and purified drug.

[0119] 177 Half-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 according to the experimental design, with 3 rats in each group. Each group was injected with 300 μCi of one of the above-mentioned chelated samples through the tail vein, and blood samples were collected at different time points after the injection of the antibody. The time points were selected at 0.5 hours, 1 hour, 17 hours, 24 hours, 48 ​​hours, and 72 hours after the injection for blood sampling and γ counter detection. The activity of the radioactive drug in the blood at different time points was converted according to the results of the γ counter, and the activity of different Fc mutants was calculated. 177 The half-life of Lu-antibody in animal blood is shown in Table 5.

[0120] Table 5 Different 177 Results of half-life determination of Lu-labeled Fc mutants in rats

[0121] From the above results, it can be seen that after 177 The half-life of Lu-labeled antibodies in rat blood remained basically unchanged or decreased, and the mutants with reduced half-life also meant that the radioactive substances could be cleared more quickly in the body.

[0122] These Fc mutations are 177 The effect on the half-life of antibodies after Lu labeling is different, and it is uncertain whether the radionuclide-labeled antibodies prepared on this basis will affect their uptake in animals.

[0123] Example 3 Testing the Effects of Different Fc Mutants on the Binding of Molecules to FcγRs and C1q The Fc fragment can bind to cells expressing FcγRs in vivo, and can also bind to proteins in the complement system such as C1q, which may eventually cause the antibody molecules to bind to normal cells and tissues due to the mediation of Fc. If labeled with radionuclides, it will inevitably form stronger toxic side effects on normal tissues, so it is necessary to remove or partially remove these effects by modifying the amino acids on Fc. In this example, we also used the Fc mutant from the patent application number 202280030415.6 to construct the antibody αTrop2-ABD Fc for comparative studies, and the summarized information is as follows. The antibodies in Table 6 were expressed and purified according to the method of Example 1 for further affinity determination.

[0124] Table 6 Names, sequences and Fc mutation information of expressed antibodies used for FcγRs and C1q binding studies

[0125] Taking αTrop2-WT Fc as the reference benchmark, the changes in the binding affinity of different mutants to different FcγR proteins and C1q complement protein were compared. The mutation combinations of αTrop2-Fc v8, αTrop2-Fc v9, αTrop2-Fc v10 and αTrop2-Fc v11 antibodies that affect the binding to FcγRs and C1q overlapped with the mutation positions of some other antibodies in the Fc region. The affinity test of the antibody Fc region affecting the binding to FcγRs and C1q was not performed on αTrop2-Fc v8, αTrop2-Fc v9, αTrop2-Fc v10 and αTrop2-Fc v11. The test results are shown in Table 7.

[0126] Table 7 Compared with αTrop2-WT Fc, the percentage change of affinity of different mutants to FcγRs and C1q

[0127] Note: "(↓)" indicates the percentage of affinity decrease compared with αTrop2-WT Fc, such as "396.3 (↓)" indicates that the affinity of the antibody with L234A and L235A mutation combination in the Fc region for CD64 receptor protein decreased by 396.3% compared with αTrop2-WT Fc; "No binding" indicates that the experimental method could not detect the obvious binding of the corresponding antibody mutant to the receptor protein, which also means that the experimental method showed that the corresponding antibody mutant completely lost the ability to bind to the receptor.

[0128] According to the experimental results, the following inferences can be drawn: (1) Antibody mutants that have completely lost the 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 (such as the liver, etc.), while the radioactive uptake in tumor tissues will be higher or remain unchanged.

[0129] (2) Among these antibody variants with Fc and FcγRs and C1q removed, under the conditions of containing the same combination of mutation sites that affect half-life, the uptake of these mutants in normal tissues and / or tumor tissues in animals after labeling with radionuclides may be the same, for example, αTrop2-Fc v6, αTrop2-Fc v7 and the control molecule αTrop2-ABD Fc, αTrop2-Fcv8 and αTrop2-Fc v10, αTrop2-Fc v11 and αTrop2-Fc v12, αTrop2-Fc v14 and αTrop2-Fc v15 have the same tissue uptake in animals after labeling with radionuclides.

[0130] In the next step, we will verify the above speculation through specific and extensive experiments.

[0131] Example 4 Imaging test of antibody molecules containing different mutation combinations in tumor-bearing mice 4.1 Antibody conjugation and labeling The antibody molecules in Table 6 were coupled and 177 Lu labeling, preparation 177 Lu-antibody radiopharmaceuticals for in vivo imaging studies in tumor-bearing mice.

[0132] 4.2 Establishment of animal tumor model In the experiment, balb / c nude mice were subcutaneously inoculated with 5×10 6 The number of MDA-MB-468 cells was resuspended in 1:1 PBS and Matrigel (0.1 mL / cell). The tumor growth was observed regularly. When the tumor grew to an average volume of 180-220 mm 3 (About 200mm 3 ) were randomly divided into groups according to tumor size and mouse body weight.

[0133] 4.3 Single Photon Emission Tomography and Data Processing Will 177Lu-antibody was diluted to 7.4MBq (200μCi) / 200μL with saline, and each tumor-bearing mouse was injected with 7.4 MBq (200μCi) of the drug through the tail vein. At 24, 48, 72, 96, and 120 hours after administration, the mice were continuously anesthetized with isoflurane, and prone on the examination bed for single photon emission tomography (model: IVIS spectrum, manufacturer: MILabs) scanning. The acquisition method was static 10 min single photon emission tomography and medium-resolution whole-body computed tomography. During the process, the animal weight, injection dose, injection time, and residual dose were recorded according to the record sheet, and the injection dose measurement time and residual dose measurement time were recorded respectively. After scanning, the data was reconstructed, and the PMOD software was used to analyze and outline the tumor, liver, and kidney tissues, quantify the drug distribution, save the images and data, and further statistically analyze the data.

[0134] PMOD software was used to analyze and outline the radioactive uptake results of tumor, liver and kidney tissues at different time points. The experimental results are shown in Table 8.

[0135] Table 8 Uptake of different radiolabeled antibody mutants in tumors, liver and kidneys at different time points (%ID / g)

[0136]

[0137] According to the above results, it can be seen that the uptake of radioactive drugs in tumor tissue caused by these different Fc mutants is different. Among the 11 mutants from αTrop2-Fc v5 to αTrop2-Fc v15, αTrop2-Fc v5, αTrop2-Fc v6 and αTrop2-Fc v7 showed better or comparable radioactive uptake in tumor tissues with other molecules at different time points. Among them, the tumor uptake of the αTrop2-Fc v5 molecule was optimal at all detected time points and was better than the control molecule αTrop2-ABD Fc, which is completely opposite to the speculation in Example 3 and is an unexpected result. Moreover, in addition to the low level of tumor uptake in the mutants αTrop2-Fc v8 to αTrop2-Fc v15, some molecules also showed long-term 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 molecules, which is completely opposite to our speculation in Example 3.

[0138] In summary, not all mutants that reduce or completely eliminate binding to FcγRs, C1q, and FcRn have ideal uptake in tumors and livers; in the series of Fc mutation combinations tested, the three combinations of L234A, L235A, H435Q or L234A, L235A, P329G, H435Q or 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 which the L234A, L235A, H435Q mutation combination has more advantages in high tumor uptake than the control mutant molecule αTrop2-ABD Fc.

[0139] Figures 1 to 12 The above results are more intuitively reflected in that the tumor tissue is circled with a white oval dotted line.

[0140] 4.4 αTrop2-Fc v5 and αTrop2-ABD Fc 89 Zr labeling and positron emission tomography testing To compare the differences in tumor uptake between αTrop2-Fc v5 and αTrop2-ABD Fc labeled with different radionuclides, we used 89 The chelating agent used was p-SCN-Bn-deferoxamine (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., catalog number HY-134797), coupled and 89 The specific steps of the Zr labeling method are as follows: 4 mg of antibody was dissolved in 0.1 M NaHCO3, pH 8.5-9.5 solution, 5 equivalents of p-SCN-Bn-deferoxamine chelating agent was added, and the antibody was concentrated using an ultrafiltration tube after reacting at room temperature overnight. 2 mg of the concentrated antibody was mixed with 90 MBq of 89 Zr was mixed, the pH was adjusted to 7.2, and incubated at room temperature for 60 minutes. 89 Zr reaction, incubate at room temperature for 15 min. Purify using gel filtration on disposable PD10 columns.

[0141] The labeled molecules were injected into the mouse model bearing MDA-MB-468 tumors through the tail vein. Micro-PET / CT (model: Super Nova, manufacturer: Pingsheng Medical Technology (Kunshan) Co., Ltd.) was used to measure the tumor lesion %ID / cc. Small animal positron emission tomography scans were performed at 24, 48, 72, 96, 120 and 144 hours to analyze the radioactive uptake of tumor tissues at multiple time points, such as Fig.13 As shown. After comparison, it was found that89 The radioactivity uptake of tumor tissue by Zr-αTrop2-Fc v5 molecule was superior to that of 89 The Zr-αTrop2-ABD Fc molecule further demonstrated the superior efficacy of the combination of L234A, L235A, and H435Q mutations.

[0142] Example 5 Testing of thiol site-directed coupling The sulfhydryl coupling of αTrop2-ABD Fc and αTrop2-Fc v5 with DOTA chelator was performed according to the following brief method. First, 10 equivalents of tris(2-carboxyethyl)phosphine (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., Catalog No.: HY-W011500) were used to reduce the disulfide bonds of the antibody at 37°C for 1 hour. Then, 4 equivalents of the chelator Maleimide-DOTA (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., Catalog No.: HY-133540) dissolved in DMSO were added to the solution. After incubation at 37°C for 12 hours, the chelator bound to the sulfhydryl group of the antibody, and the coupled sample was then purified with PBS using an ultrafiltration centrifuge tube.

[0143] The products of αTrop2-ABD Fc and αTrop2-Fc v5 coupling, αTrop2-ABD Fc-Mal-DOTA and αTrop2-Fc v5-Mal-DOTA, were tested by LC-MS according to the following steps: 1) Sample processing Take a 2 mg / mL sample, desalt it, and incubate it with PNGase F (Cat. No.: P0704, Manufacturer: New England Biotechnology Co., Ltd.) at 37°C for 1 hour to remove sugars. After the incubation, put it into a sample vial and test it on the machine.

[0144] 2) Liquid quality testing The samples were separated by ultra-high performance liquid chromatography (ULPLC) system (model: Vanquish UPLC, brand: Thermo Fisher). Phase A was 0.1% formic acid-water solution, phase B was 0.1% formic acid-acetonitrile solution, the samples were loaded by an automatic sampler, and then gradient separated by a chromatographic column (model and specification: ACQUITY UPLC BEH C4, 2.1*50 mm, 1.7 μm, 300 Å, brand: Waters), with a flow rate of 0.30 mL / min, a detection wavelength of 280 nm, and a column temperature of 80 °C. A high-resolution mass spectrometer (model: Q ExactiveHF-X, brand: Thermo Fisher) was used for analysis time: 10 min, detection mode: positive ion, parent ion scanning range: 500~4000 m / z. The experimental results are shown in Figure 14-15 and Table 9.

[0145] Table 9 Summary of mass spectrometry results of two molecules after thiol coupling

[0146] It is easy to find from the above results that, in the case of thiol coupling, it is easier and more efficient to obtain the coupled product using the Fc v5 mutant than the positive control ABD Fc mutant, and the coupled components are relatively single.

[0147] Example 6 Single Photon Emission Tomography Results of αTrop2-Fc v5 in Tumor-Bearing Mice Using Thiol-Conjugated Method The αTrop2-Fc v5-Mal-DOTA molecule in Example 5 was 177 After Lu labeling, mice bearing MDA-MB-468 tumors were injected into the tail vein, and single photon emission tomography was performed at different time points and the tumor, liver, and kidney were circled using PMOD software. Fig.16 shown.

[0148] The uptake results of three different tissues at different time points after being processed by PMOD software are shown in Table 10: Table 10 PMOD treatment results of αTrop2-Fc v5 in tumor-bearing mice using thiol coupling method (%ID / g)

[0149] The results showed that the molecules constructed using different coupling methods 177 After Lu radiolabeling, high-value uptake can still be achieved in tumor tissue for a long time.

[0150] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein is formed by fusing at least one receptor binding fragment with an Fc domain derived from IgG; The Fc domain was mutated at the following positions: L234, L235 and H435.

2. The fusion protein according to claim 1, characterized in that The Fc domain was also mutated at the following positions: P329 and / or D265.

3. The fusion protein according to claim 1, characterized in that The IgG includes human IgG1, IgG2 or IgG4 subtypes.

4. The fusion protein according to claim 1, characterized in that 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 claim 4, characterized in that The amino acid sequence of the Fc domain before mutation is shown in SEQ ID NO:

32.

6. The fusion protein according to claim 5, characterized in that The combination of Fc domain mutation sites includes: (1) L234A, L235A and H435Q; (2) L234A, L235A, P329G and H435Q; (3) L234A, L235A, D265A and H435Q.

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

23.

8. The fusion protein according to any one of claims 1 to 7, characterized in that The receptor binding fragment and the Fc domain are connected via a hinge region.

9. The fusion protein according to claim 8, characterized in that The hinge region amino acid sequence is shown in SEQ ID NO:

35.

10. The fusion protein according to any one of claims 1 to 7, characterized in that: The receptor binding fragment includes: a ligand naturally present in the body or a truncated form thereof, a single domain heavy chain antibody, a single chain variable fragment or a bispecific antibody binding fragment.

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

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

13. A carrier, characterized in that The vector comprises the nucleic acid of claim 12.

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

15. 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 and the host cell according to claim 14 in the preparation of a radioactive protein-conjugated drug.

16. A radioactive protein-conjugated drug, characterized in that: The radioactive protein-coupled drug uses the fusion protein according to any one of claims 1 to 11 as a carrier and is coupled with a small molecule cytotoxic drug or a compound chelating a radionuclide.

17. The radioactive protein-conjugated drug according to claim 16, characterized in that: The radionuclides include but are not limited to 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 radioactive protein-conjugated drug according to claim 16, characterized in that: The method of coupling the fusion protein with the compound chelating the radionuclide includes: A: coupling with the chelating ring through a linker; B: coupling through free or reduced sulfhydryl groups; C: Coupling occurs via exposed amino groups.

19. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: 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.

20. 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, the host cell according to claim 14, the radioactive protein-conjugated drug according to any one of claims 16 to 18, or the pharmaceutical composition according to claim 19 in the preparation of products for diagnosing and / or treating tumors.

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