A radioactive protein-coupled drug and its application

Abstract

The present invention provides a radioactive protein-conjugated drug and its application, belonging to the field of biopharmaceuticals. The present invention provides a novel radioactive protein-conjugated drug, comprising at least one receptor-binding fragment fused to an Fc domain derived from human immunoglobulin G (IgG). Mutation modifications include L234, L235, and H435 of the Fc domain, and may also include P329 and / or D265. The preferred mutation combination is L234A, L235A, and H435Q. After chelating a radionuclide, the novel fusion protein provided by the present invention can be taken up at low doses in tissues such as the blood, kidneys, and liver, and rapidly cleared. It can also be taken up at high doses and for extended periods in tumor tissues, significantly improving the safety and efficacy of radiopharmaceutical therapy.

Description

Technical Field

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

[0002] Radiopharmaceuticals are increasingly being used in the treatment of various tumor types as a safe and effective treatment. Drugs that preferentially bind to cancer cells or accumulate through physiological mechanisms reach the body or local lesions, and then irradiate the lesions to kill cancer cells. Furthermore, these radionuclides can emit imageable photons, allowing for non-invasive observation of the biodistribution of therapeutic drugs.

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

[0004] Radionuclide-conjugated drugs combine the advantages of precise targeting and potent killing. Using specific monoclonal antibodies carrying a radionuclide payload to target cancer cells, they concentrate the release of energy within a region several times the cell's diameter, killing cancer cells while minimizing damage to nearby normal tissues. They offer excellent efficacy, minimal adverse reactions, and the ability to prolong patient survival. They are crucial for the treatment of advanced cancers and offer unique advantages in early diagnosis, staging, and treatment evaluation.

[0005] However, there are still many problems that limit its translation 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 energy, penetration, and half-life of the radionuclides; 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 due to the molecule's large molecular weight (approximately 150 kDa) and long half-life, the labeled molecule was absorbed for a long time and in high doses in tissues such as the liver, heart, spleen, and lungs.

[0007] Large proteins (>60 kDa) are typically not filtered by the kidneys and therefore not excreted in the urine. They are primarily metabolized by the liver, the primary organ that processes these large molecules. When these large proteins are formulated into radiopharmaceuticals, they accumulate significantly in the liver and circulate in the blood for extended periods, potentially leading to the following potential side effects and safety concerns.

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

[0009] 2. Due to their large molecular weight, these macromolecules remain in the bloodstream for a prolonged period. This can lead to prolonged drug accumulation in the bloodstream, increasing potential toxicity to blood and bone marrow cells. Hematotoxicity may manifest as leukopenia and thrombocytopenia, while bone marrow damage may lead to decreased hematopoietic function and even serious complications such as myelosuppression.

[0010] Patent application number 202280030415.6, which addresses the mutational engineering of antibody Fc fragments, has discovered a combined mutant that achieves high-dose absorption in tumor tissue while ensuring low-dose absorption in the liver and blood. However, this mutant still has some indicators that need to be improved, such as low biodistribution in tumor tissue, which may affect drug efficacy. Therefore, it is necessary to research more superior molecular mutants to further enhance the distribution of radiopharmaceuticals 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 comprising at least one receptor binding fragment fused to an IgG-derived Fc domain;

[0013] The Fc domain was mutated at the following positions: L234, L235, and H435.

[0014] Specifically, the Fc domain may also be mutated at the following positions: P329 and / or D265.

[0015] Specifically, the IgG includes human IgG1, IgG2 or IgG4 subtype.

[0016] Preferably, the IgG is human IgG1.

[0017] 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.

[0018] More specifically, the amino acid sequence of the Fc domain before mutation is shown in SEQ ID NO: 32.

[0019] SEQ ID NO:32:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0020] Preferably, the Fc domain mutation sites include at least one of L234A, L235A, D265A, P329G and / or H435Q.

[0021] In certain specific embodiments of the present invention, the amino acid sequence of the Fc domain after mutation is as follows:

[0022] SEQ ID NO:17:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0023] SEQ ID NO:18: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0024] SEQ ID NO:19: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0025] SEQ ID NO:20: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0026] SEQ ID NO:21: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0027] SEQ ID NO:22: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0028] SEQ ID NO:23: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0029] SEQ ID NO:24: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0030] SEQ ID NO:25: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0031] SEQ ID NO:26: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0032] SEQ ID NO:27:

[0033] APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0034] SEQ ID NO:28: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0035] SEQ ID NO:29:APEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCQVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLPG;

[0036] SEQ ID NO:30: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0037] SEQ ID NO:31: APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0038] SEQ ID NO:34:APEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG.

[0039] Further preferably, the combination of Fc domain mutation sites includes:

[0040] (1) L234A, L235A and H435Q;

[0041] (2) L234A, L235A, P329G and H435Q;

[0042] (3) L234A, L235A, D265A and H435Q.

[0043] 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.

[0044] Specifically, the receptor binding fragment and the Fc domain are connected via a hinge region.

[0045] In certain specific embodiments of the present invention, the hinge region amino acid sequence is shown in SEQ ID NO: 35.

[0046] SEQ ID NO:35:EPKSSCTHTCPPCP.

[0047] 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.

[0048] Preferably, the receptor binding fragment is a single domain heavy chain antibody.

[0049] 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:

[0050] SEQ ID NO:1: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0051] SEQ ID NO:2: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0052] SEQ ID NO:3:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0053] SEQ ID NO:4:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0054] SEQ ID NO:5: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0055] SEQ ID NO: 6: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0056] SEQ ID NO:7: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0057] SEQ ID NO:8: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0058] SEQ ID NO:9:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0059] SEQ ID NO:10:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0060] SEQ ID NO:11:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0061] SEQ ID NO:12:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0062] SEQ ID NO:13:

[0063] EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVQVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLQQDWLNGKEYKCQVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG;

[0064] SEQ ID NO:14:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPPCAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVAV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0065] SEQ ID NO:15: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSCTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCAVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPG;

[0066] SEQ ID NO:16: EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0067] SEQ ID NO:33:EVQLVESGGGLVQPGGSLRLSCAASGGTFSRYYMGWFRQAPGKAREFVSGISESGGSTLYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAPKWYSGDFMDHLTYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNQYTQKSLSLSPG.

[0068] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned fusion protein.

[0069] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid.

[0070] In another aspect, the present invention provides a host cell comprising the above nucleic acid or vector.

[0071] Specifically, the host cell is a prokaryotic host cell or a eukaryotic host cell, and 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).

[0072] In 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.

[0073] 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 radionuclides.

[0074] Specifically, the radionuclides include but are not limited to 111 In, 223 Ra, 67Ga, 68 Ga, 44 Sc, 90 Y. 177 Lu, 225 Ac, 212 Bi, 213 Bi, 212 Pb, 227 Th, 64 Cu, 67 Cu, 89 Zr.

[0075] Specifically, the methods for coupling the fusion protein with the compound chelating radionuclides include:

[0076] A: coupling with the chelate ring through a linker;

[0077] B: coupling via free or reduced sulfhydryl groups;

[0078] C: Coupling occurs via exposed amino groups.

[0079] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein, nucleic acid, vector or host cell.

[0080] Specifically, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0081] More specifically, the excipients include, but are not limited to, at least one of ethanol, gentisic acid, vitamin C, vitamin E, resveratrol, quercetin, glutathione and / or lipoic acid.

[0082] 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.

[0083] In 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.

[0084] Specifically, the tumors include but are not limited to: squamous cell papilloma, adenoma, fibroma, lipoma, rhabdomyomas, lymphangiomas, chondromas, gliomas, ganglioneuromas, meningiomas, hydatidiform moles, squamous cell tumors, adenocarcinomas, fibrosarcomas, liposarcoma, angiosarcomas, malignant mesenchymal tumors, malignant lymphomas, leukemias, invasive hydatidiform moles, malignant melanomas, lymphomas, neuroendocrine tumors and sarcomas, leiomyomas, hemangiomas, gastric stromal tumors, gastric sarcomas, gastric cancer, colorectal polyps, 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, eczematous carcinoma, schwannoma, meningioma, glioma, congenital tumors (epidermoid cyst, dermoid cyst, teratoma), cavernous hemangioma, angioreticuloma, metastasis, granuloma, blood system tumors, multiple myeloma, nervous system tumors, urinary system tumors, head and neck malignant tumors.

[0085] In another aspect, the present invention provides a treatment method, comprising administering to a subject a composition comprising the above-mentioned fusion protein, nucleic acid, vector, host cell, radioactive protein-conjugated drug or pharmaceutical composition.

[0086] Compared with the prior art, the present invention has the following advantages:

[0087] 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.

[0088] 2. The novel mutant provided by the present invention can achieve long-term, high-dose uptake at the tumor site while maintaining low uptake in normal tissues, enabling the drug to achieve a better tumor treatment effect without damaging normal organs.

[0089] 3. Compared to unmodified molecules and molecular structures reported in other patents, the fusion protein structure of this invention exhibits significant differences in both configuration and sequence. The uptake of the radiopharmaceutical in tumors and normal tissues is significantly different from that of previously reported mutants. This unique and innovative structure provides a new therapeutic strategy for the field of radiopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Figure 3 is the single photon emission tomography result of αTrop2-Fc v5 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0091] Figure 2Figure 3 is the single photon emission tomography result of αTrop2-Fc v6 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0092] Figure 3 Figure 3 is the single photon emission tomography result of αTrop2-Fc v7 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0093] Figure 4 Figure 3 is the single photon emission tomography result of αTrop2-Fc v8 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0094] Figure 5 Figure 3 is the single photon emission tomography result of αTrop2-Fc v9 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0095] Figure 6 Figure 3 is the single photon emission tomography result of αTrop2-Fc v10 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0096] Figure 7 Figure 3 is the single photon emission tomography result of αTrop2-Fc v11 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0097] Figure 8 Figure 3 is the single photon emission tomography result of αTrop2-Fc v12 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0098] Figure 9 Figure 3 is the single photon emission tomography result of αTrop2-Fc v13 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0099] Figure 10 Figure 3 is the single photon emission tomography result of αTrop2-Fc v14 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0100] Figure 11 Figure 3 is the single photon emission tomography result of αTrop2-Fc v15 molecule in tumor-bearing mice. The white oval dotted line is the circled tumor tissue.

[0101] Figure 12 Single photon emission tomography results of αTrop2-ABD Fc molecules in tumor-bearing mice, with the white oval dotted line indicating the circled tumor tissue.

[0102] Figure 13 for 89Results of radioactive uptake measurement of Zr-labeled molecules in tumor tissue. The white oval dotted line represents the circled tumor tissue.

[0103] Figure 14 This is the deconvoluted image of αTrop2-Fc v5.

[0104] Figure 15 This is the deconvoluted image of αTrop2-ABD Fc.

[0105] Figure 16 Single photon emission tomography results of αTrop2-Fc v15-Mal-DOTA molecule in tumor-bearing mice, with the white oval dotted line indicating the circled tumor tissue. DETAILED DESCRIPTION

[0106] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0107] Definition of terms

[0108] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs.

[0109] 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.

[0110] 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).

[0111] The peptide sequences described herein are written according to common 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.

[0112] The term "antibody" as used herein refers to immunoglobulins, which have a specific molecular structure. Differences in the amino acid composition and order of arrangement within the constant region of their heavy chains result in distinct antigenicity. Based on this characteristic, immunoglobulins can be divided into five classes, also known as immunoglobulin isotypes: IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are, in order, μ, δ, γ, α, and ε.

[0113] Igs of the same class can be further divided into different subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be further divided into IgG1, IgG2, IgG3, and IgG4 subclasses.

[0114] Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five classes of Ig has either kappa or lambda chains.

[0115] In the molecular structure of antibodies, the sequences of 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, also known as the Fv region; while the remaining amino acid sequences near the C-terminus show relative stability, and this part belongs to the constant region.

[0116] The variable region contains three hypervariable regions, or HVRs, and four relatively conserved framework regions, or FRs. These three hypervariable regions play a decisive role in the formation of antibody specificity and are therefore also called complementarity determining regions, or CDRs.

[0117] The term "Fc" refers to a fragment within an antibody molecule that does not affect antigen binding but is also susceptible to crystallization. Analysis of the three-dimensional structure of Fc provides a clear understanding of 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, are able to specifically bind to cell surface receptors such as FcγRs and FcRn, thereby enabling the protein to exert a range of specific biological effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and achieve a longer half-life.

[0118] Fc can bind to a variety of FcγRs, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRII is further divided into three subclasses: FcγRIIa (CD32a), FcγRIIb (CD32b), and FcγRIIc (CD32c). FcγRIII is further divided into two subclasses: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Furthermore, Fc can bind to complement system proteins such as C1q and may ultimately bind to the cell membrane. These Fc receptors are expressed on various normal immune cells. If antibody-conjugated radionuclide drugs bind to these receptors through the antibody Fc, they may cause damage to normal tissues.

[0119] 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).

[0120] In this case, the term "half-life" specifically refers to the "half-life of a drug," which refers to the time required for plasma drug concentration to decrease by half. It is a key parameter reflecting the rate of drug elimination from the body. Drug clearance from the blood primarily depends on the body's metabolic (primarily liver) and excretion (primarily kidney) functions. Half-life is a key indicator of drug clearance from the blood. Drugs with long half-lives are eliminated slowly from the body. If used continuously over a long period of time, the drug tends to accumulate in the body. Nuclear drugs with long half-lives may cause liver damage, such as hepatocyte necrosis and inflammatory reactions, leading to drug-induced hepatitis. Some long-half-life drugs may interfere with the normal secretion and excretion of bile, causing bile accumulation in the liver. Cholestasis can further damage the intrahepatic bile ducts and, in severe cases, lead to liver fibrosis. Radiopharmaceuticals with long half-lives also significantly damage the bone marrow. These drugs act on hematopoietic stem cells in the body over a long period of time, disrupting their DNA structure and preventing normal division, 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.

[0121] The term "single-domain antibody," also known as "nanobody," "VHH," or "heavy-chain antibody (HCAb)," refers to antibodies found in camelids or small sharks that lack the CH1 domain required for light chain pairing. Consequently, they consist of only two heavy chains, each with a single variable antigen-binding (VHH) domain. Their small size (only 15 kDa) results in low serum persistence or rapid renal clearance, hindering their use in diagnostic screening and therapeutic applications, as the glomerular filtration threshold is approximately 60 kDa.

[0122] The term "mutant" refers to a new molecule that is different from an original polypeptide or protein sequence and that is 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 to 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 replacements. "Mutant" in the present invention 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% identity to 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 can be more easily determined by comparing sequence information using a known computer program for sequence comparison (e.g., Clustal package version 1.83). A variant can comprise an amino acid sequence 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 for another aliphatic residue, such as substitutions of Ile, Val, Leu, or Ala for each other, or substitutions of one polar residue for 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 hydrophobicity 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.).

[0123] Table 1 lists exemplary amino acid substitutions.

[0124] Table 1 Exemplary amino acid substitutions

[0125]

[0126] 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.

[0127] A "fusion protein" is a novel protein with multiple functions produced by linking the coding sequences of two or more different genes through genetic engineering techniques so that they are expressed in the same reading frame. Examples include the fusion of a cytokine with an antibody Fc, the fusion of an antibody binding fragment with an antibody Fc, and the fusion of the extracellular domain of a cell surface receptor with an antibody Fc.

[0128] "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.

[0129] A "host cell" is a cell capable of accepting a foreign gene (target gene) and expressing it internally 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 therapeutics. CHO cells are capable of correctly folding proteins and undergoing complex glycosylation modifications, allowing the protein therapeutics they produce to more closely resemble native proteins in the human body in terms of structure and function. Insect cells such as Sf9 and Sf21 cells can express foreign genes using the baculovirus expression vector system (BEVS).

[0130] "Vector" includes nucleic acid vectors, such as DNA vectors, RNA vectors, viruses, and the like. The expression vectors described herein may contain polynucleotide sequences and, for example, additional sequence elements 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 efficient transcription of the 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 nourseothricin).

[0131] The term "affinity" as used herein 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 partner (e.g., an antigen). Unless otherwise specified, "binding affinity" referred to herein refers to internal binding affinity, which reflects the strength of a 1:1 interaction between the two members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is typically quantified by the dissociation constant (KD). This affinity can be measured and estimated using standard methods known in the art, including those described herein.

[0132] "Conjugation" refers to the joining or linking of two or more objects together. When referring to chemical or biological compounds, conjugation can refer to a covalent linkage between two or more chemical or biological compounds.

[0133] Protein-conjugated drugs are a special type of drug that typically involves chemically or biologically conjugating a pharmacological component (such as a cytokine, protein toxin, cytotoxic compound, radionuclide, or nucleotide) to a targeting molecule. These molecules can be monoclonal antibodies, antibody fragments, or recombinant proteins.

[0134] This type of conjugated drug combines the characteristics of the active 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 type of drug that combines radionuclides with targeted protein drugs through specific chemical linkers. This combination enables the drug to utilize the targeting properties of protein drugs to precisely deliver radionuclides to diseased cells or tissues, thereby exerting a diagnostic or therapeutic effect.

[0135] The term "chelation" refers to the process of combining radioactive metal ions with chelating agents through coordination bonds to form a stable, structurally defined complex. Chelating agents are typically organic compounds containing multiple coordinating atoms. These coordinating atoms are typically 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). The oxygen 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.

[0136] When radioactive metal ions are present, the coordinating atoms in the chelator molecule form coordination bonds with the metal ions. Different chelators can alter the physical and chemical properties of radiopharmaceuticals. For example, the hydrophilicity or hydrophobicity of the chelator can affect the distribution of the radiopharmaceutical in the body. Hydrophilic chelators can make radiopharmaceuticals more easily dispersed and transported in aqueous environments such as blood, while hydrophobic chelators may direct the radiopharmaceutical to more areas such as adipose tissue. Furthermore, the size, charge, and other properties of the chelated radiopharmaceutical can also be altered by the chelator used. These properties have a significant impact on drug penetration through physiological barriers (such as the blood-brain barrier) and cellular uptake.

[0137] 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 to a chelating agent that binds radioactive metal ions, the radiopharmaceutical can specifically bind to tumor cells in vivo, enabling precise imaging or treatment of the tumor. This targeted chelation strategy can reduce the damage of radiopharmaceuticals to normal tissues, improving their therapeutic efficacy and safety.

[0138] The term "radiopharmaceutical" refers to radionuclide preparations or labeled compounds used for clinical diagnosis or treatment. These drugs contain radionuclides that emit radiation, such as alpha, beta, 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.

[0139] The term "radiochemical purity", also known as radiochemical purity, refers to the percentage of radionuclides in a radioactive sample that exist in a certain chemical form among the total radionuclides. This value reflects the proportion of radionuclides that exist in a specific chemical form among the total radionuclides.

[0140] The term "uptake" refers to the process by which a radionuclide-conjugated drug 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 up nutrients, but in this context, it applies specifically to radionuclide-conjugated drugs. For example, after being injected into the body, a radionuclide-conjugated drug must reach diseased cells (such as tumor cells) and enter these cells using a specific mechanism. This process of entry into the cell is called uptake.

[0141] 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 component of a drug's dynamic changes within the body and is closely related to its efficacy and safety. Drugs are more likely to reach and distribute to tissues and organs with abundant blood flow. For example, organs such as the heart, liver, and kidneys have high blood flow, and drugs are generally distributed more rapidly in these areas. Drugs have different affinities for targets in different tissues, which can lead to selective accumulation of drugs in certain tissues.

[0142] The term "subject" refers to an organism, such as a human, who is receiving treatment for a particular disease or condition as described herein.

[0143] The term "%ID / g" refers to the percentage of the injected dose of the radiopharmaceutical per gram of tissue. By measuring %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.

[0144] Example 1 Preparation of Antibody Samples

[0145] 1.1 Construction of antibody expression vector

[0146] After optimizing the amino acid codons of the antibody sequence (SEQ ID NO: 36) specifically binding to the human Trop2 target using a human host cell expression system, a nucleotide sequence encoding a protein expression signal peptide was added and gene synthesis was performed. The synthesized target gene was then cloned into the vector pTT5 (ampicillin-resistant) via 5' EcoRI and 3' HindIII restriction enzymes. After completion of the construction, transformation was performed. Clones were selected for sequencing, and cells with correct sequencing results were cultured, plasmids were extracted, and concentrations were determined.

[0147] 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.

[0148] Table 2 Names, sequences, and Fc mutation information of expressed antibodies used for half-life studies

[0149]

[0150] 1.2 Cell culture and antibody expression

[0151] The extracted plasmids were transfected into cells and the antibodies were expressed as follows:

[0152] (1) Determine the cell density. The viability should be greater than 95%. Use preheated HEK293 culture medium to adjust the density of HEK293 cells (purchased from ATCC, catalog number CRL-1573.3) to 3×106 cells / mL, shake gently and aliquot the cells, making sure the volume of cells in the flask does not exceed 1 / 3 of the flask specifications, and place it in a shaker for later use.

[0153] (2) Calculate the volume of the transfection buffer Opti-MEM based on the volume of the transfected cells, which is 1 / 10 of the transfection system; calculate the amount of transfection reagent PEI (purchased from Shanghai Haoyuan Biotechnology Co., Ltd., product number HY-K2014), the ratio of which is 9 μg / mL transfected cells; calculate the total amount of transfected DNA, the ratio of which is 1 μg / mL transfected cells.

[0154] The specific transfection process is as follows:

[0155] To a 50mL centrifuge tube, add 10% Opti-MEM (purchased from Beijing Zhongsheng Aobang Biotechnology Co., Ltd., Cat. No. 03.18001A) to the transfection system. Add the plasmid, mix thoroughly, filter, and let stand for 5 minutes. Add PEI to the DNA suspension, gently mix (invert 2-3 times), and let stand for 15-20 minutes. Then, gently add the complex to the aliquoted cells while gently shaking the shaker. Incubate the transfected cells in a shaker at 37°C. Add 293 serum-free feed solution on days 1, 3, and 5 after transfection. Protein purification is performed after 7 days of cell culture.

[0156] 1.3 Antibody Purification

[0157] (1) Sample preparation

[0158] 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.

[0159] (2) Antibody purification

[0160] The cell culture supernatant after centrifugation was purified using protein G (purchased from Shanghai Beyotime Biotechnology Co., Ltd., Catalog No. P2017) affinity purification method to obtain monoclonal antibodies. The operation steps are as follows.

[0161] 1) Connection system: Select a protein G column of appropriate specifications based on the expression level and connect it to the purification system;

[0162] 2) Water balance: Wash with ultrapure water for 3 CV to replace the 25% ethanol storage solution;

[0163] 3) Equilibrate the column: Equilibrate the column with 5 CV of equilibration buffer (50 mM Tris, 100 mM NaCl, pH 8.0);

[0164] 4) Sample loading: Adjust the flow rate to load the sample;

[0165] 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 sample loading flow rate.

[0166] 6) Elution: Elution buffer (100mM Glycine, 10mM NaCl, pH 3.0) was used to collect the antibody.

[0167] 7) Neutralization: Add 2M Tris, pH 8.0 to neutralize the eluted antibody;

[0168] 8) Desalting: Prepare a PD10 desalting column and rinse 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.

[0169] Example 2 Testing the Effects of Different Fc Mutants on Molecular Half-Life

[0170] 2.1 Testing the FcRn binding affinity of different Fc mutants

[0171] The equilibrium dissociation constant (KD) of the above-mentioned antibody samples of the present invention binding to human FcRn protein was determined using biofilm interferometry (BLI) assay. The BLI affinity determination steps are briefly as follows:

[0172] (1) Immobilized ligand

[0173] The antibody sample was diluted to 5 µg / mL using PBS running buffer and immobilized on the ProA sensor for 300 s. The ligand sample was diluted to 5 µg / mL using PBS running buffer and immobilized on the His1K sensor for 180 s.

[0174] (2) Analyte preparation and detection

[0175] Human FcRn protein (purchased from Sino Biological Inc., Cat. No. CT009-H08H) was diluted to 200 nM in running buffer. The binding time was 120 s, and the dissociation time was 180 s. Antibody samples were diluted to 50 nM in running buffer and serially diluted 2-fold. The binding time was 120 s, and the dissociation time was 180 s.

[0176] 3) Experimental results analysis

[0177] The results were analyzed using Data Analysis 12.0 software to obtain the association rate, dissociation rate and affinity constant (see Table 3).

[0178] Table 3 Determination of the affinity of different Fc mutants for FcRn

[0179]

[0180] 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.

[0181] Affinity measurements of different Fc variants for FcRn revealed that the introduction of the H435Q mutation, or a combination of H435A, H435Q, H310A, and H310Q mutations into the Fc region, completely abolished 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 than αTrop2-WT Fc.

[0182] 2.2 In vivo testing of the effects of different Fc mutants on antibody half-life

[0183] 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.

[0184] Blood samples were collected at various time points after antibody injection, including 0.5, 1, 2, 4, 8, 24, 48, 72, 96, and 168 hours after injection, for ELISA testing. The measured antibody concentration over time was 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).

[0185] Table 4 Results of half-life determination of naked antibodies with different Fc mutants in rats

[0186]

[0187] 2.3 Antibodies 177 Lu labeling and half-life testing of labeled substances in animal blood

[0188] 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 Biopharmaceutical Technology Co., Ltd., Cat. No. HY-128890) and react overnight at room temperature. Centrifuge and replace with PBS to remove unreacted DOTA-NHS-ester.

[0189] Chelation of radionuclides: Open the metal bath reactor and preheat to 40°C. Dilute the conjugated antibody to 5 mg / mL with PBS solution to prepare the precursor solution. Take 37 MBq (1 mCi) 177 LuCl3 solution (50 pmol) was added, and then 4 times the equivalent of the precursor solution was added, and the amount of sodium vitamin C buffer was supplemented to 50 μL. The reaction was carried out at 40 ° C and 200 rpm for 2 h to obtain 177 Lu-labeled antibody molecules exist in solution.

[0190] Use 1% sodium citrate solution as the developing solvent to perform thin layer chromatography 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.

[0191] 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 with 3 rats in each group according to the experimental design. Each group was injected with 300 μCi of one of the above-mentioned chelated samples through the tail vein. 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. Blood was collected and tested with a γ counter. The activity of the radioactive drug in the blood at different time points was converted according to the results of the γ counter. The activity of different Fc mutants was calculated. 177 The half-life of Lu-antibodies in animal blood is shown in Table 5.

[0192] Table 5 Different 177 Results of half-life determination of Lu-labeled Fc mutants in rats

[0193]

[0194] From the above results, it can be seen that after 177 The Lu-labeled antibodies showed a half-life that remained basically unchanged or decreased in rat blood. The mutants with reduced half-life also meant that the radioactive substances could be cleared more quickly in the body.

[0195] These Fc mutations are 177 The effect of Lu labeling on the half-life of antibodies is different, and it is uncertain whether the radionuclide-labeled antibodies prepared on this basis will affect their uptake in animals.

[0196] Example 3 Testing the Effects of Different Fc Mutants on the Binding of Molecules to FcγRs and C1q

[0197] 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 molecule 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. 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.

[0198] Table 6 Names, sequences, and Fc mutation information of expressed antibodies used for FcγRs and C1q binding studies

[0199]

[0200] Using αTrop2-WT Fc as a reference, the changes in the binding affinity of different mutants to different FcγR proteins and C1q complement protein were compared. The mutation combinations in the Fc region of αTrop2-Fc v8, αTrop2-Fc v9, αTrop2-Fc v10, and αTrop2-Fc v11 antibodies that affect binding to FcγRs and C1q overlap with the mutation positions in the Fc region of some other antibodies. Therefore, the affinity test of the antibody Fc region affecting 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.

[0201] Table 7 Percentage change in affinity of different mutants for FcγRs and C1q compared with αTrop2-WT Fc

[0202]

[0203] Note: "(↓)" indicates the percentage of affinity reduction compared to αTrop2-WT Fc. For example, "396.3 (↓)" indicates that the affinity of the antibody with the L234A and L235A mutations in the Fc region for the CD64 receptor protein decreased by 396.3% compared to αTrop2-WT Fc. "No binding" indicates that no significant binding between the corresponding antibody mutant and the receptor protein was detected using this experimental method, which also indicates that the corresponding antibody mutant completely lost the ability to bind to the receptor using this experimental method.

[0204] Based on the experimental results, the following inferences can be drawn:

[0205] (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 the αTrop2-WT Fc molecule 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.

[0206] (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.

[0207] In the next step, we will verify the above speculation through specific and extensive experiments.

[0208] Example 4 Imaging test of antibody molecules containing different mutation combinations in tumor-bearing mice

[0209] 4.1 Antibody Conjugation and Labeling

[0210] 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.

[0211] 4.2 Establishment of animal tumor model

[0212] In the experiment, 5×10 6 The number of MDA-MB-468 cells was resuspended in PBS and Matrigel at a ratio of 1:1 (0.1 mL / cell). Tumor growth was observed regularly. The tumors 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.

[0213] 4.3 Single Photon Emission Tomography and Data Processing

[0214] Will 177 Luminescent antibody was diluted with saline to 7.4 MBq (200 μCi) / 200 μL, and each tumor-bearing mouse was injected intravenously with 7.4 MBq (200 μCi) of the drug. At 24, 48, 72, 96, and 120 hours after drug administration, mice were anesthetized with isoflurane and placed in a prone position on an examination table for single-photon emission tomography (SPECT) scanning (IVIS Spectrum, MILabs). Acquisition methods included static 10-minute SPECT scans and medium-resolution whole-body computed tomography. Animal weight, injection dose, injection time, and residual dose were recorded using a record sheet. The injection dose and residual dose measurement times were also recorded. Data were reconstructed after scanning, and PMOD software was used to analyze and delineate tumor, liver, and kidney tissues, quantify drug distribution, and save images and data for further statistical analysis.

[0215] 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.

[0216] Table 8 Uptake of different radiolabeled antibody mutants in tumors, liver, and kidneys at different time points (% ID / g)

[0217]

[0218]

[0219] The above results indicate that the different Fc mutants induce different radiopharmaceutical uptake in tumor tissue. In a comparison of the 11 mutants, αTrop2-Fc v5 to αTrop2-Fc v15, αTrop2-Fc v5, αTrop2-Fc v6, and αTrop2-Fc v7, all exhibited superior or comparable radioactive uptake in tumor tissue to that of the other molecules at different time points. The αTrop2-Fc v5 molecule exhibited optimal tumor uptake at all tested time points and was superior to the control molecule, αTrop2-ABD Fc. This is an unexpected result, completely contrary to the assumption in Example 3. Moreover, in addition to the low level of tumor uptake among 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 contrary to our speculation in Example 3.

[0220] In summary, not all mutants that reduce or completely eliminate binding to FcγRs, C1q, and FcRn have ideal uptake in tumors and livers. Among 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 properties 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 has an advantage over the control mutant molecule αTrop2-ABD Fc in terms of high tumor uptake.

[0221] Figures 1 to 12 The above results are more intuitively demonstrated, in which the tumor tissue is circled with a white oval dotted line.

[0222] 4.4 αTrop2-Fc v5 and αTrop2-ABD Fc 89 Zr labeling and positron emission tomography testing

[0223] To compare the differences in tumor uptake between αTrop2-Fc v5 and αTrop2-ABD Fc labeled with different radionuclides, we used 89The antibody was labeled and visualized with Zr. The chelating agent used was p-SCN-Bn-deferoxamine (purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., product number HY-134797). 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 The Zr reaction was incubated at room temperature for 15 minutes and purified by gel filtration on a disposable PD10 column.

[0224] The labeled molecules were injected into the tail vein of mice bearing MDA-MB-468 tumors. Micro-PET / CT (Super Nova, manufactured by Pingsheng Medical Technology (Kunshan) Co., Ltd.) was used to measure 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 in the tumor tissue at various time points. Figure 13 As shown. After comparison, it was found that 89 The radioactive uptake of Zr-αTrop2-Fc v5 in tumor tissue was superior to that in 89 The Zr-αTrop2-ABD Fc molecule further demonstrated the superior efficacy of the combination of L234A, L235A, and H435Q mutations.

[0225] Example 5 Testing of thiol site-directed coupling

[0226] αTrop2-ABD Fc and αTrop2-Fc v5 were conjugated to the thiol groups of the antibody using the DOTA chelator according to the following simplified 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 thiol groups of the antibody. The conjugated sample was then purified with PBS using an ultrafiltration centrifuge tube.

[0227] The αTrop2-ABD Fc and αTrop2-Fc v5 conjugated products, αTrop2-ABD Fc-Mal-DOTA and αTrop2-Fc v5-Mal-DOTA, were analyzed by LC-MS according to the following steps:

[0228] 1) Sample processing

[0229] After desalting, a 2 mg / mL sample was incubated with PNGase F (Cat. No. P0704, Manufacturer: New England Biotechnology Co., Ltd.) at 37°C for 1 hour to remove sugars. After the incubation, the sample was placed in a sample vial and tested on the instrument.

[0230] 2) Liquid quality testing

[0231] The test samples were separated using an ultra-high performance liquid chromatography system (model: Vanquish UPLC, brand: Thermo Fisher). Phase A consisted of 0.1% formic acid-water solution, and phase B consisted of 0.1% formic acid-acetonitrile solution. The test samples were loaded via an autosampler and then gradient separated on a chromatographic column (model and specifications: ACQUITY UPLC BEH C4, 2.1*50 mm, 1.7 μm, 300 Å, brand: Waters) at 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 for 10 minutes, with positive ion detection and a parent ion scan range of 500–4000 m / z. The experimental results are shown in the table. Figure 14-15 and Table 9.

[0232] Table 9 Summary of mass spectrometry analysis of two molecules after thiol coupling

[0233]

[0234] From the above results, it is easy to find that when using 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 simple.

[0235] Example 6 Single Photon Emission Tomography Results of αTrop2-Fc v5 in Tumor-Bearing Mice Using Thiol-Conjugated αTrop2

[0236] The αTrop2-Fc v5-Mal-DOTA molecule in Example 5 was 177 Lu-labeled mice bearing MDA-MB-468 tumors were injected into the tail vein, and single photon emission tomography was performed at different time points. The tumor, liver, and kidney were circled using PMOD software. Figure 16 shown.

[0237] The uptake results of three different tissues at different time points after being processed by PMOD software are shown in Table 10:

[0238] Table 10 PMOD treatment results of αTrop2-Fc v5 using thiol coupling in tumor-bearing mice (%ID / g)

[0239]

[0240] 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.

[0241] 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 amino acid sequence of the Fc domain before mutation is shown in SEQ ID NO: 32; 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; 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; the amino acid sequence of the combination (3) is shown in SEQ ID NO: 23; The receptor binding fragment and the Fc domain are connected via a hinge region, and the amino acid sequence of the hinge region is shown in SEQ ID NO: 35; The receptor binding fragment is a single domain heavy chain antibody, and the amino acid sequence of the single domain heavy chain antibody is shown in SEQ ID NO: 36; The amino acid sequence of the fusion protein containing the combination (1) is shown in SEQ ID NO: 5; The amino acid sequence of the fusion protein containing the combination (2) is shown in SEQ ID NO: 6; The amino acid sequence of the fusion protein containing the combination (3) is shown in SEQ ID NO:

7.

2. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein of claim 1.

3. A carrier, characterized in that The vector comprises the nucleic acid of claim 2.

4. A host cell, characterized in that The host cell comprises the nucleic acid of claim 2 or the vector of claim 3.

5. Use of the fusion protein according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3, and the host cell according to claim 4 in the preparation of a radioactive protein-conjugated drug.

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

7. The radioactive protein-conjugated drug according to claim 6, 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.

8. The radioactive protein-conjugated drug according to claim 7, characterized in that The methods for coupling the fusion protein to the compound chelating radionuclides include: A: coupling with the chelate ring through a linker; B: coupling via free or reduced sulfhydryl groups; C: Coupling occurs via exposed amino groups.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: the fusion protein according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3 or the host cell according to claim 4.

10. Use of the fusion protein of claim 1, the nucleic acid of claim 2, the vector of claim 3, the host cell of claim 4, the radioactive protein-conjugated drug of any one of claims 6 to 8, or the pharmaceutical composition of claim 9 in the preparation of a product for diagnosing and / or treating tumors, wherein the tumors are gastric cancer, colon cancer, breast cancer, rectal cancer, or head and neck malignancies.

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