Bispecific antibody and use thereof

Abstract

A bispecific antibody and a use thereof. The bispecific antibody comprises: a first binding region having Ly6G6D-binding activity and a second binding region having 4-1BB-binding activity, wherein the first binding region comprises a Fab region, the second binding region comprises VHH, and the first binding region is linked to the second binding region. The 4-1BB×Ly6G6D bispecific antibody enables 4-1BB to exhibit a Ly6G6D-dependent agonistic effect in the presence of Ly6G6D-positive tumor cells, thus can activate tumor-specific immune cells, and minimizes systemic toxicity while exerting an anti-tumor effect. The bispecific antibody is expected to provide a new therapeutic option for cancer patients and has high clinical value.

Description

A bispecific antibody and its application Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a bispecific antibody and its applications. Background Technology

[0002] MSI-H subtype colorectal cancer has shown a high response rate to PD-1 inhibitor monotherapy. However, patients with the more common Non-MSI-H subtype typically respond poorly to immunotherapy monotherapy, with only a small percentage achieving disease stability and not exhibiting tumor shrinkage. Therefore, further exploration of combination therapy strategies is needed to improve efficacy.

[0003] 4-1BB agonist antibodies (such as urelumab and utomilumab) may cause systemic toxicity, especially hepatotoxicity, when used alone. In contrast, utomilumab has lower toxicity, but its 4-1BB agonist activity is relatively weak. To reduce the systemic toxicity of 4-1BB antibodies, developing bispecific antibodies is one possible solution.

[0004] LY6G6D protein is a membrane protein belonging to the lymphocyte antigen 6 (LY6) family, possessing important biological functions and potential applications. TCGA RNA-seq data analysis revealed that LY6G6D is significantly upregulated in colorectal tumor tissues, particularly in Non-MSI-H (MSS and MSI-L) colorectal tumors, while its expression is absent or significantly lower in normal tissues. Therefore, Ly6G6D is a potential therapeutic target for Non-MSI-H colorectal tumors. Currently, the main investigational drugs targeting Ly6G6D are CD3×Ly6G6D bispecific antibodies, but clinically, there is a need for more innovative drugs targeting Ly6G6D with different mechanisms to provide patients with more treatment options. Therefore, the development of bispecific antibodies targeting Non-MSI-H subtypes of colorectal cancer with good efficacy and safety is still necessary, as this has significant clinical value. Summary of the Invention

[0005] This invention aims to address, at least to some extent, one of the technical problems in the existing technology regarding the limited number of combination therapy strategies for Non-MSI-H subtype colorectal cancer, and the limited efficacy and safety. The bispecific antibody provided in the embodiments of this invention can effectively and specifically bind to Ly6G6D and / or 4-1BB, enabling 4-1BB to exhibit Ly6G6D-dependent agonistic activity in the presence of Ly6G6D-positive tumor cells. This achieves antitumor effects while minimizing systemic toxicity, thus improving both efficacy and safety.

[0006] Therefore, in a first aspect, the present invention provides a bispecific antibody. In some embodiments, the bispecific antibody includes: a first binding region having Ly6G6D binding activity, and a second binding region having 4-1BB binding activity; wherein the first binding region comprises a Fab region, the second binding region comprises a VHH region, and the first binding region and the second binding region are linked together.

[0007] Currently, activation of monoclonal antibodies targeting 4-1BB has shown clinical efficacy, but its effectiveness is still limited by systemic toxicity. The 4-1BB×Ly6G6D bispecific antibody designed in this application enables 4-1BB to exhibit Ly6G6D-dependent agonistic activity in the presence of Ly6G6D-positive tumor cells. This allows for the activation of tumor-specific immune cells, exerting anti-tumor effects while minimizing systemic toxicity, thus effectively treating Non-MSI-H subtype colorectal cancer.

[0008] In some embodiments, the bispecific antibody may further include at least one of the following additional technical features:

[0009] In some implementations, the bispecific antibody is a symmetrical antibody.

[0010] In some implementations, the Fab region includes a first heavy chain variable region, a CH1 region, a light chain variable region, and a CL region, wherein the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 region, and the C-terminus of the light chain variable region is connected to the N-terminus of the CL region.

[0011] In some embodiments, the first heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, wherein (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO:1, (2) H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, and (3) H-CDR3 has the amino acid sequence shown in SEQ ID NO:3.

[0012] In some embodiments, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, wherein (1) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, (2) L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, and (3) L-CDR3 has the amino acid sequence shown in SEQ ID NO:6.

[0013] Optionally, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it;

[0014] In some embodiments, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0015] In some embodiments, the CH1 region comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0016] In some embodiments, the CL region comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0017] In some embodiments, the VHH includes a second heavy chain variable region comprising H-CDR1, H-CDR2, and H-CDR3, wherein (1) the H-CDR1 has the amino acid sequence shown in SEQ ID NO: 11, 14, 17, 20, or 23; and (2) the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 12, 15, 18, 21, or 24; and (3) the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 13, 16, 19, 22, or 25.

[0018] In some embodiments, the second heavy chain variable region comprises H-CDR1, H-CDR2, and H-CDR3, wherein,

[0019] (1) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:11, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:12, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:13; or

[0020] (2) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:14, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:15, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:16; or

[0021] (3) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:17, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:18, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:19; or

[0022] (4) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:20, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:21, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:22; or

[0023] (5) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:23, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:24, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:25.

[0024] In some implementations, the CDR sequences described herein are defined by any one or a combination of systems such as Kabat, Chothia, AbM, Contact, or IMGT.

[0025] In some implementations, the CDR sequences described in the text are defined by the Kabat system. Specific sequences are shown in Table 1.

[0026] In some embodiments, the VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 26 to 30, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0027] In some implementations, the first binding region further includes an Fc region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof.

[0028] In some implementations, the N-terminus of the Fc region is connected to the C-terminus of the Fab region, and the C-terminus of the Fc region is connected to the N-terminus of the VHH.

[0029] In some implementations, the N-terminus of the Fc region is connected to the C-terminus of the CH1 region, and the C-terminus of the Fc region is connected to the N-terminus of the VHH.

[0030] In some implementations, the Fc region is derived from a human IgG antibody or a mutant thereof.

[0031] In some implementations, the Fc region is derived from human IgG1 or IgG4 antibodies or mutants thereof.

[0032] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0033] In some embodiments, the bispecific antibody further includes a linker peptide, wherein the N-terminus of the Fc region is linked to the C-terminus of the CH1 region, the C-terminus of the Fc region is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the VHH.

[0034] In some embodiments, the linker peptide includes a flexible linker peptide.

[0035] In some embodiments, the amino acid sequence of the linker peptide is (GGS)n, where n is an integer greater than or equal to 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and more preferably 2.

[0036] In some embodiments, the amino acid sequence of the linker peptide is as shown in SE ID NO:32.

[0037] In some embodiments, the bispecific antibody comprises a heavy chain and a light chain, wherein,

[0038] The heavy chain comprises any one of the amino acid sequences shown in SEQ ID NO:33-37 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it;

[0039] The light chain comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. According to some specific embodiments of the invention, the bispecific antibody has two identical fused heavy chains and two identical light chains.

[0040] In a second aspect, the present invention provides an isolated nucleic acid molecule. In some embodiments, said nucleic acid molecule comprises a nucleotide sequence encoding the bispecific antibody described in the first aspect. The nucleic acid molecule according to embodiments of the present invention is capable of efficiently encoding the aforementioned bispecific antibody.

[0041] In a third aspect, the present invention provides a nucleic acid construct. In some embodiments, the nucleic acid construct comprises the isolated nucleic acid molecule described in the second aspect. Further, the nucleic acid construct comprises one or more control sequences operatively linked to the nucleic acid molecule, said one or more control sequences directing the production of the antibody or antigen-binding fragment thereof described in the present invention in a suitable host cell, said one or more control sequences being selected from: promoters, enhancers, stop signals, signal peptides, leader sequences, transcription terminators, and any group thereof.

[0042] In a fourth aspect, the present invention provides an expression vector. In some embodiments, the expression vector carries the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, or expresses the bispecific antibody described in the first aspect. When the above-mentioned nucleic acid molecule is linked to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements need to be operably linked. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned bispecific antibody can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of bispecific antibodies.

[0043] In some embodiments, the expression vector is selected from plasmids, granules, viruses, mini-chromosomes, and artificial chromosomes.

[0044] In some implementations, the expression vector is a eukaryotic expression vector.

[0045] In some embodiments, the eukaryotic expression vector is selected from pCRII, pCR3 and pcDNA3.4, pB SII, pET 15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual.

[0046] In a fifth aspect, the present invention provides a recombinant cell. In some embodiments, the recombinant cell carries the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or expresses the bispecific antibody described in the first aspect. According to embodiments of the present invention, the recombinant cell can efficiently express the bispecific antibody under suitable conditions, the bispecific antibody being able to effectively bind to Ly6G6D and / or 4-1BB, and further, the bispecific antibody having good preventive and / or therapeutic effects on Ly6G6D and / or 4-1BB-mediated diseases.

[0047] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma fissicerum, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this invention are preferably mammalian cells, including HEK-293 cells, HELA cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0048] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the bispecific antibody described in this application. Those skilled in the art will readily understand that suitable conditions for bispecific antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the bispecific antibody based on the specific environment of their laboratory.

[0049] In a sixth aspect, the present invention provides a composition. In some embodiments, the composition comprises the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect.

[0050] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The bispecific antibodies of the present invention can also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other antibodies or their antigen-binding fragments, peptide inhibitors, small molecule antagonists, and / or agents that interfere with upstream or downstream signal transduction of Ly6G6D and / or 4-1BB.

[0051] In a seventh aspect, the present invention provides a medicament. In some embodiments, the medicament comprises the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. The medicament according to embodiments of the present invention is effective in preventing and / or treating Ly6G6D and / or 4-1BB-mediated diseases, such as cancer.

[0052] In some implementations, the drug also includes a pharmaceutically acceptable carrier.

[0053] In an eighth aspect of the invention, the invention provides for the use of the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect in the preparation of a medicament for the prevention, relief, and / or treatment of cancer.

[0054] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, low-grade glioma of the brain, uterine carcinosarcoma and endometrial cancer, and acute myeloid leukemia.

[0055] In some specific embodiments of the present invention, the cancer is colorectal cancer, preferably Non-MSI-H subtype colorectal cancer.

[0056] In a ninth aspect of the invention, a kit is provided. In some embodiments, the kit comprises the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cells described in the fifth aspect. As is known prior, the aforementioned bispecific antibody can specifically bind to Ly6G6D and / or 4-1BB. Furthermore, under suitable conditions, the nucleic acid molecule, nucleic acid construct, expression vector, or recombinant cells can all express the bispecific antibody. Further, the kit containing the above substances can effectively bind to Ly6G6D and / or 4-1BB, and can be used for the effective detection of Ly6G6D and / or 4-1BB. The kit can be used for scientific research, such as qualitative or quantitative detection of Ly6G6D and / or 4-1BB in biological samples, and can also be used to assess the condition of a subject, such as determining whether the subject's Ly6G6D and / or 4-1BB levels are too high or too low than normal after obtaining the subject's Ly6G6D and / or 4-1BB levels. The biological sample can be cells, tissues, blood, etc.

[0057] In some embodiments, the kit may also include reagents commonly used for detecting Ly6G6D and / or 4-1BB, such as coating solutions.

[0058] In some implementations, the kit may also include instructions for use.

[0059] In a tenth aspect of the invention, the invention provides for the use of the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cells described in the fifth aspect in the preparation of a kit for detecting Ly6G6D and / or 4-1BB.

[0060] In an eleventh aspect of the invention, the invention provides for the use of the bispecific antibody described in the first aspect, the isolated nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect, or the drug described in the seventh aspect, in the prevention, relief, and / or treatment of cancer.

[0061] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, low-grade glioma of the brain, uterine carcinosarcoma and endometrial cancer, and acute myeloid leukemia.

[0062] According to a specific embodiment of the present invention, the cancer is colorectal cancer, preferably Non-MSI-H subtype colorectal cancer.

[0063] In a twelfth aspect of the invention, a method for preventing, alleviating, and / or treating cancer is provided. In some embodiments, the method includes administering to a subject at least one of: (1) the bispecific antibody of the first aspect; (2) the isolated nucleic acid molecule of the second aspect; (3) the nucleic acid construct of the third aspect; (4) the expression vector of the fourth aspect; (5) the recombinant cells of the fifth aspect; (6) the composition of the sixth aspect; and (7) the drug of the seventh aspect.

[0064] Typically, the bispecific antibody is administered in an effective amount, i.e., an amount sufficient to achieve the desired therapeutic and / or preventative effect, for example, an amount that causes prevention or relief of symptoms associated with the disease being treated, such as diseases associated with Ly6G6D and / or 4-1BB. The effective amount of the composition administered to the subject will depend on the type and severity of the disease, as well as on individual characteristics such as general health status, age, sex, weight, and tolerance to the drug; it will also depend on the severity and type of the disease, factors that a person skilled in the art will be able to determine the appropriate dosage based on.

[0065] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, low-grade glioma of the brain, uterine carcinosarcoma and endometrial cancer, and acute myeloid leukemia.

[0066] According to a specific embodiment of the present invention, the cancer is colorectal cancer, preferably Non-MSI-H subtype colorectal cancer.

[0067] In some implementations, the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0068] In a thirteenth aspect of the invention, the invention provides for use in diagnosing whether a subject has cancer in at least one of the following: (1) the bispecific antibody of the first aspect; (2) the isolated nucleic acid molecule of the second aspect; (3) the nucleic acid construct of the third aspect; (4) the expression vector of the fourth aspect; (5) the recombinant cell of the fifth aspect; (6) the composition of the sixth aspect; and (7) the medicament of the seventh aspect.

[0069] In some implementations, the cancer includes at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, low-grade glioma of the brain, uterine carcinosarcoma and endometrial cancer, and acute myeloid leukemia.

[0070] According to a specific embodiment of the present invention, the cancer is colorectal cancer, preferably Non-MSI-H subtype colorectal cancer.

[0071] In a fourteenth aspect of the invention, a method for diagnosing whether a subject has cancer is provided. In some embodiments, the method includes detecting Ly6G6D and / or 4-1BB in a test sample using at least one of the following: (1) the bispecific antibody described in the first aspect; (2) the isolated nucleic acid molecule described in the second aspect; (3) the nucleic acid construct described in the third aspect; (4) the expression vector described in the fourth aspect; and (5) the recombinant cells described in the fifth aspect. As previously stated, the bispecific antibody of the embodiments of the present invention can effectively bind to Ly6G6D and / or 4-1BB. Therefore, the bispecific antibody can be used to detect Ly6G6D and / or 4-1BB, which mediate a variety of diseases. Therefore, the level of Ly6G6D and / or 4-1BB contained in a biological sample derived from the subject can be used to determine whether the subject has a disease mediated by Ly6G6D and / or 4-1BB. In addition, the above substances can be used to monitor the levels of Ly6G6D and / or 4-1BB in the test samples of subjects. That is, the above substances can also be used to stage the disease in subjects with Ly6G6D and / or 4-1BB-mediated diseases, and can also be used for prognostic assessment of Ly6G6D and / or 4-1BB-mediated diseases.

[0072] In some implementations, the above method may further include at least one of the following additional technical features:

[0073] In some implementations, the requirement that the Ly6G6D and / or 4-1BB levels in the test sample are not lower than the minimum standard for disease is an indication that the test sample originates from a patient with a Ly6G6D and / or 4-1BB-mediated disease. The minimum standard value can be determined through comparative analysis and verification of the Ly6G6D and / or 4-1BB levels in test samples from a large number of individuals with the Ly6G6D and / or 4-1BB-mediated disease and a large number of healthy individuals.

[0074] In some implementations, the cancer is at least one of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colorectal cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, low-grade glioma of the brain, uterine carcinosarcoma and endometrial cancer, and acute myeloid leukemia.

[0075] According to a specific embodiment of the present invention, the cancer is colorectal cancer, preferably Non-MSI-H subtype colorectal cancer.

[0076] In some implementations, the sample to be tested includes at least one of the following: tissue, cells, blood, serum, sweat, feces, and urine.

[0077] The technical effects of this invention include at least the following:

[0078] This invention provides a novel agonistic bispecific antibody (4-1BB×Ly6G6D) with a symmetrical structure, which relieves...

[0079] The limitations imposed by the systemic toxicity of 4-1BB monoclonal antibodies allow 4-1BB to exert a Ly6G6D-dependent agonistic effect in the presence of Ly6G6D-positive tumor cells, thereby activating tumor-specific immune cells. This approach maximizes anti-tumor efficacy while minimizing systemic toxicity, resulting in good efficacy and safety.

[0080] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0081] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0082] Figure 1 shows a schematic diagram of the structure of the bispecific antibody designed in Example 1 of the present invention;

[0083] Figure 2A shows the flow cytometry results of the binding of the bispecific antibody to human-4-1BB positive cells in Example 3 of the present invention;

[0084] Figure 2B shows the flow cytometry results of the binding of the bispecific antibody to cyno-4-1BB positive cells in Example 3 of the present invention;

[0085] Figure 3A shows the flow cytometry results of the binding of bispecific antibodies 72-81C6 and 72-61A3 to HEK293T-Ly6G6D positive cells in Example 4 of the present invention.

[0086] Figure 3B shows the flow cytometry results of the binding of bispecific antibodies 72-1A9, 72-1F9 and 72-71A9 to CT26-Ly6G6D positive cells in Example 4 of the present invention.

[0087] Figure 4A shows the detection results of the activation efficacy of the bispecific antibody in inducing 4-1BB in the presence of CT26-Ly6G6D target cells in Example 5 of the present invention;

[0088] Figure 4B shows the detection results of the activation efficacy of the bispecific antibody in inducing 4-1BB in the absence of CT26-Ly6G6D target cells in Example 5 of the present invention.

[0089] Figure 5A shows the detection results of the bispecific antibody in Example 6 of the present invention when the TFN-γ level was present or absent in CT26-Ly6G6D target cells.

[0090] Figure 5B shows the detection results of IL-2 levels of the bispecific antibody in CT26-Ly6G6D target cells in and out of the present invention in Example 6 of the present invention.

[0091] Figure 6A shows the analysis results of the effect of administration of bispecific antibodies 72-1A9 and 72-71A9 on the tumor volume in a mouse model of colorectal cancer in Example 7 of the present invention. The horizontal axis represents the number of days after tumor inoculation, and the vertical axis represents the tumor volume.

[0092] Figure 6B shows the analysis results of the effect of administration of bispecific antibodies 72-1F9 and 72-61A3 on the tumor volume in a mouse model of colorectal cancer in Example 7 of the present invention. The horizontal axis represents the number of days after tumor inoculation, and the vertical axis represents the tumor volume.

[0093] Figure 6C shows the analysis results of the effect of administration of bispecific antibody 72-81C6 in Example 7 of the present invention on tumor volume in colorectal cancer mice. The horizontal axis represents the number of days after tumor inoculation, and the vertical axis represents the tumor volume. Detailed Implementation

[0094] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0095] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, and immunology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0096] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0097] In this document, the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof will not be considered restrictive terms, but will be interpreted as meaning “but not limited to” or “not limited to.”

[0098] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and the plural in the context of describing the invention (especially in the context of the following claims).

[0099] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0100] The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., double Fab), as long as they exhibit the desired antigen-binding activity.

[0101] The term "complementarity-determining region" or "CDR" refers to the amino acid residue in the variable region of an antibody responsible for antigen binding. Nanobodies contain three CDRs, named CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), or the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). For a given nanobodies, those skilled in the art will readily identify the CDRs as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefrance et al., Dev. Comparat. Immunol. 27:55-77, 2003). The specific CDR sequences given in this application are all defined according to the Kabat system.

[0102] The term "frame region" or "FR" residues refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above. The FR of the variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4.

[0103] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0104] The term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0105] The terms “full-length antibody,” “intact antibody,” “all antibody,” and “all antibody” are used interchangeably in this document to refer to antibodies that have a structure substantially similar to that of natural antibodies or that have a heavy chain containing an Fc region as defined herein.

[0106] The term "natural antibody" refers to a naturally occurring immunoglobulin molecule with a different structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. Each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, from the N-terminus to the C-terminus, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain. The light chains of an antibody, based on the amino acid sequence of their constant domains, can be classified into one of two types, called kappa (κ) and lamuda (λ).

[0107] The term "antigen-binding fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to: double Fab; Fv; Fab; Fab, Fab'-SH; F(ab')2; single-chain antibody molecules (e.g., scFv, ScFab); and multispecific antibodies formed from antibody fragments.

[0108] The Fab fragment is an antigen-binding fragment produced by papain digestion of an antibody and consists of the intact L chain, a variable region domain (VH) of the H chain, and a first constant domain (CH1) of a heavy chain. Papain digestion of an antibody produces two identical Fab fragments. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, possessing divalent antigen-binding activity and still capable of cross-linking antigens. The Fab' fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain, these residues containing one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the cysteine ​​residues of the constant domain have free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with hinge cysteine ​​residues in between. Other chemical couplings of antibody fragments are also known.

[0109] "Fv" consists of a tight, non-covalently associated dimer of a heavy-chain variable region domain and a light-chain variable region domain. The folding of these two domains generates six hypervariable rings (three rings each from the H and L chains), which contribute amino acid residues to achieve antigen binding, thus giving the antibody antigen-binding specificity. However, even a single variable domain (or half of Fv, containing only three antigen-specific CDRs) can recognize and bind antigens, although its affinity is often lower than that of the intact binding site.

[0110] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes both the native sequence Fc region and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly designing the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies may include antibody populations with all Lys447 residues removed, antibody populations with Lys447 residues not removed, and antibody populations having mixtures containing and without Lys447 residues.

[0111] Light chains (LCs) derived from any vertebrate can be classified into two distinct types based on the amino acid sequence of their constant domain, referred to as κ and λ, respectively. Immunoglobulins can be classified into different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, with heavy chains referred to as α, δ, γ, ε, and μ, respectively. The γ and α classes are further subdivided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0112] The term "variable region" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using either the VH or VL domain from the antibody binding to that antigen to screen libraries of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887, 1993; Clarkson et al., Nature 352:624-628, 1991.

[0113] The term "hypervariant region" or "HVR" refers to the individual regions (complementarity-determining regions or CDRs) within the variable domains of an antibody that are hypervariable in sequence. Typically, an antibody contains six CDRs: three in the VH domain (H-CDR1, H-CDR2, H-CDR3) and three in the VL domain (L-CDR1, L-CDR2, L-CDR3).

[0114] The term "sFv" or "scFv," meaning "single-chain Fv," refers to an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the scFv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired antigen-binding structure.

[0115] The terms “single-domain antibody,” “nanobody,” and “VHH” antibody are used interchangeably. Originally described as an antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody” (i.e., “antibody lacking light chains”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)), it is a single-domain antibody fragment derived from unique antibodies in camelids (such as camels, alpacas, etc.) and sharks. Unlike traditional antibodies, nanobodies consist only of the variable region of heavy chain antibodies.

[0116] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of monoclonal antibody preparations, such variants are typically presented in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody preparations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody preparation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0117] The term "bispecific" antibody refers to an artificial antibody or antigen-binding fragment derived from two different monoclonal antibodies and capable of binding to two different epitopes. The two epitopes may be present on the same antigen, or they may be present on two different antigens.

[0118] In this article, "symmetric antibody" refers to an antibody with a symmetrical structural design. The antibody molecule maintains structural symmetry. For example, a bispecific antibody with the central axis between the two peptide chains of the Fc fragment as the line of symmetry has identical structures and sequences on both sides. Exemplarily, in some embodiments of the present invention, the specific structure of the symmetric antibody is shown in Figure 1. The C-terminus of the heavy chain in the first binding region (anti-Ly6G6D total antibody) is linked to the second binding region (anti-4-1BB nanobody VHH) via a linker. The bispecific antibody has two identical fused heavy chains (from N-terminus to C-terminus: VH-CH1-Fc-linker peptide-VHH) and two identical light chains (from N-terminus to C-terminus: VL-CL). The two identical heavy chains are linked by disulfide bonds, and the heavy and light chains are also linked by disulfide bonds, thereby forming a symmetric bispecific antibody with the molecular structure Fab-Fc-VHH(2+2).

[0119] The term "cancer" refers to or describes a physiological condition in mammals typically characterized by uncontrolled cell growth / proliferation. Cancer includes both solid tumors and non-solid tumors. Solid tumors include, but are not limited to, colorectal cancer, melanoma, breast cancer, lung cancer, head and neck cancer, bladder cancer, kidney cancer, ovarian cancer, pancreatic cancer, or prostate cancer, or their metastatic forms. Cancer may be Ly6G6D and / or 4-1BB positive. The term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive in this document.

[0120] The term "treatment" (and its grammatical variations, such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a treated individual, and can be for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some aspects, the bispecific antibodies of the present invention are used to delay the development of disease or slow its progression.

[0121] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain said nucleic acid molecules, but which are located outside chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0122] In this document, the term "construct" refers to a genetic vector containing a specific nucleic acid sequence and capable of transferring the target nucleic acid sequence into a host cell to obtain recombinant cells. The form of the construct is not particularly limited. According to embodiments of the invention, it can be at least one of plasmids, bacteriophages, artificial chromosomes, cosmids, viruses, and viral vectors, preferably viruses or viral vectors, which may be selected by those skilled in the art as needed.

[0123] In this document, the term "expression vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0124] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA, with no particular limitation on their length. The term "nucleic acid molecule" as used in this application actually includes any one or both complementary double strands.

[0125] The term "identity" or "sequence identity" refers to the matching of sequences between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences are 60% identical. Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods conveniently, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0126] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delay agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0127] The term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from a disease associated with Ly6G6D and / or 4-1BB.

[0128] The term "administration" means a method of administering a dose of a compound (e.g., the bispecific antibody of the present invention) to a subject. The compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, subcutaneously, percutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intra-articularly, prostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, intracutaneously, subcutaneously, subconjunctivally, intracysticly, intraarticularly, intraarticularly, intraarticularly, intraarticularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, intra-articularly, orally, intra-articularly, locally, by inhalation, by injection, by infusion, by continuous infusion, by direct perfusion of target cells via local perfusion, via catheter, by perfusion, in the form of an emulsion or a lipid composition. The method of administration can vary depending on a variety of factors (e.g., the compound or composition to be administered and the severity of the condition, disease, or ailment to be treated).

[0129] The amino acid sequences involved in this invention are shown in Table 1. The CDR sequences of the first and second binding regions are defined by the Kabat system. For ease of reading, the sequences marked in bold and underlined in the bispecific antibody are the VH sequences of the anti-Ly6G6D antibody, the sequences marked in italic and underlined are the VL sequences of the first binding region (anti-Ly6G6D antibody), the sequences marked in bold only are the linker peptides, and the sequences marked in underline only are the VHH sequences of the second binding region (anti-4-1BB). In addition, the bolded portions of the amino acid sequences of various 4-1BB proteins (Human, Cynomolgus) are the extracellular regions of each protein.

[0130] Table 1: Amino Acid Sequences

[0131] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0132] Example 1: Construction of a bispecific antibody expression vector

[0133] Using molecular cloning methods, the Anti-4-1BB(VHH) selection molecules 61A3, 81C6, 1A9, 1F9, and 71A9 (from an internal library) were constructed into Plasmid-X (pcDNA3.4) containing the Anti-LY6G6D selection molecule (from an internal library). The specific structure of the bispecific antibody is shown in Figure 1. The C-terminus of the full-length Anti-LY6G6D heavy chain is linked to the Anti-4-1BB(VHH) molecule. Using Plasmid-X as a backbone template, primers (Guangzhou Aiji Biotechnology Co., Ltd.) were designed to construct the bispecific antibody molecular recombinant vector. The monoclonal antibodies urelumab and ADG106 were synthesized by Suzhou Genewise Biotechnology Co., Ltd. to obtain recombinant expression plasmids. The above recombinant expression plasmids were transformed into Top 10 competent cells (Kangti Life, catalog number: KTSM103L), and the bacterial cells were plated on LB agar plates containing 100 μg / mL ampicillin. Single clones from plates were cultured in 1 mL of LB medium (containing 100 μg / mL ampicillin), and plasmids were extracted. Sequencing by Guangzhou Aiji Biotechnology Co., Ltd. confirmed their correctness. A series of verified expression vectors were extracted using the TIANGEN plasmid large-scale extraction kit and stored at -20℃ for later use. The obtained heavy chain plasmids were named Plasmid-72-61A3-H, Plasmid-72-81C6-H, Plasmid-72-1A9-H, Plasmid-72-1F9-H, and Plasmid-72-71A9-H, respectively, and the light chain plasmid was named Plasmid-72-L. The obtained monoclonal antibody urelumab heavy chain plasmid was named urelumab-H, and the light chain plasmid was named urelumab-L; the obtained monoclonal antibody ADG106 heavy chain plasmid was named ADG106-H, and the light chain plasmid was named ADG106-L.

[0134] Example 2: Bispecific Antibody Expression and Purification

[0135] Follow the instructions in the manual for the ExpiCHOS transient transexpression system (Gibco, catalog number A29127). Resuscitate and culture the ExpiCHOS host cells in culture medium until the cell density reaches approximately 6.0 × 10⁶ cells / year. 6When the cell count is 1 / mL, a certain amount of heavy and light chain plasmids of the bispecific or monoclonal antibody shown in Table 2 are mixed with the culture medium and transfected using the liposome method. The cells are labeled and cultured in a shaker at 37℃, 8% CO2, and 140 rpm. Feed is added every other day. Fermentation broth is collected on days 6-7 after transfection. The supernatant is collected and centrifuged at 5000 rpm for 30 min. The cell fermentation broth supernatant is collected, filtered, and used for purification. The collected fermentation broth supernatant is purified using a Protein A chromatography column (Protein A, nano-micro). The equilibration buffer is 20 mM PBS, 0.15 M NaCl, pH 7.4; the eluent is 0.1 M acetate-sodium acetate, pH 3.0. The protein eluent under the target absorption peak is collected. Immediately after elution, the pH is adjusted to 7.4 with 1 M Tris. After dialyzing with PBS buffer, size exclusion chromatography (SEC) and mass spectrometry are performed for identification. As shown in Table 3, the molecular weights identified by mass spectrometry are consistent with the theoretical molecular weights; the purity of the bispecific antibody samples captured by protein A in one step reached over 90% according to SEC detection.

[0136] Table 2: Names of Bispecific Antibiotic Molecular Plasmids

[0137] Table 3: SEC analysis of bispecific antibody molecules

[0138] Example 3: Detection of the binding of bispecific antibodies to 4-1BB positive cells using flow cytometry

[0139] In this embodiment, the binding ability of each bispecific antibody obtained in Example 2 to self-constructed human 4-1BB (SEQ ID NO:43) overexpressing cells HEK293-h4-1BB or cynomolgus monkey 4-1BB (SEQ ID NO:44) overexpressing cells CHO-cyno-4-1BB was detected using the FACS method. The parent cells HEK293 were purchased from ATCC, and the CHO cells were purchased from Lonza. In short, the cells were loaded at a rate of 1–2 × 10⁻⁶ cells / year. 5 Cells were seeded into 96-well plates, and 100 μL of serially diluted antibody was added to each well. After incubation at 4°C for 1 h, cells were washed with PBS. Then, 100 μL of PE-conjugated anti-human IgG antibody (purchased from Jackson ImmunoResearch, catalog number 109-115-098) was added to each well and incubated at 4°C for 30 min. After washing, the cells were evaluated using a CytoFlex (Bechman) flow cytometer.

[0140] The results are shown in Figures 2A and 2B and Table 4. The EC50 of the bispecific antibody binding to the human 4-1BB cell line ranged from 0.9726 to 75.1 nM, and the EC50 of the bispecific antibody binding to the cynomolgus monkey 4-1BB cell line ranged from 4.43 to 18.1 nM.

[0141] Table 4: EC50 (nM) of bispecific antibody binding to 4-1BB

[0142] Example 4: Detection of the binding of bispecific antibody to Ly6G6D positive cells using flow cytometry

[0143] In this embodiment, the binding ability of the bispecific antibodies 72-81C6 and 72-61A3 obtained in Example 2 to HEK293T-Ly6G6D cells overexpressing human Ly6G6D (sequence number: O95868·LY66D_HUMAN) was detected using the FACS method. The parent cells, HEK293T cells, were purchased from ATCC. The binding ability of the bispecific antibodies 72-1A9, 72-1F9, and 72-71A9 obtained in Example 2 to CT26-Ly6G6D cells overexpressing human Ly6G6D (sequence number: O95868·LY66D_HUMAN) was also detected using the FACS method. The parent cells, CT26 cells, were purchased from Nanjing Kebai Biotechnology. In short, the cells were loaded at a concentration of 1–2 × 10⁻⁶ cells / year. 5 Cells were seeded into 96-well plates, and 100 μL of serially diluted antibody was added to each well. After incubation at 4°C for 1 h, cells were washed with PBS. 100 μL of PE-conjugated anti-human IgG antibody (purchased from Jackson ImmunoResearch, catalog number 109-115-098) was added to each well and incubated at 4°C for 30 min. After washing, the cells were evaluated using a CytoFlex (Bechman) flow cytometer.

[0144] The results are shown in Figures 3A and 3B and Table 5. The bispecific antibody has a strong binding to human LY6G6D, and its EC50 ranges from 1.866 to 3.447 nM.

[0145] Table 5: EC50 (nM) of bispecific antibody binding to human LY6G6D positive cells

[0146] Example 5: Detection of Bispecific Antibody Activation Activity Using Reporter Gene Method

[0147] In this embodiment, the activation of 4-1BB reporter gene cells by the bispecific antibodies described in Example 2 in the presence of target cells was evaluated. In this embodiment, the effector cells were the HEK293-NFκB-luci cell line stably expressing human-4-1BB, and the target cells were the CT26-Ly6G6D cell line overexpressing human-Ly6G6D. The serially diluted antibodies were added to effector cells and target cells, respectively, in 384-well plates, with urelumab antibody used as a control antibody. The plates were incubated at 37°C in a 5% CO2 incubator for 8 hours. Luciferase substrate was added to generate bioluminescence, and the luminescence was measured using a microplate reader.

[0148] The results are shown in Figures 4A and 4B and Table 6. The bispecific antibody significantly enhanced the activation efficacy of 4-1BB in the presence of target cells CT26-Ly6G6D. That is, the activation of 4-1BB depends on the mediation of target cells, and the activation of EC50 is stronger than that of urelumab.

[0149] Table 6: Bispecific antibodies against 4-1BB activated EC50 (nM) in the presence of target cells

[0150] Example 6: Cytokine Release Assay to Detect Bispecific Antibody Activation Activity

[0151] In this embodiment, the activation activity of each bispecific antibody described in Example 2 on primary T cells was evaluated by detecting the release level of cytokines in the supernatant. In short, in this embodiment, CT26-Ly6G6D cells overexpressing human-Ly6G6D (sequence number: O95868·LY66D_HUMAN) were digested and then the cell density was adjusted to 1×10⁻⁶ cells using culture medium. 5 Cells / mL are kept on standby; after thawing the primary T cells from the liquid nitrogen tank, adjust the cell density to 1×10⁶ cells / mL using culture medium. 6 Prepare 100 μL of the bispecific antibody by serially diluting it with culture medium containing 0.2 μg / mL CD3 antibody. Add 100 μL of each antibody to a 96-well plate, using urelumab antibody as a control antibody. Add 20 μL of the above T cells and target cells CT26-Ly6G6D, respectively. Incubate at 37°C and 5% CO2 for 3 days. Detect the levels of cytokines IFN-γ and IL-2 in the supernatant using the CBA kit (purchased from BD). Refer to the kit's instruction manual for specific procedures.

[0152] The results are shown in Figures 5A and 5B. The bispecific antibody requires target cells to activate T cells, and its activation efficacy is stronger than that of urelumab.

[0153] Example 7: Efficacy evaluation of bispecific antibody in a mouse subcutaneous xenograft tumor model

[0154] Logarithmic growth phase mouse colorectal cancer cells MC38-Ly6G6D (MC38 cell line stably transfected with human Ly6G6D (sequence number: O95868·LY66D_HUMAN), the parent cells were purchased from Kangyuan Bochuang, and a stable Ly6G6D transfection was constructed in-house), centrifuged, and the cells were counted. The cell density was adjusted to 6.0 × 10⁶ cells / year with PBS. 6 0.1 mL / mouse was injected subcutaneously into the back of h4-1BB mice (purchased from Beijing Biocytogen, 6-8 weeks old). Tumors were allowed to develop at an average size of 80-100 mm². 3 Mice were randomly divided into 7 groups: 5 treatment groups, 1 positive control group, and 1 negative control group, with 6 mice in each group. The mice in the treatment groups received intraperitoneal administration of the antibody described in Example 2 for 2–3 weeks, twice a week. The ADG106 antibody group served as the positive control group, while the negative control group received the corresponding isotype intraperitoneally. Starting from day 0 of administration, tumor diameter and mouse weight were measured twice a week to calculate the trends in tumor volume and body weight. Tumor growth inhibition rate (TGI) was used as the evaluation index. (TGI)% = [1-T / C] × 100%, where T and C were the tumor volumes of the treatment group and the negative control group at the end of the experiment, respectively. Statistical analysis was performed using SPSS 16.0 software. One-way ANOVA was used for comparisons between groups, and P < 0.05 (*) was considered statistically significant. The experimental results are shown in Figures 6A, 6B, and 6C below. The bispecific antibodies 72-1F9, 72-61A3, 72-81C6, 72-1A9, and 72-71A9 all showed significant tumor-suppressing effects in the MC38 colorectal cancer model.

[0155] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0156] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, include: The first binding region, having Ly6G6D binding activity, and The second binding region has 4-1BB binding activity; The first bonding region includes a Fab region, the second bonding region includes a VHH region, and the first bonding region and the second bonding region are connected.

2. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody is a symmetrical antibody; Optionally, the Fab region includes a first heavy chain variable region, a CH1 region, a light chain variable region, and a CL region, wherein the C-terminus of the first heavy chain variable region is connected to the N-terminus of the CH1 region, and the C-terminus of the light chain variable region is connected to the N-terminus of the CL region. Optionally, the first heavy chain variable region includes H-CDR1, H-CDR2, and H-CDR3, wherein, (1) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:1, and (2) The H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, and (3) The H-CDR3 has the amino acid sequence shown in SEQ ID NO:3; Optionally, the light chain variable region includes L-CDR1, L-CDR2, and L-CDR3, wherein, (1) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, and (2) The L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, and (3) The L-CDR3 has the amino acid sequence shown in SEQ ID NO:6; Optionally, the first heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; Optionally, the first light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; Optionally, the CH1 region comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; Optionally, the CL region comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

3. The bispecific antibody according to claim 1 or 2, characterized in that, The VHH includes a second heavy chain variable region, which comprises H-CDR1, H-CDR2, and H-CDR3, wherein... (1) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 11, 14, 17, 20 or 23; and (2) The H-CDR2 has the amino acid sequence shown in SEQ ID NO: 12, 15, 18, 21 or 24; and (3) The H-CDR3 has the amino acid sequence shown in SEQ ID NO: 13, 16, 19, 22 or 25; Optionally, the second heavy chain variable region comprises H-CDR1, H-CDR2, and H-CDR3, wherein, (1) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:11, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:12, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:13; or (2) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:14, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:15, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:16; or (3) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:17, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:18, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:19; or (4) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:20, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:21, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:22; or (5) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:23, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:24, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:25; Optionally, the VHH comprises an amino acid sequence shown in any one of SEQ ID NO: 26 to 30, or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

4. The bispecific antibody according to any one of claims 1 to 3, characterized in that, The first binding region further includes an Fc region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof; Optionally, the N-terminus of the Fc region is connected to the C-terminus of the Fab region, and the C-terminus of the Fc region is connected to the N-terminus of the VHH. Optionally, the N-terminus of the Fc region is connected to the C-terminus of the CH1 region, and the C-terminus of the Fc region is connected to the N-terminus of the VHH. Optionally, the Fc region is derived from a human IgG antibody or a mutant thereof; Optionally, the Fc region is derived from human IgG1 or IgG4 antibodies or mutants thereof; Optionally, the Fc region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

5. The bispecific antibody according to any one of claims 1 to 4, characterized in that, The bispecific antibody further includes a linker peptide, wherein the N-terminus of the Fc region is linked to the C-terminus of the CH1 region, the C-terminus of the Fc region is linked to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is linked to the N-terminus of the VHH. Optionally, the linker peptide includes a flexible linker peptide; Optionally, the amino acid sequence of the linker peptide is (GGS)n, where n is an integer greater than or equal to 1, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, more preferably 2; Optionally, the amino acid sequence of the linker peptide is as shown in SE ID NO:

32.

6. The bispecific antibody according to any one of claims 1 to 5, characterized in that, The bispecific antibody comprises a heavy chain and a light chain, wherein, The heavy chain comprises any one of the amino acid sequences shown in SEQ ID NO:33-37 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it; The light chain comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

7. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the bispecific antibody according to any one of claims 1 to 6.

8. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the isolated nucleic acid molecule as described in claim 7.

9. An expression carrier, characterized in that, The expression vector carries the isolated nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, or expresses the bispecific antibody of any one of claims 1 to 6.

10. A recombinant cell, characterized in that, The recombinant cells carry the isolated nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, the expression vector of claim 9, or express the bispecific antibody of any one of claims 1 to 6.

11. A composition, characterized in that, It comprises the bispecific antibody according to any one of claims 1 to 6, the isolated nucleic acid molecule according to claim 7, the nucleic acid construct according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 10.

12. A drug, characterized in that, The invention comprises the bispecific antibody according to any one of claims 1 to 6, the isolated nucleic acid molecule according to claim 7, the nucleic acid construct according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 10, or the composition according to claim 11.

13. Use of the bispecific antibody of any one of claims 1 to 6, the isolated nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, the expression vector of claim 9, the recombinant cell of claim 10, or the composition of claim 11 in the preparation of a medicament for the prevention, relief, and / or treatment of cancer.

14. A reagent kit, characterized in that, It comprises the bispecific antibody according to any one of claims 1 to 6, the isolated nucleic acid molecule according to claim 7, the nucleic acid construct according to claim 8, the expression vector according to claim 9, or the recombinant cell according to claim 10.

15. Use of the bispecific antibody according to any one of claims 1 to 6, the isolated nucleic acid molecule according to claim 7, the nucleic acid construct according to claim 8, the expression vector according to claim 9, or the recombinant cells according to claim 10 in the preparation of a kit for detecting Ly6G6D and / or 4-1BB.

16. Use of the bispecific antibody according to any one of claims 1 to 6, the isolated nucleic acid molecule according to claim 7, the nucleic acid construct according to claim 8, the expression vector according to claim 9, the recombinant cell according to claim 10, the composition according to claim 11, or the medicament according to claim 12 in the prevention, relief, and / or treatment of cancer.

17. A method for preventing, alleviating, and / or treating cancer, characterized in that, Including administering an effective amount of at least one of the following to the subject: (1) The bispecific antibody according to any one of claims 1 to 6; (2) The isolated nucleic acid molecule as described in claim 7; (3) The nucleic acid construct according to claim 8; (4) The expression vector according to claim 9; (5) The recombinant cells according to claim 10; (6) The composition of claim 11; and (7) The medicine according to claim 12.

18. Use of at least one of the following in diagnosing whether a subject has cancer: (1) The bispecific antibody according to any one of claims 1 to 6; (2) The isolated nucleic acid molecule as described in claim 7; (3) The nucleic acid construct according to claim 8; (4) The expression vector according to claim 9; and (5) The recombinant cells of claim 10.

19. A method for diagnosing whether a subject has cancer, characterized in that, This includes detecting Ly6G6D and / or 4-1BB in the test sample using at least one of the following: (1) The bispecific antibody according to any one of claims 1 to 6; (2) The isolated nucleic acid molecule as described in claim 7; (3) The nucleic acid construct according to claim 8; (4) The expression vector according to claim 9; and (5) The recombinant cells of claim 10.

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