Bispecific antibodies and their use

Abstract

The application provides a bispecific antibody and application thereof, which comprises a first antigen binding region having 4-1BB molecule binding activity and a second antigen binding region having TROP2 molecule binding activity. The bispecific antibody prepared by the application can simultaneously target 4-1BB and TROP2, thereby mediating T cell killing of tumor cells and having strong tumor inhibition capacity.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to bispecific antibodies and their applications, and more specifically, to the use of recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, the use of said recombinant antibodies or antigen-binding fragments or nucleic acid molecules or expression vectors or recombinant cells or compositions in the preparation of drugs, drugs, the use of said recombinant antibodies in the preparation of kits, and kits. Background Technology

[0002] Cancer is a disease that seriously threatens people's lives and health. In recent years, the incidence and mortality rates of cancer worldwide have been rising. Current cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, and macromolecular immunotherapy. However, these methods only have limited effectiveness in a portion of cancer patients, and cancer remains a difficult problem troubling human health.

[0003] Acute myeloid leukemia (AML) is a cancer caused by the uncontrolled proliferation of myeloid progenitor cells. The incidence of AML is positively correlated with age, with the highest incidence in people aged 60-70. The standard treatment for AML is chemotherapy followed by bone marrow transplantation; however, due to the inability of elderly patients to tolerate the side effects of chemotherapy, the five-year productivity of patients over 60 years of age is only 10%. Because of the heterogeneity of gene mutations in tumor cells of AML patients, small molecule targeted drugs are only applicable to a small number of patients with the corresponding gene mutations, severely limiting the applicability of this type of drug.

[0004] TROP2 is a type I transmembrane protein, officially known as human trophoblast surface glycoprotein antigen 2, which shares a high structural sequence similarity with the epithelial adhesion molecule EPCAM. Studies have found that TROP2 is highly expressed in various tumors, including pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer. Furthermore, high TROP2 expression is closely associated with shortened survival and poor prognosis in cancer patients. These results suggest that TROP2 may be a potential therapeutic target for cancer.

[0005] To date, only one TROP2-targeted drug has been approved: the TROP2 antibody-drug conjugate Trodelvy. Trodelvy consists of a TROP2 monoclonal antibody and irinotecan conjugate. Essentially, it uses the antibody to target tumors and deliver chemotherapy drugs. This drug can cause side effects such as neutropenia, diarrhea, and anemia.

[0006] In recent years, bispecific antibodies have become a research hotspot in immunotherapy. Bispecific antibodies are artificial antibodies containing two specific antigen-binding sites, capable of bridging target cells (tumor cells) and effector cells (immune cells) to produce targeted tumor-killing effects. Therefore, there is a need to further develop bispecific antibodies with low immunogenicity, low toxicity, and strong affinity. Summary of the Invention

[0007] This application is based on the inventor's discoveries and understanding of the following facts and problems:

[0008] TROP2 is a type I transmembrane protein that is expressed on the surface of myeloid cells and myeloid progenitor cells (tumor cells) in acute myeloid leukemia patients, but not on the surface of normal stem cells. Therefore, TROP2-targeted therapy is a potential treatment for myeloid cell leukemia.

[0009] The 4-1BB antibody single-chain variable fragment (ScFv) can bind to T cells. The inventors designed a bispecific antibody targeting 4-1BB and TROP2. After extensive experimental screening, they obtained the superior bispecific antibody 4-1BB×TROP2. This bispecific antibody can bind to both TROP2-positive tumor cells and T cells simultaneously, effectively mediating the killing of tumor cells by T cells and exhibiting strong anti-cancer ability.

[0010] Therefore, in a first aspect, the present invention provides a recombinant antibody. According to embodiments of the present invention, it comprises: a first antigen-binding region having 4-1BB molecule binding activity; and a second antigen-binding region having TROP2 molecule binding activity. The recombinant antibody according to embodiments of the present invention can bind to both 4-1BB and TROP2 simultaneously, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0011] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid according to embodiments of the present invention can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0012] In a third aspect, the present invention provides an expression vector. According to embodiments of the invention, the vector carries the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently express the recombinant antibody in suitable host cells. The recombinant antibody can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0013] In a fourth aspect, the present invention provides a method for preparing the recombinant antibody described in the first aspect. According to embodiments of the present invention, the method includes: introducing the expression vector described in the third aspect into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method proposed in the embodiments of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0014] In a fifth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the nucleic acid described in the second aspect, or the expression vector described in the third aspect, or is capable of expressing the recombinant antibody described in the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the invention, the recombinant cell can efficiently and extensively express the aforementioned recombinant antibody under suitable conditions. The recombinant antibody can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor-suppressive ability.

[0015] In a sixth aspect, the present invention provides a composition. According to embodiments of the present invention, it comprises: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to 4-1BB and TROP2 protein molecules, effectively mediating the killing effect of T cells on tumor cells. Compositions containing the recombinant antibody or nucleic acid molecules, expression vectors, or recombinant cells capable of expressing the recombinant antibody, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventative effects against tumors.

[0016] In a seventh aspect, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third 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 treatment or prevention of myeloid leukemia and TROP2-positive cancer. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to 4-1BB and TROP2 protein molecules, effectively mediating the killing effect of T cells on tumor cells, and the medicament comprising the series of substances of the recombinant antibody also has significant therapeutic or preventive effects against cancer.

[0017] In an eighth aspect, the present invention provides a medicament. According to embodiments of the present invention, the medicament comprises: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. As previously stated, the recombinant antibody of the embodiments of the present invention can effectively bind to the 4-1BB protein molecule on the surface of immune cells and the TROP2 protein molecule on the surface of tumor cells, effectively mediating the killing effect of T cells on tumor cells. Therefore, a medicament containing the recombinant antibody or a series of substances capable of expressing the recombinant antibody also has significant therapeutic or preventive effects against cancer.

[0018] In a ninth aspect, the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a kit for the detection of 4-1BB and / or TROP2. The recombinant antibody can bind to the 4-1BB and / or TROP2 proteins. Under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the recombinant antibody, enabling the preparation of a kit. Therefore, a kit containing the recombinant antibody or a nucleic acid molecule, expression vector, or recombinant cell capable of expressing the recombinant antibody can be used for the effective detection of 4-1BB and / or TROP2. The kit can be used in scientific research, such as for the qualitative or quantitative detection of 4-1BB and / or TROP2 proteins in biological samples.

[0019] In a tenth aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises the recombinant antibody described above. The recombinant antibody provided according to the embodiment of the invention can bind to 4-1BB and / or TROP2 proteins. Under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the recombinant antibody, and a kit can be prepared from it. Therefore, a kit containing the recombinant antibody or a nucleic acid molecule, expression vector, or recombinant cell capable of expressing the recombinant antibody can be used for the effective detection of 4-1BB and / or TROP2. The kit can be used for scientific research, such as qualitative or quantitative detection of 4-1BB and / or TROP2 proteins in biological samples, and can also be used to assess individual status, such as determining whether an individual's TROP2 level is too high or too low than normal after obtaining the individual's TROP2 level.

[0020] 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

[0021] 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:

[0022] Figure 1 This is a schematic diagram of the structure of the 4-1BB×TROP2 recombinant bispecific antibody according to an embodiment of the present invention. The linking position of the linking peptide is not shown. The first heavy chain constant region (left side) and the second heavy chain constant region (right side) of the recombinant bispecific antibody are linked by a knock-into-hole structure.

[0023] Figure 2 This is a graph showing the detection results of the binding ability of the 4-1BB×TROP2 recombinant bispecific antibody to the 4-1BB protein according to an embodiment of the present invention;

[0024] Figure 3 This is a graph showing the detection results of the binding ability of the 4-1BB×TROP2 recombinant bispecific antibody to CHO-K1-4-1BB cells according to an embodiment of the present invention;

[0025] Figure 4 This is a graph showing the detection results of the binding ability of the 4-1BB×TROP2 recombinant bispecific antibody to human peripheral blood CD8+ T cells according to an embodiment of the present invention;

[0026] Figure 5 This is a graph showing the detection results of the binding ability of the 4-1BB×TROP2 recombinant bispecific antibody to CHO-K1-TROP2 cells according to an embodiment of the present invention;

[0027] Figure 6 This is a graph showing the detection results of the binding ability of the 4-1BB×TROP2 recombinant bispecific antibody to human breast cancer MDA-MB-231 cells according to an embodiment of the present invention;

[0028] Figure 7 This is a diagram showing the detection results of activated Jurkat-NFAT-lucia-4-1BB reporter cells using the 4-1BB×TROP2 recombinant bispecific antibody according to an embodiment of the present invention.

[0029] Figure 8 This is a graph showing the detection results of the 4-1BB×TROP2 recombinant bispecific antibody promoting the killing of A375-TROP2 tumor cells by PBMCs according to an embodiment of the present invention. Detailed Implementation

[0030] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0031] Furthermore, 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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0034] In this article, "bispecific antibody" refers to a peptide chain that can specifically recognize different protein molecules, which is connected to the heavy chain constant region and / or the light chain constant region, respectively. The two chains of the heavy chain constant region are connected through a knobinto hole structure.

[0035] In this paper, the "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, in this application, it is achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (one chain has T366S, L368A, Y407V, Y349C mutations, i.e., "hole"; the other chain has T366W, S354C mutations, i.e., "knob").

[0036] In this article, "operable ligation" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0037] In a first aspect, the present invention provides a recombinant antibody comprising: a first antigen-binding region having 4-1BB molecule binding activity; and a second antigen-binding region having TROP2 molecule binding activity. The recombinant antibody according to embodiments of the present invention can bind to both 4-1BB and TROP2 simultaneously, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor-suppressive ability.

[0038] According to some specific embodiments of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:

[0039] According to some specific embodiments of the present invention, the first antigen-binding region includes a 4-1BB single-chain antibody, the 4-1BB single-chain antibody including a 4-1BB antibody heavy chain variable region and a 4-1BB antibody light chain variable region, the C-terminus of the 4-1BB antibody heavy chain variable region being connected to the N-terminus of the 4-1BB antibody light chain variable region; or the C-terminus of the 4-1BB antibody light chain variable region being connected to the N-terminus of the 4-1BB antibody heavy chain variable region.

[0040] According to some specific embodiments of the present invention, the 4-1BB antibody heavy chain variable region includes: the heavy chain CDR shown in any one of SEQ ID NO: 1-3.

[0041] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody light chain includes: the light chain CDR shown in any one of SEQ ID NO:4-6.

[0042] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody heavy chain includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0043] GGSFSGYY(SEQ ID NO: 1).

[0044] INHGGYV (SEQ ID NO: 2).

[0045] ARDYGPGNYDWYFDL (SEQ ID NO: 3).

[0046] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody heavy chain includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

[0047] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody light chain includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;

[0048] QSVSSY(SEQ ID NO: 4).

[0049] DAS (SEQ ID NO: 5).

[0050] QQRSNWPPALT (SEQ ID NO: 6).

[0051] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody heavy chain includes the amino acid sequence shown in SEQ ID NO:30.

[0052] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS (SEQ ID NO: 30).

[0053] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody light chain includes the amino acid sequence shown in SEQ ID NO:31.

[0054] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 31).

[0055] According to some specific embodiments of the present invention, the 4-1BB single-chain antibody further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the heavy chain of the 4-1BB antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the light chain of the 4-1BB antibody; or the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the light chain of the 4-1BB antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the 4-1BB antibody.

[0056] According to some specific embodiments of the present invention, the linker peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 16.

[0057] GGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0058] According to some specific embodiments of the present invention, the 4-1BB single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:13.

[0059] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK(SEQ ID NO: 13).

[0060] According to some specific embodiments of the present invention, the first antigen-binding region further includes a first heavy chain constant region, wherein the C-terminus of the 4-1BB single-chain antibody is connected to the N-terminus of the first heavy chain constant region.

[0061] According to some specific embodiments of the present invention, the first heavy chain constant region includes a first hinge region and a first Fc peptide segment.

[0062] According to some specific embodiments of the present invention, the first Fc peptide segment includes a first CH2 region and a first CH3 region, wherein the C-terminus of the first CH2 region is connected to the N-terminus of the first CH3 region.

[0063] According to some specific embodiments of the present invention, the C-terminus of the first hinge region is connected to the N-terminus of the first Fc peptide segment.

[0064] According to some specific embodiments of the present invention, the first hinge region is a hinge region fragment of human, primate or murine wild-type IgG1.

[0065] According to some specific embodiments of the present invention, the first CH2 region is a fragment of the CH2 region of human, primate or mouse wild-type IgG1.

[0066] According to some specific embodiments of the present invention, the first CH3 region has T366W and / or S354C mutations compared to the CH3 region fragment of human wild-type IgG1.

[0067] The amino acid sequence of the human wild-type IgG1 (L234A / L235A) antibody is as follows:

[0068] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33).

[0069] According to some specific embodiments of the present invention, the first antigen-binding region further includes a linker peptide 2, the N-terminus of which is connected to the C-terminus of the 4-1BB single-chain antibody, and the C-terminus of which is connected to the N-terminus of the first heavy chain constant region.

[0070] According to some specific embodiments of the present invention, the linker peptide 2 comprises the amino acid sequence shown in SEQ ID NO: 17.

[0071] GGGGS (SEQ ID NO: 17).

[0072] According to some specific embodiments of the present invention, the first heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:18.

[0073] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 18).

[0074] According to some specific embodiments of the present invention, the second antigen-binding region includes a first peptide chain and a second peptide chain, wherein the first peptide chain includes a TROP2 antibody heavy chain variable region.

[0075] According to some specific embodiments of the present invention, the TROP2 antibody heavy chain variable region includes: the heavy chain CDR shown in any one of SEQ ID NO:7-9.

[0076] According to some specific embodiments of the present invention, the variable region of the 4-1BB antibody heavy chain includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0077] GYTFTNYG (SEQ ID NO: 7).

[0078] INTYTGEP (SEQ ID NO: 8).

[0079] ARGGFGSSYWYFDV (SEQ ID NO: 9).

[0080] According to some specific embodiments of the present invention, the TROP2 antibody heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:14.

[0081] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 14).

[0082] According to some specific embodiments of the present invention, the first peptide chain further includes a second heavy chain constant region, wherein the C-terminus of the TROP2 antibody heavy chain variable region is connected to the N-terminus of the second heavy chain constant region.

[0083] According to some specific embodiments of the present invention, the second heavy chain constant region includes: a CH1 region, a second hinge region, and a second Fc peptide segment.

[0084] According to some specific embodiments of the present invention, the C-terminus of the CH1 region is connected to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is connected to the N-terminus of the second Fc peptide.

[0085] According to some specific embodiments of the present invention, the second Fc peptide segment includes a second CH2 region and a second CH3 region, wherein the C-terminus of the second CH2 region is connected to the N-terminus of the second CH3 region.

[0086] According to some specific embodiments of the present invention, the CH1 region is the CH1 region of human, primate or mouse wild-type IgG1.

[0087] According to some specific embodiments of the present invention, the second hinge region is a hinge region fragment of human, primate or murine wild-type IgG1.

[0088] According to some specific embodiments of the present invention, the second CH2 region is a fragment of the CH2 region of human, primate or mouse wild-type IgG1.

[0089] According to some specific embodiments of the present invention, the second CH3 region is a fragment of the CH3 region of human, primate or mouse wild-type IgG1.

[0090] According to some specific embodiments of the present invention, the second CH3 region has at least one of the mutations T366S, L368A, Y407V, and Y349C compared to the CH3 region fragment of human wild-type IgG1.

[0091] According to some specific embodiments of the present invention, the second heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:19.

[0092] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 19).

[0093] According to some specific embodiments of the present invention, the second peptide chain includes: a TROP2 antibody light chain variable region.

[0094] According to some specific embodiments of the present invention, the TROP2 antibody light chain variable region includes the heavy chain CDR shown in any one of SEQ ID NO:10-12.

[0095] According to some specific embodiments of the present invention, the variable region of the TROP2 antibody light chain includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0096] QDVSIA (SEQ ID NO: 10).

[0097] SAS (SEQ ID NO: 11).

[0098] QQHYITPLT (SEQ ID NO: 12).

[0099] According to some specific embodiments of the present invention, the variable region of the TROP2 antibody light chain includes: the amino acid sequence shown in SEQ ID NO:15, or the amino acid sequence shown in SEQ ID NO:15.

[0100] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 15).

[0101] According to some specific embodiments of the present invention, the second peptide chain further includes a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is connected to the N-terminus of the light chain constant region.

[0102] According to some specific embodiments of the present invention, the light chain constant region is a human-derived, primate-derived, or rodent-derived wild-type light chain constant region.

[0103] According to some specific embodiments of the present invention, the antibody light chain constant region is a human-derived Kappa light chain constant region.

[0104] According to some specific embodiments of the present invention, the light chain constant region is the light chain constant region of human wild-type IgG1.

[0105] According to some specific embodiments of the present invention, the light chain constant region includes the amino acid sequence shown in SEQ ID NO:32.

[0106] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).

[0107] According to some specific embodiments of the present invention, the first antigen-binding region and the second antigen-binding region are connected by a knot-into-hole structure.

[0108] According to some specific embodiments of the present invention, the knock-into-hole structure is formed by the T366W and / or S354C mutations in the first CH3 region and at least one of the T366S, L368A, Y407V, and Y349C mutations in the second CH3 region.

[0109] According to some specific embodiments of the present invention, the first peptide chain and the second peptide chain are connected by inter-disulfide bonds.

[0110] According to some specific embodiments of the present invention, the recombinant antibody comprises the amino acid sequences shown in SEQ ID NO:20-22.

[0111] The first antigen-binding region includes the following amino acid sequence:

[0112] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPSLESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIKGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:20).

[0113] The second antigen-binding region - the first peptide chain has the amino acid sequence shown below:

[0114] QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQ GSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 21).

[0115] The second antigen-binding region—the second peptide chain—has the following amino acid sequence:

[0116] DIQLTQSPSSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 22).

[0117] In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant antibody described in the first aspect. The recombinant antibody encoded by the nucleic acid molecule according to embodiments of the present invention can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor-suppressive ability.

[0118] According to some specific embodiments of the present invention, the nucleic acid has the nucleotide sequences shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25.

[0119] Nucleic acid molecules encoding the first antigen-binding region include the following nucleotide sequences:

[0120]

[0121] Nucleic acid molecules encoding the second antigen-binding region—the first peptide chain—include the following nucleotide sequences:

[0122]

[0123] The gene encoding the second antigen-binding region—the first peptide chain—has the following nucleotide sequence:

[0124] (SEQ ID NO:25).

[0125] It should be noted that those skilled in the art will understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0126] In a third aspect, the present invention provides an expression vector carrying the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently express the recombinant antibody in suitable host cells. The recombinant antibody can simultaneously bind to 4-1BB and TROP2, effectively mediating the precise and efficient killing of tumor cells by T cells, and exhibiting strong tumor suppressor capabilities.

[0127] In a fourth aspect, the present invention provides a method for preparing the recombinant antibody described in the first aspect, comprising: introducing the expression vector described in the third aspect into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method proposed according to some specific embodiments of the present invention can effectively obtain the recombinant antibody, which can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability. According to some specific embodiments of the present invention, the cells are not particularly limited; prokaryotic cells or eukaryotic cells can be used. When the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is higher.

[0128] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0129] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific embodiments of the present invention, when the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is higher.

[0130] According to some specific embodiments of the present invention, the eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0131] In a fifth aspect, the present invention provides a recombinant cell carrying the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect, or expressing the recombinant antibody described in the first aspect. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and massively express the aforementioned recombinant antibody under suitable conditions. The recombinant antibody can simultaneously bind to 4-1BB and TROP2, effectively mediating the killing effect of T cells on tumor cells and exhibiting strong tumor suppressor ability.

[0132] 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, 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 BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0133] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the recombinant antibody described in this application. Those skilled in the art will readily understand that suitable conditions for recombinant 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. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the recombinant antibody based on the specific environment of their laboratory.

[0134] In a sixth aspect, the present invention provides a composition comprising: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, or the recombinant cell described in the fifth aspect. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to 4-1BB and TROP2 protein molecules and promote T cells to precisely and efficiently kill tumor cells. Compositions containing the recombinant antibody, such as food compositions and pharmaceutical compositions, also have significant therapeutic or preventive effects on tumors.

[0135] It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0136] In a seventh aspect, the present invention provides the use of the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third 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 treatment or prevention of myeloid leukemia and TROP2-positive cancer. As previously stated, the recombinant antibody of the embodiments of the present invention can effectively bind to 4-1BB and TROP2 protein molecules, prompting T cells to precisely and efficiently kill tumor cells. Therefore, medicaments containing the recombinant antibody and a series of other substances also have significant therapeutic or preventive effects against tumors.

[0137] According to some specific embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0138] According to some specific embodiments of the present invention, the TROP2-positive cancers include at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer.

[0139] In an eighth aspect, the present invention provides a medicament comprising: the recombinant antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the composition described in the sixth aspect. As previously described, the recombinant antibody of the embodiments of the present invention can effectively bind to 4-1BB and TROP2 protein molecules, prompting T cells to precisely and efficiently kill tumor cells. Therefore, medicaments containing the recombinant antibody and a series of other substances also have significant therapeutic or preventive effects against cancer.

[0140] According to some specific embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0141] According to some specific embodiments of the present invention, the drug is used to treat or prevent myeloid leukemia and TROP2-positive cancer.

[0142] According to some specific embodiments of the present invention, the TROP2-positive cancers include at least one of the following: pancreatic cancer, breast cancer, colon cancer, bladder cancer, oral squamous cell carcinoma, and ovarian cancer.

[0143] According to some specific embodiments of the present invention, a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient are included.

[0144] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0145] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0146] The medicaments of this invention contain a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the medicaments of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they are prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The medicaments are preferably manufactured under aseptic conditions.

[0147] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0148] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0149] In a ninth aspect, the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of a kit for the detection of 4-1BB and / or TROP2. The recombinant antibody can bind to the 4-1BB and / or TROP2 proteins. Under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the recombinant antibody, enabling the preparation of a kit. Therefore, a kit containing the recombinant antibody or a nucleic acid molecule, expression vector, or recombinant cell capable of expressing the recombinant antibody can be used for the effective detection of 4-1BB and / or TROP2. The kit can be used in scientific research, such as for the qualitative or quantitative detection of 4-1BB and / or TROP2 proteins in biological samples.

[0150] In a tenth aspect, the present invention provides a kit comprising the recombinant antibody, nucleic acid molecule, expression vector, or recombinant cell described above. The recombinant antibody provided according to embodiments of the present invention can bind to 4-1BB and / or TROP2 proteins. Under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the recombinant antibody, enabling the preparation of a kit. Therefore, a kit containing the recombinant antibody or a nucleic acid molecule, expression vector, or recombinant cell capable of expressing the recombinant antibody can be used for the effective detection of 4-1BB and / or TROP2. The kit can be used for scientific research, such as qualitative or quantitative detection of 4-1BB and / or TROP2 proteins in biological samples, and can also be used to assess individual status, such as determining whether an individual's TROP2 level is excessively high or low compared to normal levels after obtaining the individual's TROP2 level.

[0151] According to some specific embodiments of the present invention, the kit is used to detect 4-1BB and / or TROP2.

[0152] The embodiments will be described in detail below. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0153] Example 1: Preparation of 4-1BB×TROP2 bispecific antibody

[0154] This embodiment demonstrates the production of bispecific antibodies. The specific experimental procedures are as follows: ExpiCHO cells (purchased from Thermo Fisher) were cultured in ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001) and the cell concentration was adjusted to 6 × 10⁶ cells / year. 6 / mL, to obtain ExpiCHO cell solution. The pTT5 vector (synthesized by Suzhou Genewiz) containing the three-strand coding genes (as shown in SEQ ID NO: 23, 24, and 25, respectively) encoding the first and second antigen-binding regions was added to 2mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain solution A. The first antigen-binding region coding gene includes nucleotide sequences encoding the 4-1BB single-chain antibody (SEQ ID NO: 13), linker peptide 1 (SEQ ID NO: 16), linker peptide 2 (SEQ ID NO: 17), and the first heavy chain constant region (SEQ ID NO: 18). The second antigen-binding region-first peptide chain coding gene includes nucleotide sequences encoding the TROP2 heavy chain variable region (SEQ ID NO: 14) and the second heavy chain constant region (SEQ ID NO: 19). The second antigen-binding region-second peptide chain coding gene includes encoding the TROP2 light chain variable region and the light chain constant region. 160 μL of ExpiFectamineCHO transfection reagent (Thermo Fisher, A29130) was added to 2 mL of OptiSFM medium to obtain solution B. Solution A and solution B were then mixed to obtain the transfection mixture, which was added to 50 mL of ExpiCHO cell solution within 5 minutes. After culturing at 37°C and 5% CO2 for 1 day, 8 mL of feed and 300 μL of Lenhancer (Thermo Fisher, A29130) were added, and the cells were transferred to 32°C and 5% CO2 for 9 days. The culture supernatant was harvested, with 8 mL of feed added on day 5. The bispecific antibody was affinity purified from the culture supernatant using a Protein A purification column (GE) to obtain antibody 4-1BB×GPC3. Antibody 4-1BB×TROP2 has the amino acid sequences shown in SEQ ID NO:20 (first antigen-binding region), SEQ ID NO:21 (second antigen-binding region - first peptide chain), and SEQ ID NO:22 (second antigen-binding region - second peptide chain).

[0155] The gene encoding the first antigen-binding region includes the nucleotide sequence shown below:

[0156]

[0157] The second antigen-binding region—the gene encoding the first peptide chain—includes the nucleotide sequence shown below:

[0158]

[0159] The second antigen-binding region—the gene encoding the second peptide chain—includes the nucleotide sequence shown below:

[0160] (SEQ ID NO:25).

[0161] Example 2: Identification of the binding affinity between the 4-1BB×TROP2 bispecific antibody and the 4-1BB protein.

[0162] ELISA was used to detect the binding properties of the 4-1BB×TROP2 antibody obtained in Example 1. 4-1BB-strep tagII protein was coated into 96-well plates, and the intensity of the signal after antibody addition was used to determine the binding properties between the antibody and 4-1BB.

[0163] Dilute 4-1BB-strep tagII protein (produced in our laboratory) to 2 μg / mL with PBS buffer and add 100 μL / well to each well of a 96-well plate. Incubate overnight at 4°C. Remove the PBS buffer from the 96-well plate, wash 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20), then add 200 μL / well of PBS / 10% BSA and incubate at 37°C for 2 hours for blocking. Remove the blocking buffer, wash 6 times with PBST, and then dilute the 4-1BB×TROP2 antibody to the appropriate concentration with 100 μL / well of PBST / 0.05% BSA. Incubate at 37°C for 1 hour. After removing the reaction mixture, the plate was washed 6 times with PBST. HRP (horseradish peroxidase)-labeled rabbit anti-human IgG secondary antibody (Boster Biological, BA1070) was diluted 100 μL / well with PBST / 0.05% BSA and incubated at 37°C for 1 h. After incubation, the plate was washed 6 times with PBST, and 80 μL / well of TMB (tetramethylbenzidine) was added. The plate was incubated at room temperature for 3 min, and the reaction was terminated by adding 80 μL / well of 4M sulfuric acid. The absorbance was read at 450 nm using a microplate reader. Specific experimental results are as follows: Figure 2 As shown, the antibody of the present invention can bind to 4-1BB-strep tagII.

[0164] Example 3: Identification of the binding ability of 4-1BB×TROP2 bispecific antibody to CHO-K1-4-1BB cells

[0165] This embodiment uses flow cytometry to detect the binding characteristics of the 4-1BB×TROP2 bispecific antibody described in Example 1, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and CHO-K1-4-1BB cells. The specific experimental procedures are as follows:

[0166] HEK293T cells were processed at a rate of 5 × 10 5Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-4-1BB-IRES-puro (containing the coding sequence of 4-1BB protein (SEQ ID NO: 26) inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The resulting 4-1BB protein had the amino acid sequence shown in SEQ ID NO: 27. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was then added to a solution containing 1×10⁻⁶ cells. 4 In a 6-well plate, polybrene (Sigma) was added to CHO-K1 cells at a final concentration of 4 μg / mL, and the cells were cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were CHO-K1-4-1BB cells.

[0167]

[0168] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQK RGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:27).

[0169] CHO-K1-4-1BB cells were diluted to 1×10⁻⁶ with PBS. 6 / mL, add 90μL / tube to a 1.5mL EP tube, add 10μL / tube of rat serum, and block at 4℃ for 30min. Add a series of concentration gradients (0.1, 1, 10, 30, 100, 300μg / mL) of 4-1BB×TROP2 bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) 10μL / tube, and incubate at 4℃ for 30min. After incubation, add 1mL of PBS to the EP tube, centrifuge at 100g for 5min at 4℃, discard the supernatant, and wash the pellet once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μL / tube of PBS, add 1μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry. Specific experimental results are shown below. Figure 3 As shown, the bispecific antibody 4-1BB×TROP2 of the present invention can bind to CHO-K1-4-1BB cells.

[0170] Example 4: Identification of the binding ability of bispecific antibodies to human peripheral blood CD4+ T cells

[0171] This embodiment uses flow cytometry to detect the binding characteristics of the 4-1BB×TROP2 bispecific antibody described in Example 1, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human peripheral blood CD4+ T cells. The specific experimental procedures are as follows:

[0172] Human peripheral blood mononuclear cells (SaiLi Bio) were diluted with PBS to a concentration of 5 × 10⁻⁶. 6 / mL, add 90μL / tube to a 1.5mL EP tube, add 10μL / tube of rat serum, and block at 4℃ for 30min; after blocking, add a series of concentration gradients (10 -3 10 -2 10 -1 10 0 10 1 10 210 μL / tube of 4-1BB×TROP2 bispecific antibody (μg / mL) and hIgG (control IgG1, Biolegend, QA16A12) were added and incubated at 4℃ for 30 min. Then, 1 mL of PBS was added to the EP tube, and the cells were centrifuged at 4℃ and 100g for 5 min. The supernatant was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded again. The cells were resuspended in 100 μL / tube of PBS, and 1 μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) and 1 μL / tube of FITC-labeled mouse anti-human CD4 antibody (Invitrogen, OKT4) were added. The cells were incubated at 4℃ in the dark for 30 min. The cells were washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. Specific experimental results are shown below. Figure 4 As shown, this demonstrates that the bispecific antibody of the present invention can bind to human peripheral blood T cells.

[0173] Example 5: Identification of the binding ability of bispecific antibodies to CHO-K1-TROP2 cells

[0174] This embodiment uses flow cytometry to detect the binding characteristics of the 4-1BB×TROP2 bispecific antibody described in Example 1, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and CHO-K1-TROP2 cells. The specific experimental procedures are as follows:

[0175] HEK293T cells were processed at a rate of 5 × 10 5 Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-TROP2-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for TROP2 protein (SEQ ID NO: 28) between the EcoRI and BamHI restriction sites), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The obtained TROP2 protein has the amino acid sequence shown in SEQ ID NO: 29. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was added to a solution containing 1 × 10⁻⁶ cells / mL of DMEM. 4In a 6-well plate, CHO-K1 cells were incubated with Sigma polybrene at a final concentration of 4 μg / mL for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were designated CHO-K1-TROP2 cells.

[0176]

[0177]

[0178] MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPT AGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL(SEQ ID NO:29).

[0179] CHO-K1-TROP2 cells were diluted to 1×10⁻⁶ with PBS. 6 / mL, add 90μL / tube to a 1.5mL EP tube, add 10μL / tube of rat serum, and block at 4℃ for 30min. Add a series of concentration gradients (10... -4 10 -3 10 -2 10 -1 10 0 10 1 10 24-1BB×TROP2 bispecific antibody (μg / mL) and 10 μL / tube of human IgG (control IgG1, Biolegend, QA16A12) were added and incubated at 4℃ for 30 min. After incubation, 1 mL of PBS was added to the EP tube, and the cells were centrifuged at 4℃ and 100g for 5 min. The supernatant was discarded, and the cells were washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100 μL / tube of PBS. After resuspending, 1 μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) was added, and the cells were incubated at 4℃ in the dark for 30 min. The cells were washed twice with PBS, centrifuged, and the supernatant was discarded. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. The specific experimental results are as follows: Figure 5 As shown, the bispecific antibody 4-1BB×TROP2 of the present invention can bind to CHO-K1-TROP2 cells.

[0180] Example 6: Identification of the binding ability of bispecific antibodies to human breast cancer MDA-MB-231 cells.

[0181] This embodiment uses flow cytometry to detect the binding characteristics of the 4-1BB×TROP2 bispecific antibody described in Example 1, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human breast cancer MDA-MB-231 cells. The specific experimental procedures are as follows:

[0182] Human breast cancer MDA-MB-231 cells were diluted to 1×10⁻⁶ with PBS. 6 / mL, add 90μL / tube to a 1.5mL EP tube, add 10μL / tube of rat serum, and block at 4℃ for 30min. Add a series of concentration gradients (0.1, 1, 10, 30, 100, 300μg / mL) of 4-1BB×TROP2 bispecific antibody and human IgG (control IgG1, Biolegend, QA16A12) 10μL / tube, and incubate at 4℃ for 30min. After incubation, add 1mL of PBS to the EP tube, centrifuge at 100g for 5min at 4℃, discard the supernatant, and wash the pellet once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μL / tube of PBS, add 1μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry. Specific experimental results are shown below. Figure 6As shown, the bispecific antibody 4-1BB×TROP2 of the present invention can bind to human breast cancer MDA-MB-231 cells.

[0183] Example 7: Identification of bispecific antibody-induced activation of Jurkat-NFAT-lucia-4-1BB reporter cells

[0184] This embodiment utilizes the Jurkat-NFAT-lucia-4-1BB reporter system method to identify the ability of the 4-1BB×TROP2 bispecific antibody to crosslink TROP2 on the surface of target cells and 4-1BB on the surface of effector cells, thereby promoting T cell activation. The relative strength of the chemiluminescent signal (RLU) is used to determine the ability of the bispecific antibody to bridge target cells and T cells, thereby activating T cells.

[0185] HEK293T cells were processed at a rate of 5 × 10 5 Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-4-1BB-IRES-puro (containing the coding sequence of 4-1BB protein (SEQ ID NO: 26) inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The resulting 4-1BB protein had the amino acid sequence shown in SEQ ID NO: 27. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was then added to a solution containing 1×10⁻⁶ cells. 4 Jurkat-NFAT-lucia cells were placed in 6-well plates with Sigma polybrene at a final concentration of 4 μg / mL and cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were Jurkat-NFAT-lucia-4-1BB cells.

[0186] (1) Dilute CHO-K1-TROP2 cells to 1×10⁻⁶ using complete RPMI-1640 medium. 5 Add 100 μL / well to a 96-well plate.

[0187] (2) Dilute the 4-1BB×TROP2 bispecific antibody to 500 μg / mL, 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 160 ng / mL, 32 ng / mL and 6.4 ng / mL respectively with complete RPMI-1640 medium and add it to a 96-well plate with an addition volume of 20 μL / well.

[0188] (3) Jurkat-NFAT-lucia-4-1BB cells were diluted to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 5 Add 80 μL / well to a 96-well plate.

[0189] (4) The reaction system obtained in step (3) is incubated in a 37°C, 5% CO2 incubator for 24 h.

[0190] (5) Take 50 μL of culture supernatant and add it to a 96-well plate. Then add luciferase substrate to the plate at a volume of 50 μL / well.

[0191] (6) Use a multi-functional microplate reader to detect chemiluminescence.

[0192] Specific experimental results are as follows Figure 7 As shown, the bispecific antibody 4-1BB×TROP2 of the present invention can bridge target cells and T cells, and promote T cell activation.

[0193] Example 8: Bispecific antibody promotes PBMC killing of tumor cells

[0194] This embodiment examines the effect of the bispecific antibody obtained in Example 1 on the killing of A375-TROP2 tumor cells by PBMCs. The detection was performed by constructing a reaction system consisting of tumor cells, PBMCs, and different concentrations of bispecific antibody. The specific experimental procedures are as follows:

[0195] HEK293T cells were processed at a rate of 5 × 10 5Seed cells into 6-well plates and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. A mixture of pLVX-EF1a-TROP2-IRES-puro (the pLVX-EF1a-IRES-puro vector contains an insertion of the coding sequence for TROP2 protein (SEQ ID NO: 28) between the EcoRI and BamHI restriction sites), pMD2G, and psPAX2 vector (3 μg total) was added to 200 μL of serum-free DMEM medium in a 2:1:1 ratio. Then, 12 μg of polyetherimide (PEI, Polysciences Ltd.) was added. The obtained TROP2 protein has the amino acid sequence shown in SEQ ID NO: 29. After mixing and standing for 16 min, the entire mixture was added to a six-well plate containing HEK293T cells. After 6 h of culture, the medium was discarded, and fresh complete DMEM medium was added. 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm Millipore filter to obtain the viral supernatant. All of the viral supernatant was added to a solution containing 1 × 10⁻⁶ cells / mL of DMEM. 4 A375 cells (purchased from Pronosai, CL-0014) were added to 6-well plates with a final concentration of 4 μg / mL of polybrene (Sigma) and cultured for 12 h. The supernatant was then discarded, and fresh complete DMEM medium was added. The resulting cells were A375-TROP2 cells.

[0196] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well and calibrate the plate.

[0197] (2) Dilute A375-TROP2 cells to 2×10⁻⁶ using complete RPMI-1640 medium. 5 / mL, added to the RTCA plate obtained in step (1) at a volume of 50μL / well, and then the cell coefficient was detected for 24h at 37℃ and 5% CO2 using the xCELLigenceRTCA TP device;

[0198] (3) Dilute the 4-1BB×TROP2 bispecific antibody and CD27×4-1BB bispecific antibody (produced in our laboratory) with complete RPMI-1640 medium to a series of concentration gradients (0.32, 1.6, 8, 40, 200, 1000 ng / mL) and add them to the RTCA plate obtained in step (2) at a volume of 20 μL / well;

[0199] (4) Dilute PBMC (SaiLi Biotechnology) to 1.25 × 10⁻⁶ using complete RPMI-1640 medium. 6Add 80 μL / well to the RTCA plate obtained in step (3);

[0200] (5) The reaction system obtained in step (4) was subjected to cell coefficient detection at 37°C and 5% CO2 for 48 hours using an xCELLigence RTCA TP instrument.

[0201] Specific experimental results are as follows Figure 8 As shown, the bispecific antibody of the present invention can promote the killing of TROP2-positive tumor cells by PBMCs.

[0202] The experimental results above show that the bispecific antibody obtained in this invention can bind to T cells and tumor cells, bridge T cells and tumor cells, and promote T cells to kill tumor cells.

[0203] 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.

[0204] 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 recombinant antibody, characterized in that, include: The first antigen-binding region has 4-1BB molecule binding activity; and The second antigen-binding region has TROP2 molecule binding activity. The first antigen-binding region includes a 4-1BB single-chain antibody, which includes a 4-1BB antibody heavy chain variable region and a 4-1BB antibody light chain variable region. The C-terminus of the 4-1BB antibody heavy chain variable region is connected to the N-terminus of the 4-1BB antibody light chain variable region; or the C-terminus of the 4-1BB antibody light chain variable region is connected to the N-terminus of the 4-1BB antibody heavy chain variable region. The 4-1BB antibody heavy chain variable region includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively. The 4-1BB antibody light chain variable region includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. The first antigen-binding region further includes a first heavy chain constant region, wherein the C-terminus of the 4-1BB single-chain antibody is connected to the N-terminus of the first heavy chain constant region. The first heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:

18. The second antigen-binding region includes a first peptide chain and a second peptide chain. The first peptide chain includes a TROP2 antibody heavy chain variable region, wherein the TROP2 antibody heavy chain variable region includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. The first peptide chain further includes a second heavy chain constant region, wherein the C-terminus of the TROP2 antibody heavy chain variable region is connected to the N-terminus of the second heavy chain constant region, and the second heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:

19. The second peptide chain includes: a TROP2 antibody light chain variable region, wherein the TROP2 antibody light chain variable region includes CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; the second peptide chain further includes a light chain constant region, wherein the C-terminus of the TROP2 antibody light chain variable region is connected to the N-terminus of the light chain constant region; and the light chain constant region includes the amino acid sequence shown in SEQ ID NO:

32. The first peptide chain and the second peptide chain are connected by inter-disulfide bonds, and the first antigen-binding region and the second antigen-binding region are connected by a knock-in-hole structure.

2. The recombinant antibody according to claim 1, characterized in that, The variable region of the 4-1BB antibody heavy chain includes the amino acid sequence shown in SEQ ID NO:

30.

3. The recombinant antibody according to claim 1, characterized in that, The variable region of the 4-1BB antibody light chain includes the amino acid sequence shown in SEQ ID NO:

31.

4. The recombinant antibody according to claim 1, characterized in that, The 4-1BB single-chain antibody further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the heavy chain of the 4-1BB antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the light chain of the 4-1BB antibody; or the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the light chain of the 4-1BB antibody, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the 4-1BB antibody.

5. The recombinant antibody according to claim 4, characterized in that, The linker peptide 1 comprises the amino acid sequence shown in SEQ ID NO:

16.

6. The recombinant antibody according to claim 1, characterized in that, The 4-1BB single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:

13.

7. The recombinant antibody according to claim 1, characterized in that, The first antigen-binding region further includes a linker peptide 2, the N-terminus of which is connected to the C-terminus of the 4-1BB single-chain antibody, and the C-terminus of which is connected to the N-terminus of the first heavy chain constant region.

8. The recombinant antibody according to claim 7, characterized in that, The linker peptide 2 comprises the amino acid sequence shown in SEQ ID NO:

17.

9. The recombinant antibody according to claim 1, characterized in that, The TROP2 antibody heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:

14.

10. The recombinant antibody according to claim 1, characterized in that, The TROP2 antibody light chain variable region includes the amino acid sequence shown in SEQ ID NO:

15.

11. The recombinant antibody according to claim 1, characterized in that, The recombinant antibody comprises the amino acid sequence shown in SEQ ID NO:20-22.

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

13. The nucleic acid molecule according to claim 12, characterized in that, The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 23-25.

14. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 12 or 13.

15. A method for preparing the recombinant antibody according to any one of claims 1-11, characterized in that, include: The expression vector of claim 14 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the recombinant antibody.

16. The method according to claim 15, characterized in that, The cells in question are eukaryotic cells.

17. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 12 or 13, or the expression vector of claim 14, or are capable of expressing the recombinant antibody of any one of claims 1-11.

18. A composition, characterized in that, include: The recombinant antibody according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12 or 13, the expression vector according to claim 14, or the recombinant cell according to claim 17.

19. Use of the recombinant antibody of any one of claims 1-11, the nucleic acid molecule of claim 12 or 13, the expression vector of claim 14, the recombinant cell of claim 17, or the composition of claim 18 in the preparation of a medicament for the treatment or prevention of at least one of the following: breast cancer, melanoma.

20. A drug, characterized in that, include: The recombinant antibody according to any one of claims 1-11, the nucleic acid molecule according to claim 12 or 13, the expression vector according to claim 14, the recombinant cell according to claim 17, or the composition according to claim 18.

21. The medicament according to claim 20, characterized in that, The drug further includes pharmaceutically acceptable excipients.

22. The medicament according to claim 20, characterized in that, The drug is used to treat or prevent at least one of the following: breast cancer, melanoma.

23. Use of the recombinant antibody of any one of claims 1-11, the nucleic acid molecule of claim 12 or 13, the expression vector of claim 14, or the recombinant cell of claim 17 in the preparation of a kit for detecting 4-1BB and / or TROP2.

24. A reagent kit, characterized in that, The kit comprises the recombinant antibody of any one of claims 1-11, the nucleic acid molecule of claim 12 or 13, the expression vector of claim 14, or the recombinant cell of claim 17.

25. The kit according to claim 24, characterized in that, The kit is used to detect 4-1BB and / or TROP2.

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