Bispecific Antibodies and Their Applications

Abstract

The present invention provides a bispecific antibody and its application. The antibody comprises: a first antigen-binding region having CD27 molecule-binding activity; and a second antigen-binding region having CD33 molecule-binding activity. The bispecific antibody prepared by the present invention can simultaneously target CD27 and CD33, thereby mediating the killing of tumor cells by T cells and having strong tumor suppression ability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to bispecific antibodies and applications thereof, and more specifically to recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, the use of the 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 the recombinant antibodies in the preparation of kits, and kits. Background Art

[0002] Cancer is a serious threat to people's lives and health. In recent years, the incidence and mortality of cancer have continued to rise globally. Currently available cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody-targeted therapy, and macromolecular immunotherapy. However, these approaches have limited effectiveness in only some cancer patients, and cancer remains a major challenge for human health.

[0003] Acute myeloid leukemia (AML) is a cancer caused by the uncontrolled proliferation of myeloid precursor cells. The incidence of AML is positively correlated with age, with the highest incidence in people aged 60-70 years. The standard treatment for AML is chemotherapy followed by a bone marrow transplant. However, due to the inability of elderly patients to tolerate the side effects of chemotherapy, the five-year fertility rate for patients over 60 years old is only 10%. Due to the heterogeneity of genetic mutations in tumor cells of AML patients, small molecule targeted drugs can only be used in the minority of patients with corresponding genetic mutations, severely limiting the applicability of these drugs.

[0004] CD33 is a type I transmembrane protein that is expressed on the surface of myeloid cells and myeloid precursor cells (tumor cells) of patients with acute myeloid leukemia, but not on the surface of normal stem cells. Therefore, therapy targeting CD33 is a potential universal therapy for patients with acute myeloid leukemia. For example, Mylotarg is an antibody-drug conjugate consisting of a monoclonal antibody targeting CD33 and a cytotoxin calicinomycin linked to it. In essence, it uses antibodies to target tumor cells to deliver chemotherapy drugs. Clinically, it has been found that this drug can cause serious adverse reactions including hepatotoxicity (including veno-occlusive disease), infusion-related reactions (including allergic reactions), and bleeding.

[0005] Therefore, there is still a need to further develop bispecific antibodies with low immunogenicity, low toxicity and strong affinity. Summary of the Invention

[0006] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0007] CD33 is a type I transmembrane protein, whose expression is on the surface of myeloid cells and myeloid precursor cells (tumor cells) of acute myeloid leukemia patients, but not on the surface of normal stem cells. Therefore, the therapy targeting CD33 is a potentially effective therapy for myeloid cell leukemia.

[0008] The single-chain variable fragment (ScFv) of CD27 antibody can bind to T cells. The inventors designed a bispecific antibody targeting CD27 and CD33. After extensive experimental screening, the advantageous bispecific antibody CD27×CD33 was obtained. This bispecific antibody can simultaneously bind to CD33-positive tumor cells and T cells, effectively mediate the killing of tumor cells by T cells, and has strong anti-cancer ability.

[0009] Therefore, in the first aspect of the present invention, the present invention proposes a recombinant antibody. According to an embodiment of the present invention, it includes: a first antigen-binding region having CD27 molecule-binding activity; and a second antigen-binding region having CD33 molecule-binding activity. The recombinant antibody according to the embodiment of the present invention can simultaneously bind to CD27 and CD33, effectively mediate the killing of tumor cells by T cells, and has strong tumor suppression ability.

[0010] In the second aspect of the present invention, the present invention proposes a nucleic acid molecule. According to an embodiment 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 the embodiment of the present invention can simultaneously bind to CD27 and CD33, effectively mediate the killing of tumor cells by T cells, and has strong tumor suppression ability.

[0011] In the third aspect of the present invention, the present invention proposes an expression vector. According to an embodiment of the present invention, it carries the nucleic acid molecule described in the second aspect. The expression vector may include optional control sequences, and the control sequences are operably linked to the nucleic acid molecule. Among them, 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 embodiment of the present invention can highly express the recombinant antibody in a suitable host cell. The recombinant antibody can simultaneously bind to CD27 and CD33, effectively mediate the killing of tumor cells by T cells, and has strong tumor suppression ability.

[0012] In the fourth aspect of the present invention, the present invention provides a method for preparing the recombinant antibody described in the first aspect. According to an embodiment of the present invention, the method includes: introducing the expression vector described in the third aspect into a cell; culturing the cell under conditions suitable for protein expression and secretion to obtain the recombinant antibody. The method provided according to the embodiment of the present invention can effectively obtain the recombinant antibody, which can bind to both CD27 and CD33 simultaneously, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor suppression ability.

[0013] In the fifth aspect of the present invention, the present invention provides a recombinant cell. According to an embodiment of the present 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 transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can highly efficiently and abundantly express the above-mentioned recombinant antibody under suitable conditions. The recombinant antibody can bind to both CD27 and CD33 simultaneously, effectively mediate the killing effect of T cells on tumor cells, and has strong tumor suppression ability.

[0014] In the sixth aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition includes: 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 mentioned above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD27 and CD33 protein molecules, effectively mediate 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, pharmaceutical compositions, etc., also have a significant effect in treating or preventing tumors.

[0015] In the seventh aspect of the present invention, 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 drug for treating or preventing myeloid cell leukemia and CD33-positive cancers. As mentioned above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD27 and CD33 protein molecules, effectively mediate the killing effect of T cells on tumor cells. Drugs containing a series of substances including the recombinant antibody also have a significant effect in treating or preventing myeloid cell leukemia and CD33-positive cancers.

[0016] In the eighth aspect of the present invention, the present invention provides a drug. According to an embodiment of the present invention, the drug 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. According to an embodiment of the present invention, the drug is used for treating cancer. As described above, the recombinant antibody of the embodiment of the present invention can effectively bind to the CD27 protein molecule on the surface of immune cells and the CD33 protein molecule on the surface of tumor cells, and effectively mediate the killing effect of T cells on tumor cells. Therefore, the drug containing the recombinant antibody or a series of substances capable of expressing the recombinant antibody also has a significant effect on treating or preventing cancer.

[0017] In the ninth aspect of the present invention, the present invention provides the use of the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell in the preparation of a kit for detecting CD27 and / or CD33. The recombinant antibody can bind to the CD27 and / or CD33 protein. Under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can all express the recombinant antibody, and a kit can be prepared therefrom. Therefore, the kit containing the recombinant antibody or the nucleic acid molecule, expression vector or recombinant cell capable of expressing the recombinant antibody can be used to effectively detect CD27 and / or CD33. The kit can be used for scientific research, such as qualitatively or quantitatively detecting the CD27 and / or CD33 protein in a biological sample.

[0018] In the tenth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit contains the aforementioned recombinant antibody. The recombinant antibody provided by the embodiment of the present invention can bind to the CD27 and / or CD33 protein. Under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can all express the recombinant antibody, and a kit can be prepared therefrom. Therefore, the kit containing the recombinant antibody or the nucleic acid molecule, expression vector or recombinant cell capable of expressing the recombinant antibody can be used to effectively detect CD27 and / or CD33. The kit can be used for scientific research, such as qualitatively or quantitatively detecting the CD27 and / or CD33 protein in a biological sample, and can also be used to judge the individual status. For example, after obtaining the CD33 level of the individual, it can be judged whether the CD33 level is too high or too low compared with the normal level.

[0019] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, wherein:

[0021] Figure 1 It is a schematic structural diagram of a CD27×CD33 recombinant bispecific antibody according to an embodiment of the present invention. Among them, the connection position of the linker peptide is not given. The first heavy chain constant region (left side) and the second heavy chain constant region (right side) of the recombinant bispecific antibody are connected through a knob-into-hole structure;

[0022] Figure 2 It is a detection result diagram of the binding ability of the CD27×CD33 recombinant bispecific antibody according to an embodiment of the present invention to CHO-K1-CD27 cells;

[0023] Figure 3 It is a detection result diagram of the binding ability of the CD27×CD33 recombinant bispecific antibody according to an embodiment of the present invention to human peripheral blood CD8+ T cells;

[0024] Figure 4 It is a detection result diagram of the binding ability of the CD27×CD33 recombinant bispecific antibody according to an embodiment of the present invention to CHO-K1-CD33 cells;

[0025] Figure 5 It is a detection result diagram of the binding ability of the CD27×CD33 recombinant bispecific antibody according to an embodiment of the present invention to human acute myeloid leukemia HL-60 tumor cells;

[0026] Figure 6 It is a detection result diagram of the CD27×CD33 recombinant bispecific antibody activating Jurkat-NFAT-lucia-CD27 reporter cells according to an embodiment of the present invention;

[0027] Figure 7 It is a detection result diagram of the CD27×CD33 recombinant bispecific antibody promoting PBMC to kill A375-CD33 tumor cells according to an embodiment of the present invention. Detailed implementation manners

[0028] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0029] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0030] The endpoints and any values disclosed in this document for a range are not limited to the exact range or value. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0031] To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used in this document have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0032] In this document, a "bispecific antibody" refers to one obtained by connecting peptide chains that can specifically recognize different protein molecules to peptide chains of the heavy chain constant region and / or the light chain constant region, wherein the two chains of the heavy chain constant region are connected by a knob-into-hole structure.

[0033] In this document, the "knob-into-hole structure" forms knob (hole) mutations in the CH3 region of the heavy chain constant region of an antibody to facilitate the biting of heavy chains and form heterodimers. For example, in this application, it is achieved by mutating amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; T366W, S354C mutations in the other chain, i.e., "knob").

[0034] In this document, "operably linked" means connecting a foreign gene to a vector such that control elements within the vector, such as transcriptional control sequences and translational control sequences, etc., can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0035] In a first aspect of the present invention, the present invention provides a recombinant antibody, comprising: a first antigen-binding region having CD27 molecule-binding activity; and a second antigen-binding region having CD33 molecule-binding activity. The recombinant antibody according to the embodiments of the present invention can bind to CD27 and CD33 simultaneously, effectively mediate the killing of tumor cells by T cells, and has strong tumor suppression ability.

[0036] According to some specific embodiments of the present invention, the above recombinant antibody may further include at least one of the following additional technical features:

[0037] According to some specific embodiments of the present invention, the first antigen-binding region comprises a CD27 single-chain antibody, the CD27 single-chain antibody comprises a CD27 heavy-chain variable region and a CD27 light-chain variable region, and the C-terminus of the CD27 heavy-chain variable region is connected to the N-terminus of the CD27 light-chain variable region; or the C-terminus of the CD27 light-chain variable region is connected to the N-terminus of the CD27 heavy-chain variable region.

[0038] According to some specific embodiments of the present invention, the CD27 heavy-chain variable region comprises heavy-chain CDRs shown in any one of SEQ ID NO: 1-3.

[0039] According to some specific embodiments of the present invention, the CD27 light-chain variable region comprises light-chain CDRs shown in any one of SEQ ID NO: 4-6.

[0040] According to some specific embodiments of the present invention, the CD27 heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0041] GFTFSSYD (SEQ ID NO: 1).

[0042] IWYDGSNK (SEQ ID NO: 2).

[0043] ARGSGNWGFFDY (SEQ ID NO: 3).

[0044] According to some specific embodiments of the present invention, the CD27 heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;

[0045] According to some specific embodiments of the present invention, the CD27 light-chain variable region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively;

[0046] QGISRW (SEQ ID NO: 4).

[0047] AAS (SEQ ID NO: 5).

[0048] QQYNTYPRT (SEQ ID NO: 6).

[0049] According to some specific embodiments of the present invention, the variable region of the CD27 antibody heavy chain comprises: the amino acid sequence shown in SEQ ID NO: 30

[0050] EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKF KNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 30).

[0051] According to some specific embodiments of the present invention, the variable region of the CD27 antibody light chain comprises: the amino acid sequence shown in SEQ ID NO: 31 DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFS GSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK (SEQ ID NO: 31).

[0052] According to some specific embodiments of the present invention, the CD27 single-chain antibody further comprises 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 CD27 antibody heavy chain, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the CD27 antibody light chain; or the N-terminus of the linker peptide 1 is connected to the C-terminus of the variable region of the CD27 antibody light chain, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the CD27 antibody heavy chain.

[0053] According to some specific embodiments of the present invention, the linker peptide 1 comprises the amino acid sequence shown in SEQ ID NO: 16

[0054] GGGGSGGGGSGGGGS (SEQ ID NO: 16).

[0055] According to some specific embodiments of the present invention, the CD27 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO: 13

[0056] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIK(SEQ ID NO:13).

[0057] According to some specific embodiments of the present invention, the first antigen-binding region further comprises a first heavy chain constant region, wherein the C-terminus of the anti-CD27 single-chain antibody is connected to the N-terminus of the first heavy chain constant region.

[0058] According to some specific embodiments of the present invention, the first heavy chain constant region comprises a first hinge region and a first Fc peptide segment.

[0059] According to some specific embodiments of the present invention, the first Fc peptide segment comprises a first CH2 region and a first CH3 region, and the C-terminus of the first CH2 region is connected to the N-terminus of the first CH3 region.

[0060] 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.

[0061] 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.

[0062] According to some specific embodiments of the present invention, the first CH2 region is a CH2 region fragment of human, primate or murine wild-type IgG1.

[0063] According to some specific embodiments of the present invention, compared with the CH3 region fragment of human wild-type IgG1, the first CH3 region has T366W and / or S354C mutations.

[0064] The amino acid sequence of the human wild-type IgG1 (L234A / L235A) antibody is:

[0065] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:33).

[0066] According to some specific embodiments of the present invention, the first antigen-binding region further comprises a linker peptide 2, the N-terminus of the linker peptide 2 is connected to the C-terminus of the CD27 single-chain antibody, and the C-terminus of the linker peptide 2 is connected to the N-terminus of the first heavy-chain constant region.

[0067] According to some specific embodiments of the present invention, the linker peptide 2 comprises the amino acid sequence shown in SEQ ID NO: 17.

[0068] GGGGS(SEQ ID NO: 17).

[0069] According to some specific embodiments of the present invention, the first heavy-chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18.

[0070] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 18).

[0071] According to some specific embodiments of the present invention, the second antigen-binding region comprises a first peptide chain and a second peptide chain, and the first peptide chain comprises: the heavy-chain variable region of the CD33 antibody.

[0072] According to some specific embodiments of the present invention, the heavy-chain variable region of the CD33 antibody comprises: the heavy-chain CDRs shown in any one of SEQ ID NOs: 7-9.

[0073] According to some specific embodiments of the present invention, the variable region of the CD33 antibody heavy chain comprises CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively.

[0074] GYTITDSN (SEQ ID NO: 7).

[0075] IYPYNGGT (SEQ ID NO: 8).

[0076] VNGNPWLAY (SEQ ID NO: 9).

[0077] According to some specific embodiments of the present invention, the variable region of the CD33 antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14.

[0078] EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKF KNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 14).

[0079] According to some specific embodiments of the present invention, the first peptide chain further comprises a second heavy chain constant region, wherein the C-terminus of the variable region of the CD33 antibody heavy chain is connected to the N-terminus of the second heavy chain constant region.

[0080] According to some specific embodiments of the present invention, the second heavy chain constant region comprises: a CH1 region, a second hinge region, and a second Fc peptide segment.

[0081] 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 segment.

[0082] According to some specific embodiments of the present invention, the second Fc peptide segment comprises: a second CH2 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.

[0083] According to some specific embodiments of the present invention, the CH1 region is the CH1 region of a human, primate, or murine wild-type IgG1.

[0084] According to some specific embodiments of the present invention, the second hinge region is a hinge region fragment of a human, primate, or murine wild-type IgG1.

[0085] According to some specific embodiments of the present invention, the second CH2 region is a CH2 region fragment of human, primate or murine wild-type IgG1.

[0086] According to some specific embodiments of the present invention, the second CH3 region is a CH3 region fragment of human, primate or murine wild-type IgG1.

[0087] According to some specific embodiments of the present invention, the second CH3 region has at least one of the mutations T366S, L368A, Y407V, Y349C compared to the CH3 region fragment of human wild-type IgG1.

[0088] According to some specific embodiments of the present invention, the second heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 19.

[0089] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 19).

[0090] According to some specific embodiments of the present invention, the second peptide chain comprises: the light chain variable region of a CD33 antibody.

[0091] According to some specific embodiments of the present invention, the light chain variable region of the CD33 antibody comprises: the heavy chain CDRs shown in any one of SEQ ID NOs: 10 - 12.

[0092] According to some specific embodiments of the present invention, the light chain variable region of the CD33 antibody comprises the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0093] ESLDNYGIR(SEQ ID NO: 10).

[0094] AAS (SEQ ID NO: 11).

[0095] QQTKEVPWS (SEQ ID NO: 12).

[0096] According to some specific embodiments of the present invention, the light chain variable region of the CD33 antibody comprises: the amino acid sequence shown in SEQ ID NO: 15, or the amino acid sequence shown in SEQ ID NO15.

[0097] DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK (SEQ ID NO: 15).

[0098] According to some specific embodiments of the present invention, the second peptide chain further comprises a light chain constant region, wherein the C-terminus of the light chain variable region of the CD33 antibody is connected to the N-terminus of the light chain constant region.

[0099] According to some specific embodiments of the present invention, the light chain constant region is a human, primate or murine wild-type light chain constant region.

[0100] According to some specific embodiments of the present invention, the antibody light chain constant region is a human Kappa light chain constant region.

[0101] According to some specific embodiments of the present invention, the light chain constant region is a human wild-type IgG1 light chain constant region.

[0102] According to some specific embodiments of the present invention, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 32.

[0103] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).

[0104] According to some specific embodiments of the present invention, the first antigen-binding region and the second antigen-binding region are connected by a knob-into-hole structure.

[0105] According to some specific embodiments of the present invention, the knob-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, Y349C mutations in the second CH3 region.

[0106] According to some specific embodiments of the present invention, the first peptide chain and the second peptide chain are linked by an interchain disulfide bond.

[0107] According to some specific embodiments of the present invention, the recombinant antibody comprises the amino acid sequences shown in SEQ ID NO: 20-22.

[0108] The first antigen-binding region comprises the following amino acid sequence:

[0109] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKGGGGSPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20).

[0110] The second antigen-binding region - the first peptide chain has the following amino acid sequence shown:

[0111] EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:21).

[0112] The second antigen-binding region - the second peptide chain has the amino acid sequence shown below:

[0113] DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:22).

[0114] In a second aspect of the present invention, 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 the embodiments of the present invention can bind to both CD27 and CD33 simultaneously, effectively mediate the killing of tumor cells by T cells, and has strong tumor suppression ability.

[0115] 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.

[0116] The nucleic acid molecule encoding the first antigen-binding region comprises the following nucleotide sequence:

[0117]

[0118] The nucleic acid molecule encoding the second antigen-binding region - the first peptide chain comprises the following nucleotide sequence:

[0119]

[0120] The gene encoding the second antigen-binding region - the first peptide chain has the nucleotide sequence shown below:

[0121] gacattcagctgactcagagcccttccacactgagtgcttctgttggcgacagggtgaccataacttgccgagcttccgagtccttggataactacggcattaggtttctcacttggtttcaacagaagccaggcaaggctccaaagctcctgatgtacgccgctagcaaccagggttctggtgtgccttctcggttctctggcagcgggagcgggacagaattcacactgacaatatcatccttgcagccagatgacttcgccacttattactgtcagcagaccaaggaggtgccctggagcttcggccaggggaccaaagtggaggtcaagcgcaccgtggctgctccaagcgttttcatttttccaccaagtgatgagcaactgaagtcaggaacagccagcgtggtgtgtctcttgaacaatttctatccaagagaggcaaaagtgcagtggaaggtggataatgctcttcagagcggtaactcacaggagtctgtaaccgaacaggactccaaagactccacttactccctctcctccaccctcactctgagtaaagccgactacgagaaacacaaagtttacgcctgcgaggtcacccatcagggcttgtccagccctgtgaccaagtcctttaacagaggcgagtgc(SEQ ID NO:25).

[0122] It should be noted that for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that it actually includes either any one of the complementary double strands or both. For convenience, in this specification and claims, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and the disclosure of one means the disclosure of the other.

[0123] In a third aspect of the present invention, there is provided an expression vector carrying the nucleic acid molecule described in the second aspect. The expression vector may include optional control sequences which are operably linked to the nucleic acid molecule. Among them, the control sequences are one or more control sequences capable of directing the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can highly express the recombinant antibody in a suitable host cell. The recombinant antibody can bind to both CD27 and CD33 simultaneously, effectively mediating the precise and efficient killing of tumor cells by T cells and having strong tumor suppression ability.

[0124] In a fourth aspect of the present invention, there is provided a method for preparing the recombinant antibody described in the first aspect, including: introducing the expression vector described in the third aspect into a cell; culturing the cell 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. The recombinant antibody can bind to both CD27 and CD33 simultaneously, effectively mediating the killing effect of T cells on tumor cells and having strong tumor suppression ability. According to some specific embodiments of the present invention, the cell is not particularly limited, and both prokaryotic cells and eukaryotic cells can be used. When the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is relatively high.

[0125] According to some specific embodiments of the present invention, the cell is a eukaryotic cell.

[0126] According to some specific embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some specific embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is relatively high.

[0127] According to some specific embodiments of the present invention, the eukaryotic cell does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0128] In a fifth aspect of the present invention, there is provided 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 transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can highly express the above-mentioned recombinant antibody in large quantities under suitable conditions. The recombinant antibody can bind to both CD27 and CD33 simultaneously, effectively mediating the killing effect of T cells on tumor cells and having strong tumor suppression ability.

[0129] It should be noted that the recombinant cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. 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, Schizosaccharomyces pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells. In some embodiments, the recombinant cells of the present 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.

[0130] It should be noted that the "suitable conditions" described in the specification of the present application refer to the conditions suitable for the expression of the recombinant antibody of the present application. It is easily understood by those skilled in the art that the conditions suitable for the expression of the recombinant antibody include, but are not limited to, appropriate transformation or transfection methods, appropriate transformation or transfection conditions, healthy host cell states, appropriate host cell densities, suitable cell culture environments, and suitable cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibody according to the specific environment of the laboratory.

[0131] In the sixth aspect of the present invention, the present invention provides a composition, comprising: the recombinant antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect or the recombinant cell of the fifth aspect. As mentioned above, the recombinant antibody of the embodiment of the present invention can effectively bind to CD27 and CD33 protein molecules and promote the accurate and efficient killing of tumor cells by T cells. Compositions containing the recombinant antibody, such as food compositions, pharmaceutical compositions, etc., also have a significant effect on treating or preventing tumors.

[0132] It should be noted that the composition includes combinations separated in time and / or space, as long as they can act together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole, or separately administered to the subject. When the components contained in the composition are separately administered to the subject, the individual components can be administered to the subject simultaneously or sequentially.

[0133] In the seventh aspect of the present invention, 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 drug for treating or preventing myeloid cell leukemia and CD33-positive cancers. As described above, the recombinant antibody of the embodiments of the present invention can effectively bind to CD27 and CD33 protein molecules, prompting T cells to accurately and efficiently kill tumor cells. Therefore, drugs containing a series of substances including the recombinant antibody also have a significant effect on treating or preventing tumors.

[0134] According to some specific embodiments of the present invention, the above use may further include at least one of the following additional technical features:

[0135] The CD33-positive cancers include at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0136] In the eighth aspect of the present invention, the present invention provides a drug 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 described above, the recombinant antibody of the embodiments of the present invention can effectively bind to CD27 and CD33 protein molecules, prompting T cells to accurately and efficiently kill tumor cells. Therefore, drugs containing a series of substances including the recombinant antibody also have a significant effect on treating or preventing cancers.

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

[0138] According to some specific embodiments of the present invention, it includes a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

[0139] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0140] As used herein, "pharmaceutically acceptable" ingredients are those that are suitable for humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for administering a therapeutic agent, including various excipients and diluents.

[0141] The medicament of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode. The dosage forms of the medicament of the present invention are injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. The said medicament should be manufactured under aseptic conditions.

[0142] The effective amount of the active ingredient described in the present invention may vary with the administration mode, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The said factors include but are not limited to: the pharmacokinetic parameters of the said active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the administration route, etc. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0143] The pharmaceutically acceptable carriers described in the present invention include (but are not limited to): water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the administration mode, which are well-known to those of ordinary skill in the art.

[0144] According to some specific embodiments of the present invention, the medicament is used for treating or preventing myeloid cell leukemia and CD33-positive cancers.

[0145] According to some specific embodiments of the present invention, the CD33-positive cancers include at least one of the following: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0146] In the ninth aspect of the present invention, the present invention provides the use of the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell in the preparation of a kit for detecting CD27 and / or CD33. The recombinant antibody can bind to the CD27 and / or CD33 protein. Under suitable conditions, the nucleic acid molecule, expression vector or recombinant cell can all express the recombinant antibody, and a kit can be prepared therefrom. 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 to effectively detect CD27 and / or CD33. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD27 and / or CD33 proteins in biological samples.

[0147] In the tenth aspect of the present invention, the present invention provides a kit, which contains the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell. According to the recombinant antibody provided by the embodiments of the present invention, it can bind to CD27 and / or CD33 protein. Under appropriate conditions, the nucleic acid molecule, expression vector or recombinant cell can all express the recombinant antibody, and a kit can be prepared therefrom. 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 to effectively detect CD27 and / or CD33. The kit can be used for scientific research, such as qualitatively or quantitatively detecting CD27 and / or CD33 protein in a biological sample, and can also be used to judge the individual status. For example, after obtaining the CD33 level of the individual, it can be judged whether the CD33 level is too high or too low compared with the normal level.

[0148] According to some specific embodiments of the present invention, the kit is used to detect CD27 and / or CD33.

[0149] The following will introduce the embodiments in detail. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0150] Example 1 Preparation of CD27×CD33 Bispecific Antibody

[0151] In this example, the production of the bispecific antibody was carried out. The specific experimental operations were as follows: ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher, A2910001), and the cell concentration was adjusted to 6×10 6 / mL to obtain an ExpiCHO cell solution. Add the pTT5 vector (synthesized by Genewiz Suzhou) containing the three-chain encoding genes encoding the first antigen-binding region and the second antigen-binding region (shown in SEQ ID NO: 23, 24, and 25 respectively) to 2 mL of OptiSFM medium (Thermo Fisher, 12309019) to obtain Solution A. Among them, the first antigen-binding region encoding gene includes the nucleotide sequence encoding the CD27 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 encoding gene includes the nucleotide sequence encoding the CD33 antibody 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 encoding gene includes the CD33 antibody light-chain variable region and the light-chain constant region. Add 160 μL of ExpiFectamine CHO transfection reagent (Thermo Fisher, A29130) to 2 mL of OptiSFM medium to obtain Solution B. Then mix Solution A and Solution B to obtain a transfection mixture, and add all of the transfection mixture to 50 mL of the ExpiCHO cell solution within 5 minutes. After culturing for 1 day at 37 °C and 5% CO2, add 8 mL of Feed (Thermo Fisher, A29130) and 300 μL of Enhancer (Thermo Fisher, A29130), and transfer to 32 °C and 5% CO2 for culturing for 9 days, and harvest the culture supernatant. Add 8 mL of Feed on the 5th day. Affinity purify the bispecific antibody from the culture supernatant using a Protein A purification column (GE) to obtain the antibody CD27×CD33. The antibody CD27×CD33 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).

[0152] The first antigen-binding region encoding gene includes the nucleotide sequence shown as follows:

[0153]

[0154] The first antigen-binding region - the first peptide chain-encoding gene comprises the nucleotide sequence shown below:

[0155]

[0156] The first antigen-binding region - the second peptide chain-encoding gene comprises the nucleotide sequence shown as follows:

[0157] gacattcagctgactcagagcccttccacactgagtgcttctgttggcgacagggtgaccataacttgccgagcttccgagtccttggataactacggcattaggtttctcacttggtttcaacagaagccaggcaaggctccaaagctcctgatgtacgccgctagcaaccagggttctggtgtgccttctcggttctctggcagcgggagcgggacagaattcacactgacaatatcatccttgcagccagatgacttcgccacttattactgtcagcagaccaaggaggtgccctggagcttcggccaggggaccaaagtggaggtcaagcgcaccgtggctgctccaagcgttttcatttttccaccaagtgatgagcaactgaagtcaggaacagccagcgtggtgtgtctcttgaacaatttctatccaagagaggcaaaagtgcagtggaaggtggataatgctcttcagagcggtaactcacaggagtctgtaaccgaacaggactccaaagactccacttactccctctcctccaccctcactctgagtaaagccgactacgagaaacacaaagtttacgcctgcgaggtcacccatcagggcttgtccagccctgtgaccaagtcctttaacagaggcgagtgc(SEQ ID NO:25).

[0158] Example 2 Identification of the binding ability of the bispecific antibody to CHO-K1-CD27 cells

[0159] In this example, flow cytometry was used to detect the binding characteristics of the bispecific antibody, and the strength of the signal after the addition of the bispecific antibody was used to judge the binding characteristics of the bispecific antibody and CHO-K1-CD27 cells. The specific experimental operations are as follows:

[0160] HEK293T cells were seeded at 5×10 5Seed cells in a six-well plate at a density of [cell number] cells / well and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. Add the pLVX-EF1a-CD27-IRES-puro (the coding sequence of CD27 protein (SEQ ID NO:26) is inserted between the EcoRI and BamHI restriction enzyme sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (a total of 3 μg) in a ratio of 2:1:1 to 200 μL of serum-free DMEM medium, and then add 12 μg of polyetherimide (PEI, Polysciences, Inc.). The obtained CD27 protein has the amino acid sequence shown in SEQ ID NO:27. Mix well and let stand for 16 min, then add all the liquid to the six-well plate seeded with HEK293T cells. After 6 h of culture, discard the medium and add fresh complete DMEM medium. After 48 h of transfection, collect the cell culture supernatant and filter it through a 0.45 μm filter (Millipore) to obtain the virus supernatant. Add all the virus supernatant to a six-well plate containing 1×10 4 CHO-K1 cells, add polybrene (Sigma) at a final concentration of 4 μg / mL, and culture for 12 h. Subsequently, discard all the supernatant and add fresh complete DMEM medium. The resulting cells are CHO-K1-CD27 cells.

[0161] ATGGCCAGACCTCACCCTTGGTGGCTTTGTGTCTTGGGTACTTTGGTGGGCCTCTCTGCAACACCTGCTCCAAAGTCCTGCCCCGAGAGACATTATTGGGCACAAGGCAAGCTTTGTTGTCAGATGTGCGAACCTGGAACATTTCTGGTGAAAGATTGCGACCAACACAGGAAAGCTGCTCAGTGCGACCCATGTATCCCAGGAGTATCTTTCTCTCCAGACCATCATACCCGACCTCATTGCGAGAGCTGCAGGCACTGTAACAGTGGGCTGTTGGTGCGGAACTGTACAATAACAGCTAACGCCGAGTGTGCCTGTCGAAACGGATGGCAGTGCCGGGACAAAGAGTGCACAGAATGCGACCCTCTGCCCAATCCTTCTCTGACTGCACGTTCCTCCCAGGCATTGTCTCCCCACCCACAGCCAACACATCTCCCCTATGTGTCTGAGATGCTGGAAGCCAGGACCGCTGGTCATATGCAAACCCTGGCAGACTTTCGCCAGCTGCCTGCTAGGACTCTGTCTACCCATTGGCCTCCTCAAAGGAGCCTTTGCTCCAGCGATTTCATTCGCATATTGGTGATCTTTTCCGGCATGTTTCTTGTCTTTACATTGGCCGGGGCTCTCTTTCTGCATCAGCGTAGGAAATATCGGTCCAACAAGGGCGAAAGTCCCGTTGAGCCAGCTGAACCATGCCACTATAGCTGTCCAAGAGAGGAAGAGGGATCAACCATACCCATTCAGGAGGATTACAGGAAGCCCGAGCCTGCCTGTTCCCCT(SEQ ID NO:26).

[0162] MARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP(SEQ ID NO:27).

[0163] Dilute CHO-K1-CD27 cells with PBS to 1×10 6 / mL, add 90 μL per tube to 1.5 mL EP tubes, add 10 μL per tube of rat serum to them, and block at 4°C for 30 min. Add a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of CD27×CD33 bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12) 10 μL per tube, and incubate at 4°C for 30 min. After incubation, add 1 mL of PBS to the EP tubes, centrifuge at 4°C and 100 g for 5 min, discard all the supernatant, and wash the precipitate with PBS once again. After centrifugation, discard all the supernatant, resuspend the cells with 100 μL per tube of PBS, and add 1 μL per tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) to the resuspended cells, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and discard all the supernatant after centrifugation. Resuspend the cells with 200 μL per tube of PBS, and detect with a flow cytometer. The specific experimental results are as Figure 2 shown, further indicating that the bispecific antibody CD27×CD33 of the present invention can bind to CHO-K1-CD27 cells.

[0164] Example 3 Identification of the binding ability of the bispecific antibody to human peripheral blood T cells

[0165] In this example, flow cytometry was used to detect the binding characteristics of the CD27×CD33 bispecific antibody in Examples 1 and 2 above, and the strength of the signal after the addition of the bispecific antibody was used to judge the binding characteristics of the bispecific antibody and human peripheral blood CD8 + T cells. The specific experimental operations are as follows:

[0166] Dilute human peripheral blood mononuclear cells (Saili Bio) with PBS to 5×10 6 / mL, add it to a 1.5 mL EP tube at a volume of 90 μL / tube, add 10 μL / tube of rat serum to it, and incubate at 4°C for 30 min; after the blocking, add 10 μL / tube of a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of the CD27×CD33 bispecific antibody and hIgG (control IgG1, Biolegend, QA16A12). After incubating at 4°C for 30 min, add 1 mL of PBS to the EP tube, centrifuge at 4°C and 100 g for 5 min, discard all the supernatant, wash the precipitate once with PBS, centrifuge and discard all the supernatant, resuspend the cells with 100 μL / tube of PBS, add 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 CD8 antibody (Invitrogen, OKT8), and incubate in the dark at 4°C for 30 min. Wash twice with PBS, centrifuge and discard all the supernatant. Resuspend the cells with 200 μL / tube of PBS and detect with a flow cytometer. The specific experimental results are as Figure 3 shown, indicating that the CD27×CD33 bispecific antibody of the present invention can bind to human peripheral blood T cells.

[0167] Example 4 Identification of the Binding Ability of the Bispecific Antibody to CHO-K1-CD33 Cells

[0168] In this example, flow cytometry was used to detect the binding characteristics of the CD27×CD33 bispecific antibody used in the above example, and the strength of the signal after the addition of the bispecific antibody was used to judge the binding characteristics of the bispecific antibody and CHO-K1-CD33 cells. The specific experimental operations are as follows:

[0169] HEK293T cells were seeded at 5×10 5Seed six-well plates with cells at a density of 4 per well and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. Add the pLVX-EF1a-CD33-IRES-puro vector (the coding sequence of CD33 protein (SEQ ID NO: 28) is inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (a total of 3 μg) in a ratio of 2:1:1 to 200 μL of serum-free DMEM medium, and then add 12 μg of polyetherimide (PEI, Polysciences, Inc.). The resulting CD33 protein has the amino acid sequence shown in SEQ ID NO: 29. After mixing, let it stand for 16 min, and then add all the liquid to the six-well plate seeded with HEK293T cells. After culturing for 6 h, discard the medium and add fresh complete DMEM medium for culture. After 48 h of transfection, collect the cell culture supernatant and filter it through a 0.45 μm filter (Millipore) to obtain the virus supernatant. Add all the virus supernatant to a six-well plate containing

[0170] CHO-K1 cells, add polybrene (Sigma) at a final concentration of 4 μg / mL, and culture for 12 h. Subsequently, discard all the supernatant and add fresh complete DMEM medium. The resulting cells are CHO-K1-CD33 cells.

[0171] MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ(SEQ ID NO:29).

[0172] Dilute CHO-K1-CD33 cells with PBS to 1×10 6 / mL, add 90 μL / tube to 1.5 mL EP tubes, add 10 μL / tube of rat serum to them, and block at 4 °C for 30 min. Add 10 μL / tube of a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) of the CD27×CD33 bispecific antibody and human IgG (control IgG1, Biolegend, QA16A12) to them, and incubate at 4 °C for 30 min. After incubation, add 1 mL of PBS to the EP tubes, centrifuge at 4 °C and 100 g for 5 min, discard all the supernatant, and wash the precipitate with PBS once more. After centrifugation, discard all the supernatant, resuspend the cells with 100 μL / tube of PBS, and add 1 μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) to it after resuspension, and incubate at 4 °C in the dark for 30 min. Wash twice with PBS, and discard all the supernatant after centrifugation. Resuspend the cells with 200 μL / tube of PBS, and detect with a flow cytometer. The specific experimental results are as Figure 4 shown, further showing that the bispecific antibody CD27×CD33 of the present invention can bind to CHO-K1-CD33 cells.

[0173] Example 5 Identification of the binding ability of the bispecific antibody to human acute myeloid leukemia HL-60 tumor cells

[0174] In this example, flow cytometry was used to detect the binding characteristics of the CD27×CD33 bispecific antibody described in the above example, and the strength of the signal after the addition of the bispecific antibody was used to judge the binding characteristics of the bispecific antibody and human acute myeloid leukemia HL-60 tumor cells. The specific experimental operations are as follows:

[0175] Dilute HL-60 cells with PBS to 1×10 6 / mL, add 90 μL / tube to 1.5 mL EP tubes, add 10 μL / tube of rat serum to them, and incubate at 4°C for 30 min. Add 10 μL / tube of CD27×CD33 bispecific antibody and human IgG antibody (IgG1, Biolegend, QA16A12) at a series of concentration gradients (0.1, 1, 10, 30, 100, 300 μg / mL) respectively, and incubate at 4°C for 30 min. After incubation, add 1 mL of PBS to the EP tubes, centrifuge at 4°C and 100 g for 5 min, discard all the supernatant, and wash the precipitate with PBS once more. After centrifugation, discard all the supernatant, resuspend the cells with 100 μL / tube of PBS, and add 1 μL / tube of Alexa-647-labeled rat anti-human Fc antibody secondary antibody (Biolegend, M1310G05) to it after resuspension, and incubate at 4°C in the dark for 30 min. Wash twice with PBS, and discard all the supernatant after centrifugation. Resuspend the cells with 200 μL / tube of PBS and detect them with a flow cytometer. The specific experimental results are as Figure 5 shown, further indicating that the bispecific antibody CD27×CD33 of the present invention can bind to human acute myeloid leukemia HL-60 cells.

[0176] Example 6: Identification of the activation of Jurkat-NFAT-lucia-CD27 reporter cells by bispecific antibody

[0177] In this example, the Jurkat-NFAT-lucia-CD27 reporter system method was used to identify the ability of the CD27×CD33 bispecific antibody to crosslink CD33 on the surface of target cells and CD27 on the surface of effector cells, and thus activate T cells. The strength of the relative chemiluminescence signal (RLU) was used to judge the ability of the bispecific antibody to activate T cells by bridging CD33-positive target cells and T cells.

[0178] HEK293T cells were seeded at 5×10 5Seed cells in a six-well plate at a density of cells per well and culture overnight in DMEM medium without antibiotics. Discard the medium before transfection and add 1 mL of fresh DMEM medium without antibiotics. Add the pLVX-EF1a-CD27-IRES-puro (the coding sequence of CD27 protein (SEQ ID NO: 26) is inserted between the EcoRI and BamHI restriction sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (3 μg in total) to 200 μL of serum-free DMEM medium at a ratio of 2:1:1. Then add 12 μg of polyetherimide (PEI, Polysciences, Inc.). The obtained CD27 protein has the amino acid sequence shown in SEQ ID NO: 27. After mixing, let it stand for 16 min, and then add all the liquid to the six-well plate seeded with HEK293T cells. After culturing for 6 h, discard the medium and add fresh complete DMEM medium for culture. After 48 h of transfection, collect the cell culture supernatant and filter it through a 0.45 μm filter (Millipore) to obtain the virus supernatant. Add all the virus supernatant to a six-well plate containing 1×10 4 Jurkat-NFAT-lucia cells, add polybrene (Sigma) at a final concentration of 4 μg / mL, and culture for 12 h. Subsequently, discard all the supernatant and add fresh complete DMEM medium. The resulting cells are Jurkat-NFAT-lucia-CD27 cells.

[0179] (1) Dilute the CHO-K1-CD33 cells described in Example 4 to 1×10 5 / mL with complete RPMI-1640 medium, add them to a 96-well plate, and the added volume is 100 μL per well.

[0180] (2) Dilute the above CD27×CD33 bispecific antibody and the negative control human IgG antibody (IgG1, Biolegend, QA16A12) 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 them to the 96-well plate, and the added volume is 20 μL per well.

[0181] (3) Dilute Jurkat-NFAT-lucia-CD27 cells to 1.25×10 5 / mL with complete RPMI-1640 medium, add them to the 96-well plate, and the added volume is 80 μL per well.

[0182] (4) Incubate the reaction system obtained in step (3) in a 37 °C, 5% CO2 incubator for 24 h.

[0183] (5) 50 μL of the culture supernatant was aspirated and added to a 96-well plate, and then the luciferase substrate (Invivogen) was added to the plate at a volume of 50 μL / well.

[0184] (6) Chemiluminescence was detected using a multifunctional microplate reader.

[0185] The specific experimental results are as Figure 6 shown, further indicating that the bispecific antibody CD27×CD33 of the present invention can bridge CD33-positive target cells and T cells, promoting T cell activation.

[0186] Example 7: Bispecific Antibody Promotes PBMC Killing of CD33-Positive Tumor Cells

[0187] In this example, the effect of the CD27×CD33 bispecific antibody obtained in Example 1 on the killing of A375-CD33 tumor cells by PBMC was detected by constructing a reaction system of the tumor cells + PBMC + different concentrations of the bispecific antibody. The specific experimental operations are as follows:

[0188] HEK293T cells were seeded in a six-well plate at 5×10 5 cells / well and cultured overnight in DMEM medium without antibiotics. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without antibiotics was added. The pLVX-EF1a-CD33-IRES-puro (the coding sequence of CD33 protein (SEQ ID NO: 28) was inserted between the EcoRI and BamHI restriction enzyme sites of the pLVX-EF1a-IRES-puro vector), pMD2G, and psPAX2 vectors (a total of 3 μg) were added to 200 μL of serum-free DMEM medium at a ratio of 2:1:1, and then 12 μg of polyetherimide (PEI, Polysciences Co., Ltd.) was added. The obtained CD33 protein has the amino acid sequence shown in SEQ ID NO: 29; after mixing, it was allowed to stand for 16 min, and then all the liquid was added to the above six-well plate seeded with HEK293T cells. After culturing for 6 h, the medium was discarded and fresh complete DMEM medium was added for culture. After 48 h of transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the virus supernatant. All the virus supernatant was added to a six-well plate containing 1×10 4 A375 cells (purchased from Procell, CL-0014), and polybrene (Sigma) was added at a final concentration of 4 μg / mL and cultured for 12 h. Subsequently, all the supernatant was discarded and fresh complete DMEM medium was added. The obtained cells were A375-CD33 cells.

[0189] (1) Add complete RPMI-1640 medium to a 16-well RTCA plate at a volume of 50 μL / well, and calibrate it on the machine;

[0190] (2) Dilute A375-CD33 cells with complete RPMI-1640 medium to 2×10 5 / mL, and add them to the RTCA plate obtained in step (1) at a volume of 50 μL / well respectively. Then, use the xCELLigence RTCA TP device to detect the cell index for 24 h under the conditions of 37 °C and 5% CO2;

[0191] (3) Dilute the CD27×CD33 bispecific antibody and the control 4-1BB×CD33 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), with an added volume of 20 μL / well;

[0192] (4) Dilute PBMC (Saile Biotech) with complete RPMI-1640 medium to 1.25×10 6 cells / mL, and add them to the RTCA plate obtained in step (3), with an added volume of 80 μL / well;

[0193] (5) Detect the cell index of the reaction system obtained in step (4) for 48 h using the xCELLigence RTCA TP device at 37 °C and 5% CO2.

[0194] The specific experimental results are as Figure 7 shown, further indicating that the CD27×CD33 bispecific antibody of the present invention can promote the killing of CD33-positive tumor cells by PBMC.

[0195] It can be seen from the above experimental results that the bispecific antibody obtained in the present invention can bind to T cells and tumor cells, bridge T cells and tumor cells, and promote the activation of T cells and tumor cells.

[0196] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0197] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill 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, Comprising: A first antigen-binding region having CD27 molecule-binding activity; And A second antigen-binding region having CD33 molecule-binding activity, wherein The first antigen-binding region comprises a CD27 single-chain antibody, the CD27 single-chain antibody comprises a CD27 heavy-chain variable region and a CD27 light-chain variable region, the C-terminus of the CD27 heavy-chain variable region is connected to the N-terminus of the CD27 light-chain variable region, the CD27 heavy-chain variable region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 respectively, and the CD27 light-chain variable region comprises CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively; The first antigen-binding region further comprises a first heavy-chain constant region, wherein the C-terminus of the CD27 single-chain antibody is connected to the N-terminus of the first heavy-chain constant region, the first heavy-chain constant region comprises a first hinge region and a first Fc peptide segment, the C-terminus of the first hinge region is connected to the N-terminus of the first Fc peptide segment, the first Fc peptide segment comprises a first CH2 region and a first CH3 region, the C-terminus of the first CH2 region is connected to the N-terminus of the first CH3 region, the first hinge region is a hinge region fragment of primate-derived or murine-derived wild-type IgG1, the first CH2 region is a CH2 region fragment of primate-derived or murine-derived wild-type IgG1, and the first CH3 region has a T366W and / or S354C mutation compared to the CH3 region fragment of human-derived wild-type IgG1; The second antigen-binding region includes a first peptide chain and a second peptide chain, and the first peptide chain is linked to the second peptide chain by an interchain disulfide bond. The first peptide chain includes: a CD33 antibody heavy chain variable region, and the CD33 antibody heavy chain variable region includes CDR1, CDR2, and CDR3 sequences 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 CD33 antibody heavy chain variable region is linked to the N-terminus of the second heavy chain constant region, and the second heavy chain constant region includes: a CH1 region, a second hinge region, and a second Fc peptide segment. The C-terminus of the CH1 region is linked to the N-terminus of the second hinge region, and the C-terminus of the second hinge region is linked to the N-terminus of the second Fc peptide segment. The second Fc peptide segment includes: a second CH2 and a second CH3 region, wherein the C-terminus of the second CH2 region is linked to the N-terminus of the second CH3 region. The CH1 region is a CH1 region of a primate-derived or murine-derived wild-type IgG1, the second hinge region is a hinge region fragment of a primate-derived or murine-derived wild-type IgG1, the second CH2 region is a CH2 region fragment of a primate-derived or murine-derived wild-type IgG1, the second CH3 region is a CH3 region fragment of a primate-derived or murine-derived wild-type IgG1, and the second CH3 region has at least one of the mutations T366S, L368A, Y407V, and Y349C compared to the CH3 region fragment of a human-derived wild-type IgG1; The second peptide chain includes: a CD33 antibody light chain variable region, and the CD33 antibody light chain variable region includes CDR1, CDR2, and CDR3 sequences 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 CD33 antibody light chain variable region is linked to the N-terminus of the light chain constant region, and the light chain constant region is a primate-derived or murine-derived wild-type light chain constant region.

2. The recombinant antibody according to claim 1, wherein The CD27 antibody heavy chain variable region includes: the amino acid sequence shown in SEQ ID NO: 30; Optionally, the CD27 antibody light chain variable region includes: the amino acid sequence shown in SEQ ID NO:

31.

3. The recombinant antibody according to claim 2, wherein, The CD27 single-chain antibody further includes a linker peptide 1, wherein the N-terminus of the linker peptide 1 is linked to the C-terminus of the CD27 antibody heavy chain variable region, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the CD27 antibody light chain variable region; or the N-terminus of the linker peptide 1 is linked to the C-terminus of the CD27 antibody light chain variable region, and the C-terminus of the linker peptide 1 is linked to the N-terminus of the CD27 antibody heavy chain variable region.

4. The recombinant antibody according to claim 3, characterized in that, The linker peptide 1 includes the amino acid sequence shown in SEQ ID NO:

16.

5. The recombinant antibody according to claim 3, characterized in that, The CD27 single-chain antibody includes the amino acid sequence shown in SEQ ID NO:

13.

6. The recombinant antibody according to claim 1, wherein The first hinge region is a hinge region fragment of a human-derived wild-type IgG1.

7. The recombinant antibody according to claim 1, wherein The first CH2 region is a fragment of the CH2 region of human wild-type IgG1.

8. The recombinant antibody according to claim 1, characterized in that, The first antigen-binding region further comprises a linker peptide 2, the N-terminus of which is connected to the C-terminus of the CD27 single-chain antibody, and the C-terminus of which is connected to the N-terminus of the first heavy-chain constant region.

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

17.

10. The recombinant antibody according to claim 1, characterized in that, The first heavy-chain constant region comprises the amino acid sequence shown in SEQ ID NO:

18.

11. The recombinant antibody according to claim 1, wherein, The CD33 antibody heavy-chain variable region comprises: the amino acid sequence shown in SEQ ID NO:

14.

12. The recombinant antibody according to claim 1, wherein The CH1 region is the CH1 region of human wild-type IgG1.

13. The recombinant antibody according to claim 1, characterized in that, The second hinge region is a fragment of the hinge region of human wild-type IgG1.

14. The recombinant antibody according to claim 1, characterized in that, The second CH2 region is a fragment of the CH2 region of human wild-type IgG1.

15. The recombinant antibody according to claim 1, wherein The second CH3 region is a fragment of the CH3 region of human wild-type IgG1.

16. The recombinant antibody according to claim 1, wherein, Compared with the CH3 region fragment of human wild-type IgG1, the second CH3 region has at least one of the mutations of T366S, L368A, Y407V, and Y349C.

17. The recombinant antibody according to claim 1, wherein The second heavy-chain constant region comprises the amino acid sequence shown in SEQ ID NO:

19.

18. The recombinant antibody according to claim 1, characterized in that, The CD33 antibody light-chain variable region comprises: the amino acid sequence shown in SEQ ID NO:

15.

19. The recombinant antibody according to claim 1, characterized in that, The antibody light-chain constant region is the human Kappa light-chain constant region.

20. The recombinant antibody according to claim 1, characterized in that The antibody light-chain constant region is the light-chain constant region of human wild-type IgG1.

21. The recombinant antibody according to claim 1, wherein The antibody light-chain constant region comprises the amino acid sequence shown in SEQ ID NO:

32.

22. The recombinant antibody according to claim 1, wherein The first antigen-binding region and the second antigen-binding region are connected by a knob-into-hole structure.

23. The recombinant antibody according to claim 22, characterized in that, The knob-into-hole structure is formed by the mutation of T366W and / or S354C in the first CH3 region and at least one of the mutations of T366S, L368A, Y407V, and Y349C in the second CH3 region.

24. The recombinant antibody according to claim 22, wherein, The recombinant antibody comprises the amino acid sequences shown in SEQ ID NO: 20-22.

25. A nucleic acid molecule, characterized in that, The nucleic acid encodes the recombinant antibody according to any one of claims 1 to 24.

26. The nucleic acid molecule according to claim 25, wherein The nucleic acid molecule has the nucleotide sequences shown in SEQ ID NO: 23-25.

27. An expression vector, characterized in that, Carry the nucleic acid molecule according to claim 25 or 26.

28. A method for preparing a recombinant antibody according to any one of claims 1-24, characterized in that, Comprising: Introduce the expression vector according to claim 27 into a cell; Cultivate the cell under conditions suitable for protein expression and secretion to obtain the recombinant antibody.

29. The method according to claim 28, wherein The cell is a eukaryotic cell.

30. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule according to claim 25 or 26, or the expression vector according to claim 27, or is capable of expressing the recombinant antibody according to any one of claims 1-24.

31. A composition, characterized in that, Comprising: The recombinant antibody according to any one of claims 1 to 24, the nucleic acid molecule according to claim 25 or 26, the expression vector according to claim 27, or the recombinant cell according to claim 30. Use of the recombinant antibody according to any one of claims 1-24, the nucleic acid molecule according to claim 25 or 26, the expression vector according to claim 27, the recombinant cell according to claim 30 or the composition according to claim 31 in the preparation of a medicament for the treatment of malignant melanoma.

33. A drug, characterized in that, Comprising: The recombinant antibody according to any one of claims 1-24, the nucleic acid molecule according to claim 25 or 26, the expression vector according to claim 27, the recombinant cell according to claim 30 or the composition according to claim 31.

34. The drug according to claim 33, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

35. The medicament according to claim 33, characterized in that, The medicament is used for the treatment of malignant melanoma.

36. Use of the recombinant antibody according to any one of claims 1-24, the nucleic acid molecule according to claim 25 or 26, the expression vector according to claim 27 or the recombinant cell according to claim 30 in the preparation of a kit for detecting CD27 and / or CD33.

37. A kit, characterized in that, The kit contains the recombinant antibody according to any one of claims 1-24, the nucleic acid molecule according to claim 25 or 26, the expression vector according to claim 27 or the recombinant cell according to claim 30.

38. The kit according to claim 37, wherein The kit is used for detecting CD27 and / or CD33.

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