Anti-human HER2 / CCR4 bispecific antibody and application thereof

Abstract

The invention relates to an anti-HER2 / CCR4 bispecific antibody and an application thereof, and provides a bispecific antibody which can target an HER2 target spot and a CCR4 target spot at the same time, the bispecific antibody comprises a first antigen binding region, a second antigen binding region and an Fc structural domain, the first antigen binding region has HER2 binding activity, the second antigen binding region has CCR4 binding activity, and the Fc structural domain has Fc binding activity. And the Fc structural domain is an Fc structural domain of a Trastuzumab monoclonal antibody. The bispecific antibody can be combined with HER2 with high specificity and high affinity and can be combined with CCR4 with high specificity and relatively low affinity; effect cells are promoted to kill tumor cells and Treg cells in a tumor microenvironment, exhaustion of peripheral blood Treg cells caused by a CCR4 monoclonal antibody is effectively avoided, and good anti-cancer activity and higher safety are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a bispecific antibody against human HER2 / CCR4 and an application thereof. Background Art

[0002] HER2 is a 180KDa transmembrane glycoprotein that is overexpressed on the surface of a variety of tumor cells, including 20%-30% of breast cancer, about 20% of stomach and 12-15% of gallbladder. The amplification of the HER2 gene or the overexpression of the HER2 protein plays an important role in the occurrence, development and metastasis of malignant tumors, so HER2 is considered an important target for anti-tumor therapy. In 1998, the U.S. Food and Drug Administration (FDA) approved the recombinant humanized anti-HER2 monoclonal antibody Trastuzumab (trade name Herceptin) for the treatment of HER2-positive advanced breast cancer. Herceptin significantly prolongs the survival of patients with HER2-positive advanced tumors and is regarded as a milestone in the history of tumor treatment. However, with the widespread use of Herceptin, about 70% of patients are resistant to Herceptin, including primary resistance and secondary resistance.

[0003] CCR4 is a surface marker of Treg. In mice and humans, Treg cells preferentially express CCR4 compared with conventional T cells. CCR4 is a receptor for two chemokines, CCL17 and CCL22, both of which are produced by tumor cells, tumor-associated macrophages, and dendritic cells. The interaction between CCR4 and chemokines leads to the recruitment of Treg cells into the tumor microenvironment, which in turn promotes local tumor growth and enhances tumor metastasis in peripheral blood or lymphoid organs, which is associated with poor prognosis. Blocking CCR4 can reduce the number of intertumor Treg cells and improve antitumor immunity. Targeting CCR4 is expected to become a new strategy for immunotherapy of malignant tumors. Given that most Treg-targeting agents cannot distinguish between tumor-associated Treg cells and Treg cells in peripheral blood, targeting tumor-infiltrating Treg cells while retaining systemic Treg cells is the main obstacle to effective tumor immunotherapy. Finding new and promising tools to target Treg cells can be a major research area for future immunosuppressive therapy. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an anti-human HER2 / CCR4 bispecific antibody and its application.

[0005] The technical solution adopted by the present invention is: an anti-human HER2 / CCR4 bispecific antibody, comprising a first antigen binding fragment binding to HER2, a second antigen binding fragment binding to CCR4 and an Fc domain.

[0006] Preferably, the first antigen-binding region comprises the Fab segment of an anti-Trastuzumab monoclonal antibody, the second antigen-binding region comprises the Fab segment of an anti-Moglizumab monoclonal antibody, and the Fc domain is the Fc domain of a Trastuzumab monoclonal antibody.

[0007] Preferably, the heavy chain variable region of the first antigen-binding region is connected to the heavy chain variable region of the second antigen-binding region via a connecting peptide, and the light chain variable region of the first antigen-binding region is connected to the light chain variable region of the second antigen-binding region via a connecting peptide.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO.1, the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region of the second antigen-binding region is as shown in SEQ ID NO.6; the amino acid sequence of the connecting peptide is as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO.8; and the amino acid sequence of the Fc domain is as shown in SEQ ID NO.9.

[0009] Preferably, the C-terminus of the Fab region of the second antigen-binding fragment is connected to the N-terminus of the Fc region of the first antigen-binding fragment, and the C-terminus of the Fab region of the first antigen-binding fragment of the antibody is connected to the N-terminus of the Fab region of the second antigen-binding fragment of the bispecific antibody.

[0010] An engineered bacterium capable of expressing a bispecific antibody against human HER2 / CCR4 is constructed by constructing a plasmid capable of expressing a first antigen binding fragment heavy chain variable region-connecting peptide-second antigen binding fragment heavy chain variable region, and a plasmid capable of expressing a first antigen binding fragment light chain variable region-connecting peptide-second antigen binding light chain variable region, which are transfected into an expression vector strain to obtain an engineered bacterium expressing a bispecific antibody.

[0011] Preferably, the plasmid expressing the first antigen-binding fragment heavy chain variable region-connecting peptide-second antigen-binding fragment heavy chain variable region is H1-pAb20-hCHIgG1 plasmid or H2-pAb20-hCHIgG1 plasmid, and the sequences are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively;

[0012] The plasmid expressing the first antigen-binding fragment light chain variable region-connecting peptide-second antigen-binding light chain variable region is L1-pAb20-hCK plasmid or L2-pAb20-hCK plasmid, and the sequences are shown in SEQ ID NO.21 and SEQ ID NO.22 respectively.

[0013] Preferably, the plasmid expressing the heavy chain variable region is mixed with the plasmid expressing the light chain variable region at a ratio of 1:3 and transfected into 293F cells.

[0014] The method for preparing a bispecific antibody against human HER2 / CCR4 comprises culturing the engineered bacteria, placing the bacteria in a CO2 shaking incubator at 170 rpm and 37°C, introducing 5% CO2 during the culturing process, collecting the protein expressed by the engineered bacteria, and separating and purifying the bispecific antibody against human HER2 / CCR4.

[0015] Use of anti-human HER2 / CCR4 bispecific antibodies in the preparation of drugs.

[0016] Preferably, it is used for preparing anti-tumor drugs; or for preparing drugs for treating or preventing immune diseases, wherein the immune disease is graft-versus-host disease.

[0017] The advantages and positive effects of the present invention are: providing an anti-human HER2 / CCR4 bispecific antibody that can bind to HER2 with high specificity and high affinity, and bind to CCR4 with high specificity and relatively low affinity, promote effector cells to kill tumor cells and Treg cells in the tumor microenvironment, effectively avoid the depletion of peripheral blood Treg cells caused by CCR4 monoclonal antibodies, and have good anti-cancer activity and higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthesis of HER2 / CCR4 bispecific antibodies;

[0019] Figure 2 SDS-PAGE electrophoresis of HER2×CCR4 bispecific antibody;

[0020] Figure 3 ELISA measured the affinity of the anti-HER2×CCR4 bispecific antibody to the HER2 protein molecule;

[0021] Figure 4 ELISA measured the affinity of anti-HER2×CCR4 bispecific antibodies to CCR4 protein molecules;

[0022] Figure 5 Flow cytometry was used to measure the binding of the anti-HER2×CCR4 bispecific antibody to the SKBR3 cell line that highly expresses HER2;

[0023] Figure 6 Flow cytometry was used to measure the binding of the anti-HER2×CCR4 bispecific antibody to the NCI-N87 cell line that highly expresses HER2;

[0024] Figure 7 Flow cytometry was used to measure the binding of anti-HER2×CCR4 bispecific antibody to Treg cells expressing CCR4;

[0025] Figure 8 The killing effect of the anti-HER2×CCR4 bispecific antibody on SKBR3 cells that highly express HER2 in vitro was measured by a standard lactate dehydrogenase (LDH) assay;

[0026] Fig. 9 The killing effect of the anti-HER2×CCR4 bispecific antibody on NCI-N87 cells that highly express HER2 in vitro was measured by a standard lactate dehydrogenase (LDH) assay;

[0027] Fig.10 The killing effect of the anti-HER2×CCR4 bispecific antibody on Treg cells expressing CCR4 in vitro was measured by a standard lactate dehydrogenase (LDH) assay;

[0028] Fig.11 The anti-HER2×CCR4 bispecific antibody was tested to inhibit the chemotaxis of Treg cells in vitro. DETAILED DESCRIPTION

[0029] The embodiments of the present invention are described below with reference to the accompanying drawings.

[0030] The present invention relates to an anti-human HER2 / CCR4 bispecific antibody and its application, and provides a bispecific antibody capable of simultaneously targeting HER2 target and CCR4 target, wherein the bispecific antibody comprises a first antigen binding region, a second antigen binding region and an Fc domain, wherein the first antigen binding region has HER2 binding activity, the second antigen binding region has CCR4 binding activity, and the Fc domain is the Fc domain of the Trastuzumab monoclonal antibody. The bispecific antibody can bind to HER2 with high specificity and high affinity, and bind to CCR4 with high specificity and relatively low affinity.

[0031] The first antigen-binding region includes the Fab segment of the Trastuzumab monoclonal antibody, specifically including the heavy chain variable region and the light chain variable region, the heavy chain variable region sequence is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.5; the second antigen-binding region includes the Fab segment of the Moglizumab monoclonal antibody, specifically including the heavy chain variable region and the light chain variable region, the heavy chain variable region sequence is shown in SEQ ID NO.2, and the light chain variable region sequence is shown in SEQ ID NO.6; the heavy chain variable region in the first antigen-binding fragment derived from the Trastuzumab monoclonal antibody is connected to the heavy chain variable region in the second antigen-binding fragment derived from the Moglizumab monoclonal antibody in the second antigen-binding fragment through connecting peptides H1 and H2, respectively, wherein the amino acid sequence of the connecting peptide H1 is shown in SEQ ID NO.3, and the amino acid sequence of the connecting peptide H2 is shown in SEQ ID NO. NO.4; the light chain variable region in the first antigen-binding fragment derived from the trastuzumab monoclonal antibody is connected to the light chain variable region in the second antigen-binding fragment derived from the moglizumab monoclonal antibody in the second antigen-binding fragment through connecting peptides L1 and L2, respectively, wherein the amino acid sequence of the connecting peptide L1 is shown in SEQ ID NO.7, and the amino acid sequence of the connecting peptide L2 is shown in SEQ ID NO.8. The amino acid sequence of the Fc domain is shown in SEQ ID NO.9.

[0032] SEQ ID NO.1

[0033] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIY

[0034] PTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGF

[0035] YAMDYWGQGTLVTVSS

[0036] SEQ ID NO.2

[0037] EVQLVESGGDLVQPGRSLRLSCAASGFIFSNYGMSWVRQAPGKGLEWVATISS

[0038] ASTYSYYPDSVKGRFTISRDNAKNSLYLQMNSLRVEDTALYYCGRHSDGNFA

[0039] FGYWGQGTLVTVSS

[0040] SEQ ID NO.3ASTKGPSVFPLAP

[0041] SEQ ID NO.4ASTKGP

[0042] SEQ ID NO.5

[0043] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASF

[0044] LYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID NO.6

[0045] DVLMTQSPLSLPVTPGEPASISCRSSRNIVHINGDTYLEWYLQKPGQSPQLLIY

[0046] KVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSLLPWTFGQGT

[0047] KVEIK

[0048] SEQ ID NO.7TVAAPSVFIFPP

[0049] SEQ ID NO.8TVAAP

[0050] SEQ ID NO.9

[0051] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF

[0052] PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT

[0053] HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

[0054] WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK

[0055] ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE

[0056] WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0057] HNHYTQKSLSLSPGK

[0058] The C-terminus of the Fab region of the second antigen-binding fragment in the bispecific antibody is connected to the N-terminus of the Fc region of the bispecific antibody via a connecting peptide, and the C-terminus of the Fab region of the first antigen-binding fragment of the bispecific antibody is connected to the N-terminus of the Fab region of the second antigen-binding fragment of the bispecific antibody via a connecting peptide. In certain embodiments of the present invention, the first antigen is HER2 and the second antigen is CCR4.

[0059] To prepare a bispecific antibody, first construct a plasmid capable of expressing the first antigen binding fragment heavy chain variable region-connector peptide-second antigen binding fragment heavy chain variable region, construct a plasmid capable of expressing the first antigen binding fragment light chain variable region-connector peptide-second antigen binding light chain variable region, transfect into the expression vector strain at a mixing ratio of 1:3, and culture the engineered bacteria under conditions suitable for protein expression and secretion. In certain embodiments of the present invention, the constructed engineered bacteria are placed in a CO2 shaking incubator at 170rmp / min and 37°C, and 5% CO2 is introduced during the culture process; the protein expressed by the cells is separated, and the bispecific antibody expressed is purified. The obtained bispecific antibody can bind to HER2 with high specificity and high affinity, bind to CCR4 with high specificity and relatively low affinity, promote effector cells to kill tumor cells and Treg cells in the tumor microenvironment, effectively reduce the exhaustion of peripheral blood Treg cells caused by CCR4 monoclonal antibodies, and has good anti-cancer activity and higher safety.

[0060] HER2 targets are specifically expressed on tumor cells, CCR4 is expressed in Treg cells in the tumor microenvironment, and some peripheral blood Treg cells also have corresponding expressions; CCR4 is a chemokine receptor, and binding with the corresponding chemokine can guide peripheral blood CCR4+Treg to be recruited into the tumor microenvironment. Depletion of CCR4+Treg in the tumor microenvironment can enhance anti-tumor immunity, while depletion of peripheral blood CCR4+Treg may enhance anti-tumor immunity, but it may also increase autoimmune reactions and affect the functions of other immune cells. In clinical applications, it is necessary to carefully weigh these potential pros and cons and find more specific targeting strategies to reduce side effects. With HER2 and CCR4 as targets, bispecific antibodies mainly deplete CCR4+Treg in the tumor microenvironment, exert anti-tumor effects, and bind to some CCR4+Treg in the peripheral blood that have the ability to chemotaxis to the tumor microenvironment. At the same time, the relatively low affinity binding to CCR4 avoids the side effects caused by excessive depletion of peripheral blood CCR4+Treg.

[0061] The structural design of the bispecific antibody is that the part targeting HER2 is on the outside and the part targeting CCR4 is on the inside. Such a structural design leads to a relatively reduced affinity for CCR4. However, the linker connecting the inner and outer parts can be long or short. Different length combinations will cause different antigen-antibody binding areas exposed at the CCR4 Fab end, resulting in different affinities, and ultimately leading to differences in anti-tumor effects. In certain embodiments of the present invention, four bispecific antibodies were made and verified by experiments. Although the overall affinity of the anti-CCR4 end was lower than that of the anti-HER2 end, the CCR4 end of XL-11 had the strongest affinity among the four bispecific antibodies, and had a stronger ability to kill CCR4+Treg in the tumor microenvironment, while the affinity of the four bispecific antibodies to peripheral blood Treg was not significantly different.

[0062] The bispecific antibody can be used in a drug for treating or preventing anti-tumor diseases, or in a drug for preventing or treating HER2 and CCR4-mediated related diseases. The bispecific antibody can be used to prepare a drug for treating or preventing immune diseases, wherein the immune disease can be graft-versus-host disease. The bispecific antibody can be used as the main active ingredient or one of the main active ingredients, and mixed with other excipients to prepare a medicament or drug.

[0063] The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.

[0064] In the following embodiments, all buffers, culture media, reagents and consumables involved are as follows: PBS buffer (pH=7.4), PBST buffer (PBS buffer + 0.05% Twen-20), carbonate coating solution (pH=9.6 Na2CO3 1.59 g + NaHCO3 2.93 g + distilled water 1000 mL), 293F complete medium (purchased from Sino Biological 293F serum-free medium), 1640 complete medium (10% serum + 1% double antibody + 1640 basal medium), DMEM complete medium (10% serum + 1% double antibody + DMEM basal medium), PEI transfection reagent (purchased from Quanshijin), protein expression-promoting feed (purchased from Sino Biological) and Protein A protein purification column (purchased from Tiandirenhe).

[0065] Example 1: Construction, expression and purification of anti-HER2×CCR4 bispecific antibody molecules

[0066] The anti-HER2×CCR4 bispecific antibody was constructed by using the Fab region of the Trastuzumab monoclonal antibody as the anti-HER2 domain of the bispecific antibody, the Fab region of the Moglizumab monoclonal antibody as the anti-CCR4 binding domain of the bispecific antibody, and the Fc domain of the Trastuzumab monoclonal antibody as the Fc domain of the bispecific antibody.

[0067] The first antigen-binding portion of the bispecific antibody specifically binds to human HER2 and is composed of the Fab region of the Trastuzumab monoclonal antibody, which is contained in the heavy chain amino acid sequence shown in SEQ ID NO: 1 and the light chain amino acid sequence shown in SEQ ID NO: 5; the second antigen-binding portion of the bispecific antibody specifically binds to human CCR4 through the Fab configuration, which is contained in the heavy chain amino acid sequence shown in SEQ ID NO: 2 and the light chain amino acid sequence shown in SEQ ID NO: 6. The Fc domain is derived from the Trastuzumab monoclonal antibody and is contained in the amino acid sequence shown in SEQ ID NO: 9.

[0068] The heavy chain variable region in the Fab arm of the first antigen-binding fragment is connected to the heavy chain variable region in the Fab arm of the second antigen-binding fragment through a connecting peptide H1 or H2; the light chain variable region in the Fab arm of the first antigen-binding fragment is connected to the light chain variable region in the Fab arm of the second antigen-binding fragment through a connecting peptide L1 or L2. The amino acid sequence of the connecting peptide H1 is shown in SEQ ID NO.3, and the amino acid sequence of the connecting peptide H2 is shown in SEQ ID NO.4; the amino acid sequence of the connecting peptide L1 is shown in SEQ ID NO.7, and the amino acid sequence of the connecting peptide L2 is shown in SEQ ID NO.8.

[0069] The heavy chain amino acid sequence of the first antigen-binding fragment of the bispecific antibody that binds to HER2 is shown in SEQ ID NO.1, and the corresponding base sequence is shown in SEQ ID NO.10;

[0070] SEQ ID NO.10

[0071] GAAGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGATTTAACATCAAGGACACATACATCCACTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAAGAATCTACCCCACAAACGGCTATACCAGGTAC GCAGACAGCGTGAAAGGAAGATTCACCATCAGCGCAGACACAAGCAAGAACACAGCATACCTGCGATGAACTCTCTGAGAGCAGAGGACACAGCCGTGTATTATTGTAGCAGGTGGGGAGGAGACGGATTTTACGCCATGGACTATTGGGGACAGGGAACACTGGTGACAGTGTCTAGC

[0072] The heavy chain amino acid sequence of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.2, and the corresponding base sequence is shown in SEQ ID NO.11;

[0073] SEQ ID NO.11

[0074] GAAGTGCAGCTGGTGGAATCTGGAGGAGATCTGGTGCAGCCAGGAAGATCTCTGAGACTGTCTTGCGCAGCAAGCGGATTCATCTTCAGCAACTACGGCATGTCTTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAACAATTAGCAGCGCAAGCACATACAGCTACT ACCCAGATAGCGTGAAGGGAAGATTCACCATCAGCAGGGACAACGCAAAGAACAGCCTGTACCTGCGATGAACTCTCTGAGAGTGGAGGACACAGCCCTGTATTATTGCGGAAGACACAGCGACGGAAACTTCGCATTTGGATATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT

[0075] The amino acid sequence of the peptide chain H1 connecting the heavy chain of the first antigen-binding fragment of the bispecific antibody that binds to HER2 and the heavy chain of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.3, and the corresponding base sequence is shown in SEQ ID NO.12;

[0076] SEQ ID NO.12GCTAGCACCAAGGGACCTTCTGTGTTCCTCTGGCCCCT

[0077] The amino acid sequence of the peptide chain H2 connecting the heavy chain of the first antigen-binding fragment of the bispecific antibody that binds to HER2 and the heavy chain of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.4, and the corresponding base sequence is shown in SEQ ID NO.13;

[0078] SEQ ID NO.13GCTAGCACCAAGGGACCT

[0079] The amino acid sequence of the light chain of the first antigen-binding fragment of the bispecific antibody that binds to HER2 is shown in SEQ ID NO.5, and the corresponding base sequence is shown in SEQ ID NO.14;

[0080] SEQ ID NO.14

[0081] GACATCCAGATGACACAGAGCCCATCTTCTCTGAGCGCAAGCGTGGGAGATAGAGTGACAATCACCTGTAGAGCCTCTCAGGACGTGAATACAGCAGTGGCTTGGTATCAGCAGAAGCCAGGAAAAGCACCAAAGCTGCTGATCTACAGCGCAAGCTTTC TGTATAGCGGAGTGCCAAGCAGATTTAGCGGATCTAGAAGCGGAACCGACTTCACACTGACAATCAGCTCTCTGCAGCCAGAAGACTTCGCAACCTACTATTGCCAGCAGCACTACACAACCCCACCAACATTTGGACAGGGAACCAAGGTGGAAATCAAA

[0082] The amino acid sequence of the light chain of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.6, and the corresponding base sequence is shown in SEQ ID NO.15;

[0083] SEQ ID NO.15

[0084] GACGTGCTGATGACACAGTCTCCACTGTCTCTGCCAGTGACACCAGGAGAACCAGCATCTATCTCTTGCAGAAGCAGCAGAAACATCGTGCACATCAACGGAGACACCTACCTCGAGTGGTATCTGCAGAAACCAGGACAGTCTCCACAGCTGCTGATCTACAAGGTG AGCAACAGGTTCAGCGGAGTGCCAGATAGATTTAGCGGAAGCGGAAGCGGAACAGACTTTACCCTGAAGATCAGCAGAGTGGAAGCAGAAGACGTGGGCGTGTATTATTGCTTCAGGGATCTCTGCTGCCTTGGACATTTGGACAGGGAACCAAGGTGGAGATCAAG

[0085] The amino acid sequence of the peptide chain L1 connecting the light chain of the first antigen-binding fragment of the bispecific antibody that binds to HER2 and the light chain of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.7, and the corresponding base sequence is shown in SEQ ID NO.16;

[0086] SEQ ID NO.16ACCGTGGCTGCCCCTAGCGTGTTCATCTTCCCTCCT

[0087] The amino acid sequence of the peptide chain L2 connecting the light chain of the first antigen-binding fragment of the bispecific antibody that binds to HER2 and the light chain of the second antigen-binding fragment of the bispecific antibody that binds to CCR4 is shown in SEQ ID NO.8, and the corresponding base sequence is shown in SEQ ID NO.17;

[0088] SEQ ID NO.17ACCGTGGCTGCCCCT

[0089] The amino acid sequence of the Fc domain of the bispecific antibody is shown in SEQ ID NO.9, and the corresponding base sequence is shown in SEQ ID NO.18;

[0090] SEQ ID NO.18

[0091] GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC

[0092] ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC

[0093] CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGC

[0094] ACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCG

[0095] TGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACG

[0096] TGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAA

[0097] ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCT

[0098] GGGGGGACCGTCAGTCTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT

[0099] GATCTCCCGGACCCCCGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG

[0100] AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT

[0101] AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT

[0102] GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT

[0103] ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC

[0104] ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC

[0105] CCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGG

[0106] TCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG

[0107] CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG

[0108] CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC

[0109] AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT

[0110] ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0111] 1.1 Anti-HER2 × CCR4 Bispecific Antibody Construction Strategy

[0112] The bispecific antibody XL-11, bispecific antibody XL-21, bispecific antibody XL-12 and bispecific antibody XL-22 were constructed respectively. The variable region of the bispecific antibody XL-11, from N-terminus to C-terminus, is the heavy chain variable region in the first antigen-binding fragment-connecting peptide H1-the heavy chain variable region in the second antigen-binding fragment and the light chain variable region in the first antigen-binding fragment-connecting peptide L1-the light chain variable region in the second antigen-binding fragment. The variable region of the bispecific antibody XL-21, from N-terminus to C-terminus, is the heavy chain variable region in the first antigen-binding fragment-connecting peptide H1-the heavy chain variable region in the second antigen-binding fragment and the light chain variable region in the first antigen-binding fragment-connecting peptide L2-the light chain variable region in the second antigen-binding fragment. The variable region of the bispecific antibody XL-12, from N-terminus to C-terminus, is the heavy chain variable region in the first antigen-binding fragment-connecting peptide H2-the heavy chain variable region in the second antigen-binding fragment and the light chain variable region in the first antigen-binding fragment-connecting peptide L1-the light chain variable region in the second antigen-binding fragment. The variable region of the bispecific antibody XL-22 is, from N-terminus to C-terminus, the heavy chain variable region in the first antigen binding fragment-connector peptide H2-heavy chain variable region in the second antigen binding fragment and the light chain variable region in the first antigen binding fragment-connector peptide L2-light chain variable region in the second antigen binding fragment. The specific connection mode of the connecting peptide is shown in Table 1. The molecular configuration of the anti-HER2×CCR4 bispecific antibody is as follows: Figure 1 shown.

[0113] Table 1

[0114] Bispecific antibody name Combination of connecting peptides XL-11 L1+H1 XL-21 L2+H1 XL-12 L1+H2 XL-22 L2+H2

[0115] 1.2 Construction of anti-HER2×CCR4 bispecific antibody plasmid

[0116] The plasmids for constructing anti-HER2×CCR4 bispecific antibodies include four plasmids in total. The plasmid names and corresponding vectors are shown in Table 2.

[0117] Table 2 Heavy chain and light chain plasmid names and corresponding vector names of anti-HER2×CCR4 bispecific antibodies

[0118]

[0119] The specifics of the antibody sequences inserted into the four plasmids for constructing the anti-HER2×CCR4 bispecific antibody are as follows:

[0120] H1-pAb20-hCHIgG1 plasmid: In the order of the heavy chain variable region of the Trastuzumab monoclonal antibody, the connecting peptide H1, and the heavy chain variable region of the Moglizumab monoclonal antibody, the above three fragments were cloned into the heavy chain vector pAb20-hCHIgG1 (Suzhou Hongxun) containing the heavy chain constant region of the antibody to obtain the H1-pAb20-hCHIgG1 plasmid expressing the heavy chain fragment of the anti-HER2×CCR4 bispecific antibody connected by the connecting peptide H1. The base sequence of the H1-pAb20-hCHIgG1 plasmid is shown in SEQ ID NO.19;

[0121] SEQ ID NO.19

[0122] GAAGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGATTTAACATCAAGGACACATACATCCACTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAAGAATCTACCCCACAAACGGCTATACCAGGTACGCAGACAGC GTGAAAGGAAGATTCACCATCAGCGCAGACACAAGCAAGAACACAGCATACCTGCGATGAACTCTCTGAGAGCAGAGGACACAGCCGTGTATTATTGTAGCAGGTGGGGAGGAGACGGATTTTACGCCATGGACTATTGGGGACAGGGAACACTGGTGACAGTGTCTAGCGCAAGCACAAAAGGACCA AGCGGTTTCCACTGGCACCAGAAGTGCAGCTGGTGGAATCTGGAGGAGATCTGGTGCAGCCAGGAAGATCTCTGAGACTGTCTTGCGCAGCAAGCGGATTCATCTTCAGCAACTACGGCATGTCTTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAACAATTAGCAGCGCAAGCACA TACAGCTACTACCCAGATAGCGTGAAGGGAAGATTCACCATCAGCAGGGACAACGCAAAGAACAGCCTGTACCTGCAGATGAACTCTCTGAGAGTGGAGGACACAGCCCTGTATTATTGCGGAAGACACAGCGACGGAAACTTCGCATTTGGATATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT

[0123] H2-pAb20-hCHIgG1 plasmid: In the order of the heavy chain variable region of the Trastuzumab monoclonal antibody, the connecting peptide H2, and the heavy chain variable region of the Moglizumab monoclonal antibody, the above three fragments were cloned into the heavy chain vector pAb20-hCHIgG1 of Suzhou Hongxun containing the heavy chain constant region of the antibody to obtain the H2-pAb20-hCHIgG1 plasmid expressing the heavy chain fragment of the anti-HER2×CCR4 bispecific antibody connected by the connecting peptide H2. The base sequence of the H2-pAb20-hCHIgG1 plasmid is shown in SEQ ID NO.20;

[0124] SEQ ID NO.20

[0125] GAAGTGCAGCTGGTGGAAAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGATTTAACATCAAGGACACATACATCCACTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAAGAATCTACCCCACAAACGGCTATACCAGGTACGCAGACAGCGTGAAAGGAAGATTCACCATCAGCGCAGACACAAGCAAGAACACAGCATACCTGCAGATGAACTCTCTGAGAGCAGAGGACACAGCCGTGTATTATTGTAGCAGGTGGGGAGGAGACGGATTTTACGCCATGGACTATTGGGGACAGGGAACACTGGTGACAGTGTCTAGCGCTAGCACCAAGGGACCTGAAGTGCAGCTGGTGGAATCTGGAGGAGATCTGGTGCAGCCAGGAAGATCTCTGAGACTGTCTTGCGCAGCAAGCGGATTCATCTTCAGCAACTACGGCATGTCTTGGGTGAGACAGGCACCAGGAAAAGGACTCGAGTGGGTGGCAACAATTAGCAGCGCAAGCACATACAGCTACTACCCAGATAGCGTGAAGGGAAGATTCACCATCAGCAGGGACAACGCAAAGAACAGCCTGTACCTGCAGATGAACTCTCTGAGAGTGGAGGACACAGCCCTGTATTATTGCGGAAGACACAGCGACGGAAACTTCGCATTTGGATATTGGGGCCAGGGAACACTGGTGACAGTGTCTTCT

[0126] L1-pAb20-hCK plasmid: In the order of the light chain variable region of the Trastuzumab monoclonal antibody, the connecting peptide L1, and the light chain variable region of the Moglizumab monoclonal antibody, the above three fragments were cloned into the light chain vector pAb20-hCK of Suzhou Hongxun containing the antibody light chain constant region to obtain the L1-pAb20-hCK plasmid expressing the light chain fragment of the anti-HER2×CCR4 bispecific antibody connected by the connecting peptide L1. The base sequence of the L1-pAb20-hCK plasmid is shown in SEQ ID NO.21;

[0127] SEQ ID NO.21

[0128] GACATCCAGATGACACAGAGCCCATCTTCTCTGAGCGCAAGCGTGGGAGATAGAGTGACAATCACCTGTAGAGCCTCTCAGGACGTGAATACAGCAGTGGCTTGGTATCAGCAGAAGCCAGGAAAAGCACCAAAGCTGCTGATCTACAGCGCAAGCTTTCTGTATAGCGGAGT GCCAAGCAGATTTAGCGGATCTAGAAGCGGAACCGACTTCACACTGACAATCAGCTCTCTGCAGCCAGAAGACTTCGCAACCTACTATTGCCAGCAGCACTACACAACCCCACCAACATTTGGACAGGGAACCAAGGGTGGAAATCAAAACAGTGGCCGCACCAAGCGTGTTTA TCTTTCCACCAGACGTGCTGATGACACAGTCTCCACTGTCTCTGCCAGTGACACCAGGAGAACCAGCATCTATCTCTTGCAGAAGCAGCAGAAACATCGTGCACATCAACGGAGACACCTACCTCGAGTGGTATCTGCAGAAACCAGGACAGTCTCCACAGCTGCTGATCTAC AAGGTGAGCAACAGGTTCAGCGGAGTGCCAGATAGATTTAGCGGAAGCGGAAGCGGAACAGACTTTACCCTGAAGATCAGCAGAGTGGAAGACGTGGGCGTGTATTATTGCTTCCAGGGATCTCTGCTGCCTTGGACATTTGGACAGGGAACCAAGGTGGAGATCAAG

[0129] L2-pAb20-hCK plasmid: In the order of the light chain variable region of the Trastuzumab monoclonal antibody, the connecting peptide L2, and the light chain variable region of the Moglizumab monoclonal antibody, the above three fragments were cloned into the light chain vector pAb20-hCK of Suzhou Hongxun containing the antibody light chain constant region to obtain the L2-pAb20-hCK plasmid expressing the light chain fragment of the anti-HER2×CCR4 bispecific antibody connected by the connecting peptide L2. The base sequence of the L2-pAb20-hCK plasmid is shown in SEQ ID NO.22;

[0130] SEQ ID NO.22

[0131] GACATCCAGATGACACAGAGCCCATCTTCTCTGAGCGCAAGCGTGGGAGATAGAGTGACAATCACCTGTAGAGCCTCTCAGGACGTGAATACAGCAGTGGCTTGGTATCAGCAGAAGCCAGGAAAAGCACCAAAGCTGCTGATCTACAGCGCAAGCTTTCTGTATAGC GGAGTGCCAAGCAGATTTAGCGGATCTAGAAGCGGAACCGACTTCACACTGACAATCAGCTCTCTGCAGCCAGAAGACTTCGCAACCTACTATTGCCAGCAGCACTACACAACCCCACCAACATTTGGACAGGGAACCAAGGTGGAAATCAAAACCGTGGCTGCCCCT GACGTGCTGATGACACAGTCTCCACTGTCTCTGCCAGTGACACCAGGAGAACCAGCATCTATCTCTTGCAGAAGCAGCAGAAACATCGTGCACATCAACGGAGACACCTACCTCGAGTGGTATCTGCAGAAACCAGGACAGTCTCCACAGCTGCTGATCTACAAGGTG AGCAACAGGTTCAGCGGAGTGCCAGATAGATTTAGCGGAAGCGGAAGCGGAACAGACTTTACCCTGAAGATCAGCAGAGTGGAAGCAGAAGACGTGGGCGTGTATTATTGCTTCAGGGATCTCTGCTGCCTTGGACATTTGGACAGGGAACCAAGGTGGAGATCAAG

[0132] 1.3 Construction of anti-HER2×CCR4 bispecific antibody expression vector

[0133] The four plasmids of the anti-HER2×CCR4 bispecific antibody were transfected into 293F cells using PEI transfection reagent by liposome transfection method at a ratio of heavy chain: light chain = 1:3. The combination of the bispecific antibody transfection plasmids is shown in Table 3.

[0134] Table 3 Combinations of anti-HER2×CCR4 bispecific antibody transfection plasmids

[0135] Bispecific antibody name Heavy chain plasmid name Light chain plasmid name Heavy chain: light chain XL-11 H1-pAb20-hCHIgG1 L1-pAb20-hCK 1:3 XL-21 H1-pAb20-hCHIgG1 L2-pAb20-hCK 1:3 XL-12 H2-pAb20-hCHIgG1 L1-pAb20-hCK 1:3 XL-22 H2-pAb20-hCHIgG1 L2-pAb20-hCK 1:3

[0136] 293F cell culture conditions and transfection expression steps are as follows:

[0137] According to 0.5×10 6 Inoculate cells in 200 ml of medium in a 1L shake flask at an inoculation rate of 10 cells / ml. Incubate in a shaker incubator at 37°C, 160 rpm, and 5% carbon dioxide concentration to ensure that the cells can double in number every 24 hours and do not clump. Subculture 293F cells to a density of 1×10 cells / ml before transfection. 6 cells / ml, and cultured for 4 hours in a shaking incubator at 37°C, 160rpm, and 5% carbon dioxide concentration. Pipette 200ug of plasmid (filtered and sterilized) and add it to 10ml of PBS, vortex mix for 3 seconds, and mix thoroughly. Add 600ul of filter-sterilized PEI solution to the PBS / DNA mixture. After the PEI-DNA mixture is allowed to stand at room temperature for 20 minutes, the DNA / PEI mixture is added to the cells. Culture for 5 days at 37°C, 5% CO2, and 80% humidity. Add expression-promoting supplements at 24h after transfection, and then add the above supplements every 24h until the sample is collected. Obtain the supernatant on the 5th day after transfection, centrifuge the supernatant at 4°C, 2000rpm, and filtrate the supernatant with a 0.22μm syringe filter.

[0138] Purify the bispecific antibody in the supernatant using a Protein A gravity column: fix the Protein A gravity column on an iron stand, remove the lower and upper plugs in turn, and drain the protective liquid of the gravity column; add 5 column volumes of balancing solution (0.5M NaCl, 20mM Na2HPO4, pH=7.0) 2-3 times to keep the filler in the same buffer system as the target protein to protect the protein; add the sample to the balanced gravity column, retain each 1ml sample for 2 minutes to ensure that the target protein is fully in contact with the medium and improve the recovery rate of the target protein, and collect the effluent; add 10-15 column volumes of washing solution (0.5M NaCl, 20mMNa2HPO4, pH=7.0), remove non-specifically adsorbed impurities, collect the washing solution; add elution solution (0.1M citric acid) with a pH of 4, and allow it to bind to the filler for 10 minutes; use 5-10 column volumes of elution solution with a pH of 2.5 to elute the target protein (0.1M citric acid), collect in sections, collect one tube for each column volume, and test them separately. The eluted components must be immediately adjusted to neutrality and neutralized with a neutralizing solution of 1 / 10 of the volume of the elution components; balance the filler with 3 column volumes of balancing solution and 5 column volumes of deionized water in turn. Store the gravity column in an equal volume of 20% ethanol at 2-8°C to prevent bacterial contamination of the filler.

[0139] The size and structure of the anti-HER2×CCR4 bispecific antibody were detected by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Figure 2 As shown, the four HER2×CCR4 bispecific antibodies were well expressed in transiently transfected 293F cells and secreted into the culture supernatant. On non-reducing SDS-PAGE, the molecular weights of the four HER2×CCR4 bispecific antibodies were all ≥250 kDa. On reducing SDS-PAGE, the molecular weights of the heavy chains were all ≈64 kDa, and the molecular weights of the light chains were all ≈36 kDa. After purification, the purity was high, and no mixed bands were observed.

[0140] Example 2: Identification of the binding activity of anti-HER2×CCR4 bispecific antibody to HER2 protein

[0141] The pH value is 9.6 carbonate coating solution (0.035mol / L NaHCO3, 0.015mol / L Na2CO3) was used to dilute the HER2 protein to 10 μg / ml, and then 100 μl / well was added to the ELISA plate; incubated at 4°C overnight; the next day, the plate was washed three times with PBST buffer (pH=7.4); 5% BSA buffer was added to each well and blocked at room temperature for 2 hours; the plate was washed three times with PBST buffer (pH=7.4); the antibody to be detected was diluted in a gradient with PBST buffer, the initial concentration of the antibody was 20000 ng / ml, and it was diluted 5 times step by step into 10 gradients, and incubated at room temperature for 1 hour; the plate was washed three times with PBST buffer (pH=7.4), and the secondary antibody HRP-labeled sheep anti-human Fc antibody (purchased from Solebo) was added, and incubated at room temperature for 1 hour; the plate was washed three times with PBST buffer (pH=7.4) and patted dry, 100 μl TMB solution (purchased from Sigma) was added to each well, and placed in the dark at 37°C for 20 minutes; 50 μl 2M The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450nm by a microplate reader. GraphPad Prism was used for data analysis, graphing and calculation of EC50.

[0142] The results are as follows Figure 3 As shown, the four anti-HER2×CCR4 bispecific antibodies were able to bind to HER2 antigen, with EC50 of 6.272 ng / ml, 10.58 ng / ml, 9.562 ng / ml and 6.149 ng / ml, respectively. Compared with Trastuzumab monoclonal antibody (EC50=3.996 ng / ml), the affinity of the four anti-HER2×CCR4 bispecific antibodies to HER2 protein was not significantly reduced, and still maintained a relatively high affinity.

[0143] Example 3: Identification of the binding activity of anti-HER2×CCR4 bispecific antibody to CCR4 protein

[0144] CCR4 protein was diluted to 10 μg / ml with carbonate coating solution (0.035 mol / L NaHCO3, 0.015 mol / L Na2CO3) at pH = 9.6, and then added to the ELISA plate at 100 μl / well; incubated at 4°C overnight; the next day, the plate was washed three times with PBST buffer (pH = 7.4); 5% BSA buffer was added to each well for blocking, and the blocking was carried out at room temperature for 2 hours; the plate was washed three times with PBST buffer (pH = 7.4); then the antibody to be detected was diluted in a gradient with PBST buffer, the initial concentration of the antibody was 20,000 ng / ml, and it was diluted 5 times step by step into 10 gradients, and incubated at room temperature for 1 hour; the plate was washed three times with PBST buffer (pH = 7.4), and the secondary antibody HRP-labeled sheep anti-human Fc antibody (purchased from Solebol) was added, and incubated at room temperature for 1 hour; the plate was washed three times with PBST buffer (pH = 7.4) and patted dry, and 100 μl was added to each well. TMB solution (purchased from Sigma) was placed at 37°C in the dark for 20 minutes; 50 μl of 2M H2SO4 stop solution was added to each well to terminate the substrate reaction, and the OD value was read at 450 nm by an enzyme reader. GraphPad Prism was used for data analysis, graphing and calculation of EC50.

[0145] The results are as follows Figure 4 As shown, the four anti-HER2×CCR4 bispecific antibodies can bind to the CCR4 antigen, and the EC50s are 21.52ng / ml, 102ng / ml, 33.34ng / ml and 1438ng / ml, respectively. The four anti-HER2×CCR4 bispecific antibodies still bind to the CCR4 protein with strong affinity, but compared with the Moglizumab monoclonal antibody (EC50=9.593ng / ml), the affinity of the four anti-HER2×CCR4 bispecific antibodies to CCR4 is reduced. Among them, XL-11, which is connected by a combination of longer connecting peptides L1 and H1, has the strongest affinity to the CCR4 protein among the four anti-HER2×CCR4 bispecific antibodies. XL-22, which is connected by a combination of shorter connecting peptides L2 and H2, has the weakest affinity to the CCR4 protein among the four anti-HER2×CCR4 bispecific antibodies. Example 4: Identification of the binding of anti-HER2×CCR4 bispecific antibody to SKBR3 cell line with high expression of HER2

[0146] After the SKBR3 cells in the T25 flask were fully grown, they were digested with 0.5% trypsin and counted with a hemocytometer. The cells were evenly divided into 1.5EP tubes, with 0.2×10 6Cells, centrifuged at 800 rpm for 5 min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Dilute the antibody to be detected to 0.15 mg / ml with 1% FBS-PBS, add it to the cells in turn, 100 μl / tube, mix by pipetting, incubate at 37°C for 1 h, centrifuged at 800 rpm for 5 min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Dilute anti-human IgG Fc-FITC fluorescent secondary antibody (Solebo, 1:100) with 1% FBS-PBS, add it to the cells, 100 μl / tube, and mix by pipetting. After incubating at 4°C for 1 h in the dark, centrifuged at 800 rpm for 5 min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Add 300 μl 1% FBS-PBS, resuspend the cells by pipetting, filter with a 40 um cell strainer and then test on the instrument.

[0147] The results are as follows Figure 5 As shown, the anti-HER2×CCR4 bispecific antibodies were able to bind to SKBR3 cells that highly expressed HER2. Compared with the Trastuzumab monoclonal antibody, the binding ability of the four anti-HER2×CCR4 bispecific antibodies to SKBR3 cells that highly expressed HER2 was not significantly reduced and still maintained a strong binding ability.

[0148] Example 5: Identification of the binding of anti-HER2×CCR4 bispecific antibody to the NCI-N87 cell line that highly expresses HER2

[0149] After the NCI-N87 cells in the T25 flask were fully grown, they were digested with 0.5% trypsin and counted with a hemocytometer. The cells were evenly divided into 1.5EP tubes, with 0.2×10 6Cells, centrifuged at 800 rpm for 5 min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Dilute the antibody to be detected to 0.15 mg / ml with 1% FBS-PBS, add it to the cells in turn, 100 μl / tube, mix by pipetting, incubate at 37°C for 1 h, discarded the supernatant after centrifugation at 800 rpm for 5 min, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Dilute anti-human IgG Fc-FITC fluorescent secondary antibody (Solebo, 1:100) with 1% FBS-PBS, add it to the cells, 100 μl / tube, and mix by pipetting. After incubating at 4°C for 1 h in the dark, centrifuged at 800 rpm for 5 min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (800 rpm, 5 min). Add 300 μl 1% FBS-PBS, resuspend the cells by pipetting, filter with a 40 um cell strainer and then test on the instrument.

[0150] The results are as follows Figure 6 As shown, the anti-HER2×CCR4 bispecific antibodies were able to bind to NCI-N87 cells that highly expressed HER2. Compared with the Trastuzumab monoclonal antibody, the binding ability of the four anti-HER2×CCR4 bispecific antibodies to NCI-N87 cells that highly expressed HER2 was not significantly reduced and still maintained a strong binding ability.

[0151] Example 6: Identification of the binding of anti-HER2×CCR4 bispecific antibody to Treg cells expressing CCR4

[0152] Place the peripheral blood in a 50mL centrifuge tube and centrifuge at 650g for 15 minutes at room temperature. Take the upper yellow plasma part and put it in a new 50mL centrifuge tube (the lower layer is blood cells, which are used for the subsequent extraction of mononuclear cells). Inactivate the plasma in a 56℃ water bath for 30 minutes. After the plasma is inactivated, it can be seen that the plasma is turbid. Centrifuge at 900g for 10 minutes, take the supernatant, and store it at 4℃ for later use.

[0153] Take the lower red cell sediment from the plasma extraction step, add physiological saline or PBS to the original volume, and gently blow evenly; take a 50mL centrifuge tube, add 15mL lymphocyte separation solution, and slowly add the diluted blood in the above steps to the upper layer of the lymphocyte separation solution; the speed of adding blood samples is moderate, especially at the beginning, to avoid damaging the interface between the lymphocyte separation solution and the blood sample. Centrifuge at 800g for 20min at room temperature (no brake deceleration); after centrifugation, the sample in the centrifuge tube is divided into 4 layers from top to bottom: plasma layer - buffy coat layer - human lymphocyte separation solution layer - red blood cell and granulocyte sedimentation. Carefully aspirate the buffy coat layer and about half of the liquid below it into a new centrifuge tube, add physiological saline or PBS to a volume of 40mL, mix well, and centrifuge at 300g for 10min; discard the supernatant, resuspend the sediment with 40mL of physiological saline or PBS again, and centrifuge at 300g for 10min; if the sample has more platelets, the washing step can be repeated 1-2 times. Discard the supernatant, resuspend the pellet with a small amount of buffer, take part of the cell suspension for counting and set aside.

[0154] The PBMC suspension was centrifuged and the supernatant was removed. A certain amount of Treg serum-free medium was added to adjust the PBMC concentration to 1×10 8 cells / mL; enriched antibody incubation: enriched antibody was incubated at 200uL antibody / 1-1.5×10 8 The initial PBMC ratio was added to the cell suspension, and the mixture was quickly and gently pipetted. After thorough mixing, the mixture was incubated at room temperature for 15 min. After incubation, Treg serum-free medium (1×10 8 cells / 10-40mL), centrifuge at 300g for 10min, discard the supernatant, add a certain amount of Treg serum-free medium to restore the volume of the cell suspension to the initial level; when discarding the supernatant, there is often some buffer left at the mouth of the tube. Use a pipette to absorb the residual liquid as much as possible. If necessary, wash again to avoid interference of some antibody components in the residual liquid with the subsequent binding of magnetic beads and cells. Transfer the cell suspension to a flow tube with 100uL magnetic beads / 1×10 8 Add streptavidin magnetic beads to PBMC at the initial PBMC ratio, quickly and gently blow with a pipette to evenly mix, and incubate at room temperature for 10 minutes; after incubation, magnetically absorb for 3 minutes, and collect the cell suspension that is not magnetically absorbed; if the amount of cells is small during magnetic absorption, 1 mL of Treg serum-free medium can be added to resuspend the cells and then magnetically absorb again. Add the same volume of Treg serum-free medium as in the above step to the flow tube containing magnetically absorbed cells, gently blow off the magnetic beads and then magnetically absorb again for 3 minutes, collect the liquid that is not magnetically absorbed and merge it with the cell suspension collected in the above step; repeat the magnetic absorption of the combined cell suspension several times, collect the cell suspension that is not magnetically absorbed, and use it to more completely remove non-target cells. The resulting cells are the enriched seed cells.

[0155] On day 0, prepare 30 mL of complete medium for regulatory T cells: 26 mL of Treg serum-free medium, 10% heat-inactivated autologous plasma (3 mL), 1 mL of Treg-II, and IL-2 (the final concentration of IL-2 in the complete medium is 500 IU / mL), and evenly suspend the complete medium with the seed cells and inoculate them into a T75 bottle; on day 4-5, add 30 mL of complete medium for Treg cells: 27 mL of Treg serum-free medium, 10% heat-inactivated autologous plasma (3 mL) and 500 U / mL of IL-2; on day 6-7, add 60 mL of complete medium for Treg cells: 53 mL of Treg serum-free medium, 10% heat-inactivated autologous plasma (6 mL), 1mL of Treg-III and 500U / mLIL-2; on day 8-9, add 120mL of Treg cell complete medium: 108mL of Treg serum-free medium, 10% heat-inactivated autologous plasma (12mL) and 500U / mL IL-2; on day 10-11, add 240mL of Treg cell complete medium: 216mL of Treg serum-free medium, 10% heat-inactivated autologous plasma (24mL) and 500U / mL IL-2; on day 12-13, add the remaining Treg serum-free medium, the remaining heat-inactivated autologous plasma and 500U / mL IL-2; on day 14-15, harvest Treg cells for analysis or downstream applications.

[0156] The harvested Treg cells were evenly divided into 1.5EP tubes, with 0.2×10 per tube. 6 Cells, centrifuged at 300g for 10min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (300g, 10min). Dilute the antibody to be detected to 0.15mg / ml with 1% FBS-PBS, add it to the cells in turn, 100μl / tube, mix by pipetting, incubate at 37℃ for 1h, discard the supernatant after centrifugation at 300g for 10min, resuspended with 1% FBS-PBS, and washed twice by centrifugation (300g, 10min). Dilute anti-humanIgG Fc-FITC fluorescent secondary antibody (Solebo, 1:100) with 1% FBS-PBS, add it to the cells, 100μl / tube, and mix by pipetting. After incubating at 4℃ for 1h in the dark, centrifuged at 300g for 10min, discarded the supernatant, resuspended with 1% FBS-PBS, and washed twice by centrifugation (300g, 10min). Add 300 μl 1% FBS-PBS, resuspend the cells by pipetting, filter with a 40 um cell strainer and then test on the instrument.

[0157] The results are as follows Figure 7As shown in the figure, the anti-HER2×CCR4 bispecific antibodies can bind to Treg cells expressing CCR4. Compared with the Moglizumab monoclonal antibody, the binding ability of the four anti-HER2×CCR4 bispecific antibodies to Treg cells expressing CCR4 was reduced, which may avoid the depletion of peripheral blood Treg cells caused by excessive binding of anti-HER2×CCR4 bispecific antibodies to peripheral blood Treg cells, indicating that the four anti-HER2×CCR4 bispecific antibodies have potentially better safety, and there is no significant difference between the bispecific antibodies connected by different connecting peptide combinations.

[0158] Example 7: Standard lactate dehydrogenase (LDH) assay to detect the killing effect of anti-HER2×CCR4 bispecific antibody on SKBR3 cells that highly express HER2 in vitro

[0159] After the SKBR3 cells in the T25 flask are full, they are digested with 0.5% trypsin and counted with a hemocytometer. The cells are inoculated into a 96-well plate, 200ul / well, 10,000 cells / well, and the cell density is not more than 80-90% when tested. The growth medium is aspirated from the 96-well plate, the cells are washed once with PBS, and the medium is replaced with a low-serum medium containing 1% serum, and adaptive culture is performed for 1 hour; 1.28, 32, 160, 800, 40000ng / ml gradient dilutions of the antibodies to be tested are added and incubated at 37°C for 30 minutes; 250,000 PBMCs are added to each well as the experimental group and incubated at 37°C for 2 hours; the supernatant is aspirated, the supernatant is centrifuged at 400g for 10 minutes, and the supernatant is taken and added to the corresponding 96-well plate. 20μL of 10% Triton X-100 solution is added to the cells as the maximum release. Add 20 μL Assay Buffer to cells for spontaneous release, and add 20 μL Assay Buffer to wells without cells in 1% low serum medium as background control. Adjust the medium volume of the well plate cells to a final volume of 200 μL; incubate in a CO2 incubator at 37°C for 3 h. Centrifuge the 96-well plate at 400 g for 5 min. Transfer 100 μL of cell supernatant to a new 96-well assay plate.

[0160] Add 100 μL LDH Reaction Solution to each well. Incubate the plate at 37°C for up to 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate according to the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100.

[0161] The results are as follows Figure 8As shown, the four anti-HER2×CCR4 bispecific antibodies can kill SKBR3 cells that highly express HER2 in vitro, and the killing efficiency increases with the increase of antibody concentration. Compared with the Trastuzumab monoclonal antibody, the killing efficiency of the four anti-HER2×CCR4 bispecific antibodies on SKBR3 cells that highly express HER2 in vitro is not significantly reduced, and they still have a good ability to kill HER2-positive tumor cells. Example 8: Standard lactate dehydrogenase (LDH) experiment detects the killing effect of anti-HER2×CCR4 bispecific antibodies on NCI-N87 cells that highly express HER2 in vitro

[0162] After the NCI-N87 cells in the T25 bottle are full, they are digested with 0.5% trypsin and counted with a hemocytometer. The cells are inoculated into a 96-well plate, 200ul / well, 10,000 cells / well, and the cell density does not exceed 80-90% when tested. The growth medium is aspirated from the 96-well plate, the cells are washed once with PBS, the medium is replaced with a low-serum medium with 1% serum, and the adaptive culture is carried out for 1 hour; 1.28, 32, 160, 800, 40000ng / ml gradient dilutions of the antibodies to be tested are added and incubated at 37°C for 30 minutes; 250,000 PBMCs are added to each well as the experimental group and incubated at 37°C for 2 hours; the supernatant is aspirated, the supernatant is centrifuged at 400g for 10 minutes, the supernatant is taken, and added to the corresponding 96-well plate. 20Μ10% Triton X-100 solution is added to the cells as the maximum release. Add 20 μL Assay Buffer to cells for spontaneous release, and add 20 μL Assay Buffer to wells without cells in 1% low serum medium as background control. Adjust the medium volume of the well plate cells to a final volume of 200 μL; incubate in a CO2 incubator at 37°C for 3 h. Centrifuge the 96-well plate at 400 g for 5 min. Transfer 100 μL of cell supernatant to a new 96-well assay plate.

[0163] Add 100 μL LDH Reaction Solution to each well. Incubate the plate at 37°C for up to 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate according to the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100.

[0164] The results are as follows Fig. 9As shown in the figure, the four anti-HER2×CCR4 bispecific antibodies can kill NCI-N87 cells that highly express HER2 in vitro, and the killing efficiency increases with the increase of antibody concentration. Compared with the trastuzumab monoclonal antibody, the killing efficiency of the four anti-HER2×CCR4 bispecific antibodies on NCI-N87 cells that highly express HER2 in vitro is not significantly reduced, and they still have a good ability to kill HER2-positive tumor cells.

[0165] Example 9: Standard lactate dehydrogenase (LDH) assay to detect the killing effect of anti-HER2×CCR4 bispecific antibody on Treg cells expressing CCR4 in vitro

[0166] After the primary Treg cells induced in the above T75 flask are full, count them with a hemocytometer, and inoculate the cells into a 96-well plate, 200ul / well, 10,000 cells / well, and make the cell density not exceed 80-90% when tested. Aspirate the growth medium from the 96-well plate, wash the cells once with PBS, replace the medium with a low-serum medium containing 1% serum, and adaptively culture for 1 hour; add 1.28, 32, 160, 800, 40000ng / ml gradient dilutions of the antibodies to be tested and incubate at 37°C for 30 minutes; add 250,000 PBMCs to each well as the experimental group and incubate at 37°C for 2 hours; aspirate the supernatant, centrifuge the supernatant at 400g for 10 minutes, take the supernatant, and add it to the corresponding 96-well plate. Add 20μL of 10% Triton X-100 solution to the cells as the maximum release. Add 20 μL Assay Buffer to cells for spontaneous release, and add 20 μL Assay Buffer to wells without cells in 1% low serum medium as background control. Adjust the medium volume of the well plate cells to a final volume of 200 ul; incubate in a CO2 incubator at 37°C for 3 h. Centrifuge the 96-well plate at 400 g for 5 min. Transfer 100 μL of cell supernatant to a new 96-well assay plate.

[0167] Add 100 μL LDH Reaction Solution to each well. Incubate the plate at 37°C for up to 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate according to the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100.

[0168] The results are as follows Fig.10As shown in the figure, the four anti-HER2×CCR4 bispecific antibodies can kill Treg cells expressing CCR4 in vitro, and the killing efficiency increases with the increase of antibody concentration. Compared with the Moglizumab monoclonal antibody, the killing ability of the four anti-HER2×CCR4 bispecific antibodies on Treg cells expressing CCR4 is reduced. Among them, XL-11, which is connected by the longer connecting peptides L1 and H1, has the strongest killing ability on Treg cells expressing CCR4 among the four anti-HER2×CCR4 bispecific antibodies, and XL-22, which is connected by the shorter connecting peptides L2 and H2, has the weakest killing ability on Treg cells expressing CCR4 among the four anti-HER2×CCR4 bispecific antibodies.

[0169] Example 10: Chemotaxis assay to detect the inhibitory effect of anti-HER2×CCR4 bispecific antibody on Treg cell chemotaxis in vitro

[0170] Add 600ul of SKBR3 tumor cell culture supernatant and 100ng / ml of CCL22 to a 24-well plate; aspirate 150,000 Tregs into 200ul of Treg-specific culture medium, add 0.1mg / ml of bispecific antibody, mix well by pipetting, and drop into the chamber in a circle in mid-air; place the chamber in the well plate and check that there are no bubbles between the culture medium and the chamber; let it stand for 15min to avoid aggregation during cell sedimentation, then place it in an incubator and let it stand for 4h; use a hemocytometer to count the number of cells in the lower chamber.

[0171] like Fig.11 As shown in the figure, all four anti-HER2×CCR4 bispecific antibodies can inhibit the chemotaxis of Treg cells to CCL22. Compared with Moglizumab monoclonal antibody, the ability of the four anti-HER2×CCR4 bispecific antibodies to inhibit the chemotaxis of Treg cells to CCL22 is reduced. Among them, XL-11, which is connected by a combination of longer connecting peptides L1 and H1, has the strongest ability to inhibit the chemotaxis of Treg cells to CCL22 among the four anti-HER2×CCR4 bispecific antibodies, and XL-22, which is connected by a combination of shorter connecting peptides L2 and H2, has the weakest ability to inhibit the chemotaxis of Treg cells to CCL22 among the four anti-HER2×CCR4 bispecific antibodies.

[0172] It can be seen from the above experimental results that the four bispecific antibodies targeting HER2 and CCR4 provided by the present invention all have good antigen binding activity and are high-affinity antibodies, which can reduce the binding of antibodies to peripheral blood Treg cells to a certain extent, have good killing activity against tumor cells and Treg cells in the tumor microenvironment, and inhibit the chemotaxis of Treg cells to the tumor microenvironment with high expression of CCL22, have good anti-cancer activity and equally good safety. Among them, the anti-cancer activity of XL-11 connected in combination with longer connecting peptides L1 and H1 is the best.

[0173] The present invention provides an anti-HER2 and CCR4 bispecific antibody and a concept and method for the application thereof. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bispecific antibody against human HER2 / CCR4, characterized in that: It comprises a first antigen-binding fragment that binds to HER2, a second antigen-binding fragment that binds to CCR4, and an Fc domain.

2. The anti-human HER2 / CCR4 bispecific antibody according to claim 1, characterized in that: The first antigen binding region includes the Fab segment of the Trastuzumab monoclonal antibody, the second antigen binding region includes the Fab segment of the Moglizumab monoclonal antibody, and the Fc domain is the Fc domain of the Trastuzumab monoclonal antibody.

3. The anti-human HER2 / CCR4 bispecific antibody according to claim 2, characterized in that: The heavy chain variable region of the first antigen-binding region is connected to the heavy chain variable region of the second antigen-binding region through a connecting peptide, and the light chain variable region of the first antigen-binding region is connected to the light chain variable region of the second antigen-binding region through a connecting peptide.

4. The anti-human HER2 / CCR4 bispecific antibody according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region of the first antigen binding region is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain variable region of the second antigen binding region is shown as SEQ ID NO.2, the amino acid sequence of the light chain variable region of the first antigen binding region is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region of the second antigen binding region is shown as SEQ ID NO.6; the amino acid sequence of the connecting peptide is shown as SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO.8; and the amino acid sequence of the Fc domain is shown as SEQ ID NO.

9.

5. An engineered bacterium capable of expressing the anti-human HER2 / CCR4 bispecific antibody according to any one of claims 1 to 4, characterized in that: A plasmid capable of expressing the first antigen-binding fragment heavy chain variable region-connecting peptide-second antigen-binding fragment heavy chain variable region is constructed, and a plasmid capable of expressing the first antigen-binding fragment light chain variable region-connecting peptide-second antigen-binding light chain variable region is constructed, and the plasmids are transfected into expression vector strains to obtain engineered bacteria expressing bispecific antibodies.

6. The engineered bacteria according to claim 5, characterized in that: The plasmid expressing the first antigen-binding fragment heavy chain variable region-connecting peptide-second antigen-binding fragment heavy chain variable region is H1-pAb20-hCHIgG1 plasmid or H2-pAb20-hCHIgG1 plasmid, and the sequences are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively; the plasmid expressing the first antigen-binding fragment light chain variable region-connecting peptide-second antigen-binding light chain variable region is L1-pAb20-hCK plasmid or L2-pAb20-hCK plasmid, and the sequences are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively.

7. The engineered bacteria according to claim 6, characterized in that: The plasmid expressing the heavy chain variable region was mixed with the plasmid expressing the light chain variable region at a ratio of 1:3 and transfected into 293F cells.

8. A method for preparing a bispecific antibody against human HER2 / CCR4, characterized in that: The engineered bacteria according to any one of claims 5 to 7 are cultured in a CO2 shaking incubator at 170 rpm and 37°C, 5% CO2 is introduced during the culture process, the protein expressed by the engineered bacteria is collected, and the anti-human HER2 / CCR4 bispecific antibody is obtained by separation and purification.

9. Use of the anti-human HER2 / CCR4 bispecific antibody according to any one of claims 1 to 4 in the preparation of a medicament.

10. The use according to claim 9, characterized in that: Used for preparing anti-tumor drugs; or used for preparing drugs for treating or preventing immune diseases, wherein the immune disease is graft-versus-host disease.

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