Bispecific antibody against human her2 / CCR4 and use thereof

Abstract

Provided are a bispecific antibody against human HER2 / CCR4 and a use thereof. Provided is a bispecific antibody capable of simultaneously targeting an HER2 target and a CCR4 target. 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 an HER2-binding activity, the second antigen-binding region has a CCR4-binding activity, and the Fc domain is an Fc domain of a trastuzumab monoclonal antibody. The bispecific antibody can bind to HER2 with a high specificity and a high affinity, and bind to CCR4 with a high specificity and a relatively low affinity. The bispecific antibody promotes effector cells to kill tumor cells and Treg cells in the tumor microenvironment, and effectively avoids the depletion of Treg cells in the peripheral blood caused by CCR4 monoclonal antibodies, thereby having a good anti-cancer activity and a higher safety.

Description

A bispecific antibody against human HER2 / CCR4 and its application Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a bispecific antibody against human HER2 / CCR4 and its applications. Background Technology

[0002] HER2 is a 180 kDa transmembrane glycoprotein overexpressed on the surface of various tumor cells, including 20%-30% of breast cancer, approximately 20% of gastric cancer, and 12-15% of gallbladder cancer. HER2 gene amplification or HER2 protein overexpression plays a crucial role in the development, progression, and metastasis of malignant tumors; therefore, 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 prolonged the survival of patients with HER2-positive advanced tumors, considered a milestone in the history of cancer treatment. However, with the widespread use of Herceptin, approximately 70% of patients have developed resistance, including primary and secondary resistance.

[0003] CCR4 is a surface marker of Treg cells. In mice and humans, Treg cells preferentially express CCR4 compared to 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, thereby promoting local tumor growth, enhancing tumor metastasis in peripheral blood or lymphoid organs, and being associated with poor prognosis. Blocking CCR4 can reduce the number of intratumoral Treg cells and enhance anti-tumor immunity. Targeting CCR4 holds promise as a new strategy for immunotherapy of malignant tumors. Given that most Treg-targeting agents cannot distinguish between tumor-associated Treg cells and peripheral blood Treg cells, targeting tumor-infiltrating Treg cells while preserving systemic Treg cells is currently a major obstacle to effective tumor immunotherapy. Finding new and promising tools to target Treg cells could be a major research area for future immunosuppressive therapy. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a bispecific antibody against human HER2 / CCR4 and its applications.

[0005] The technical solution adopted in this invention is: a bispecific antibody against human HER2 / CCR4, comprising a first antigen-binding fragment that binds to HER2, a second antigen-binding fragment that binds to CCR4, and an Fc domain.

[0006] Preferably, the first antigen-binding region includes the Fab segment of the anti-Trastuzumab monoclonal antibody, the second antigen-binding region includes the Fab segment of the anti-Moglizumab monoclonal antibody, and the Fc domain is the Fc domain of the 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 linker 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 linker 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 linker 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 bispecific antibodies against human HER2 / CCR4 was developed. A plasmid expressing the first antigen-binding fragment heavy chain variable region-linking peptide-second antigen-binding fragment heavy chain variable region was constructed, as well as a plasmid expressing the first antigen-binding fragment light chain variable region-linking peptide-second antigen-binding light chain variable region. These plasmids were then transfected into the expression vector strain to obtain the engineered bacterium expressing the bispecific antibody.

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

[0012] The plasmids expressing the first antigen-binding fragment light chain variable region-linking peptide-second antigen-binding light chain variable region are L1-pAb20-hCK plasmids or L2-pAb20-hCK plasmids, with sequences 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 preparation method of bispecific antibody against human HER2 / CCR4 involves culturing engineered bacteria in a CO2 shaking incubator at 170 rpm and 37°C, with 5% CO2 introduced during the culture process. The protein expressed by the engineered bacteria is collected, and the bispecific antibody against human HER2 / CCR4 is obtained by isolation and purification.

[0015] Application of anti-human HER2 / CCR4 bispecific antibodies in drug preparation.

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

[0017] The advantages and positive effects of this invention are: it provides a bispecific antibody against human HER2 / CCR4, which can bind to HER2 with high specificity and high affinity, and bind to CCR4 with high specificity and relatively low affinity, promoting effector cells to kill tumor cells and Treg cells in the tumor microenvironment, effectively avoiding the depletion of peripheral blood Treg cells caused by CCR4 monoclonal antibodies, and has good anti-cancer activity and higher safety. Attached Figure Description

[0018] Figure 1. Schematic diagram of HER2 / CCR4 bispecific antibody synthesis;

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

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

[0021] Figure 4 shows the affinity of the anti-HER2×CCR4 bisomatic antibody to the CCR4 protein molecule as measured by ELISA.

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

[0023] Figure 6 shows the binding of the anti-HER2×CCR4 bispecific antibody to the NCI-N87 cell line that highly expresses HER2, as determined by flow cytometry.

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

[0025] Figure 8 shows the killing effect of anti-HER2×CCR4 bispecific antibody on SKBR3 cells with high HER2 expression in vitro, as determined by the standard lactate dehydrogenase (LDH) assay.

[0026] Figure 9 shows the killing effect of the anti-HER2×CCR4 bispecific antibody on NCI-N87 cells with high HER2 expression in vitro, as determined by the standard lactate dehydrogenase (LDH) assay.

[0027] Figure 10 shows the killing effect of anti-HER2×CCR4 bispecific antibody on CCR4-expressing Treg cells in vitro, as determined by the standard lactate dehydrogenase (LDH) assay.

[0028] Figure 11 shows the effect of anti-HER2×CCR4 bispecific antibody on inhibiting Treg cell chemotaxis in vitro. Detailed Implementation

[0029] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0030] This invention relates to a bispecific antibody against human HER2 / CCR4 and its applications. The bispecific antibody can simultaneously target both HER2 and CCR4. It comprises a first antigen-binding region, a second antigen-binding region, and an Fc domain. 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 binds to HER2 with high specificity and high affinity, and binds to CCR4 with high specificity and relatively low affinity.

[0031] The first antigen-binding region includes the Fab fragment of the Trastuzumab monoclonal antibody, specifically comprising a heavy chain variable region and a light chain variable region. The sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the sequence of the light chain variable region is shown in SEQ ID NO.5. The second antigen-binding region includes the Fab fragment of the Moglizumab monoclonal antibody, specifically comprising a heavy chain variable region and a light chain variable region. The sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the sequence of the light chain variable region 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 linked to the heavy chain variable region in the second antigen-binding fragment derived from the Moglizumab monoclonal antibody via linker peptides H1 and H2, respectively. The amino acid sequence of linker peptide H1 is shown in SEQ ID NO.3, and the amino acid sequence of linker peptide H2 is shown in SEQ ID NO.4. As shown in NO.4, the light chain variable region of the first antigen-binding fragment derived from Trastuzumab monoclonal antibody is linked to the light chain variable region of the second antigen-binding fragment derived from Moglizumab monoclonal antibody via linker peptides L1 and L2, respectively. The amino acid sequence of linker peptide L1 is shown in SEQ ID NO.7, and the amino acid sequence of linker 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] The C-terminus of the Fab region of the second antigen-binding fragment in the bispecific antibody is linked to the N-terminus of the Fc region of the bispecific antibody via a linker peptide, and the C-terminus of the Fab region of the first antigen-binding fragment of the bispecific antibody is linked to the N-terminus of the Fab region of the second antigen-binding fragment of the bispecific antibody via a linker peptide. In some embodiments of the present invention, the first antigen is HER2 and the second antigen is CCR4.

[0033] To prepare bispecific antibodies, plasmids expressing the first antigen-binding fragment heavy chain variable region-linking peptide-second antigen-binding fragment heavy chain variable region and plasmids expressing the first antigen-binding fragment light chain variable region-linking peptide-second antigen-binding light chain variable region were first constructed. These plasmids were then transfected into expression vector strains at a 1:3 ratio. The engineered bacteria were cultured under conditions suitable for protein expression and secretion. In some embodiments of this invention, the constructed engineered bacteria were placed in a CO2 shaking incubator at 170 rpm and 37°C, with 5% CO2 purging during the culture process. The expressed proteins were then separated and purified to obtain the bispecific antibodies. The obtained bispecific antibodies exhibit high specificity and high affinity for HER2 and high specificity and relatively low affinity for CCR4, promoting the killing of tumor cells and Treg cells in the tumor microenvironment by effector cells. This effectively reduces the depletion of peripheral blood Treg cells induced by CCR4 monoclonal antibodies, demonstrating good anticancer activity and higher safety.

[0034] HER2 is specifically expressed on tumor cells, while CCR4 is expressed in tumor microenvironment Treg cells, and also in some peripheral blood Treg cells. CCR4 is a chemokine receptor; binding to its corresponding chemokines can guide the recruitment of peripheral blood CCR4+ Tregs to the tumor microenvironment. Depletion of tumor microenvironment CCR4+ Tregs can enhance anti-tumor immunity, while depletion of peripheral blood CCR4+ Tregs, although potentially enhancing anti-tumor immunity, may also lead to increased autoimmune responses and affect the function of other immune cells. In clinical applications, these potential benefits and risks need to be carefully weighed, and more specific targeting strategies should be sought to reduce side effects. Targeting HER2 and CCR4 allows bispecific antibodies to primarily deplete tumor microenvironment CCR4+ Tregs to exert anti-tumor effects, while also binding to some peripheral blood CCR4+ Tregs capable of chemotaxis into the tumor microenvironment. Simultaneously, the relatively low affinity binding to CCR4 avoids the side effects caused by excessive depletion of peripheral blood CCR4+ Tregs.

[0035] The structural design of bispecific antibodies places the HER2-targeting portion on the outside and the CCR4-targeting portion on the inside. This structural design results in a relatively lower affinity for CCR4. However, the linker connecting the inner and outer portions can be of varying lengths. Different combinations of lengths result in different antigen-antibody binding areas exposed at the CCR4 Fab end, leading to different affinities and ultimately differences in antitumor efficacy. In some embodiments of this invention, four bispecific antibodies were developed and experimentally verified. Although the overall affinity of the anti-CCR4 end was lower than that of the anti-HER2 end, XL-11 showed the strongest affinity at the CCR4 end among the four bispecific antibodies, exhibiting a stronger ability to kill CCR4+ Tregs in the tumor microenvironment. There was no significant difference in the affinity of the four bispecific antibodies for peripheral blood Tregs.

[0036] This bispecific antibody can be used in antitumor drugs for treatment or prevention, or in drugs for the prevention or treatment of HER2 and CCR4-mediated diseases. Bispecific antibodies can also be used to prepare drugs for the treatment or prevention of immune diseases, such as graft-versus-host disease. The bispecific antibody can be used as a main active ingredient or one of the main active ingredients, mixed with other excipients to prepare a drug or preparation.

[0037] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies.

[0038] In the following examples, all buffers, culture media, and reagents involved are as follows: PBS buffer (pH=7.4), PBST buffer (PBS buffer + 0.05% Twen-20), carbonate coating solution (pH=9.6 Na2CO3 1.59g + NaHCO3 2.93g + distilled water 1000mL), 293F complete medium (purchased from Sinocare 293F serum-free medium), 1640 complete medium (10% serum + 1% penicillin antibody + 1640 basal medium), DMEM complete medium (10% serum + 1% penicillin antibody + DMEM basal medium), PEI transfection reagent (purchased from TransGen), protein expression feed (purchased from Sinocare), and Protein A protein purification column (purchased from Tiandi Renhe).

[0039] Example 1: Construction, expression and purification of anti-HER2×CCR4 bispecific antibody molecule

[0040] A bispecific antibody against HER2×CCR4 was constructed by using the Fab region of the Trastuzumab monoclonal antibody as the anti-HER2 domain, the Fab region of the Moglizumab monoclonal antibody as the anti-CCR4 binding domain, and the Fc domain of the Trastuzumab monoclonal antibody as the Fc domain.

[0041] The first antigen-binding moiety 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 moiety of the bispecific antibody specifically binds to human CCR4 via the Fab configuration and 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.

[0042] The heavy chain variable region of the Fab arm of the first antigen-binding fragment is connected to the heavy chain variable region of the Fab arm of the second antigen-binding fragment via linker peptides H1 or H2, respectively; the light chain variable region of the Fab arm of the first antigen-binding fragment is connected to the light chain variable region of the Fab arm of the second antigen-binding fragment via linker peptides L1 or L2, respectively. The amino acid sequences of linker peptide H1 are shown in SEQ ID NO.3, H2 in SEQ ID NO.4, L1 in SEQ ID NO.7, and L2 in SEQ ID NO.8, respectively.

[0043] The heavy chain amino acid sequence of the first antigen-binding fragment of the bispecific antibody binding to HER2 is shown in SEQ ID NO.1, and the corresponding base sequence is shown in SEQ ID NO.10.

[0044] The heavy chain amino acid sequence of the second antigen-binding fragment of the bispecific antibody binding to CCR4 is shown in SEQ ID NO.2, and the corresponding base sequence is shown in SEQ ID NO.11;

[0045] The amino acid sequence of peptide chain H1, which connects the heavy chain of the first antigen-binding fragment of the bispecific antibody to the first antigen-binding fragment of the bispecific antibody to the second antigen-binding fragment of the CCR4, is shown in SEQ ID NO.3, and the corresponding base sequence is shown in SEQ ID NO.12.

[0046] The amino acid sequence of the peptide chain H2 connecting the heavy chain of the first antigen-binding fragment of the bispecific antibody binding to HER2 and the heavy chain of the second antigen-binding fragment of the bispecific antibody binding to CCR4 is shown in SEQ ID NO.4, and the corresponding base sequence is shown in SEQ ID NO.13.

[0047] The light chain amino acid sequence of the first antigen-binding fragment of the bispecific antibody binding to HER2 is shown in SEQ ID NO.5, and the corresponding base sequence is shown in SEQ ID NO.14.

[0048] The light chain amino acid sequence of the second antigen-binding fragment of the bispecific antibody binding to CCR4 is shown in SEQ ID NO.6, and the corresponding base sequence is shown in SEQ ID NO.15;

[0049] The amino acid sequence of peptide chain L1, which connects the light chain of the first antigen-binding fragment of the bispecific antibody to HER2 and the light chain of the second antigen-binding fragment of the bispecific antibody to CCR4, is shown in SEQ ID NO.7, and the corresponding base sequence is shown in SEQ ID NO.16.

[0050] The amino acid sequence of peptide chain L2, which connects the light chain of the first antigen-binding fragment of the bispecific antibody to HER2 and the light chain of the second antigen-binding fragment of the bispecific antibody to CCR4, is shown in SEQ ID NO.8, and the corresponding base sequence is shown in SEQ ID NO.17.

[0051] 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.

[0052] 1.1 Strategy for constructing anti-HER2×CCR4 bispecific antibodies

[0053] Bispecific antibodies XL-11, XL-21, XL-12, and XL-22 were constructed. The variable regions of bispecific antibody XL-11, from N-terminus to C-terminus, are: heavy chain variable region of the first antigen-binding fragment - linker H1 - heavy chain variable region of the second antigen-binding fragment, and light chain variable region of the first antigen-binding fragment - linker L1 - light chain variable region of the second antigen-binding fragment. The variable regions of bispecific antibody XL-21, from N-terminus to C-terminus, are: heavy chain variable region of the first antigen-binding fragment - linker H1 - heavy chain variable region of the second antigen-binding fragment, and light chain variable region of the first antigen-binding fragment - linker L2 - light chain variable region of the second antigen-binding fragment. The variable regions of bispecific antibody XL-12, from N-terminus to C-terminus, are: heavy chain variable region of the first antigen-binding fragment - linker H2 - heavy chain variable region of the second antigen-binding fragment, and light chain variable region of the first antigen-binding fragment - linker L1 - light chain variable region of the second antigen-binding fragment. The variable regions of the bispecific antibody XL-22, from the N-terminus to the C-terminus, are sequentially: the heavy chain variable region of the first antigen-binding fragment - linker peptide H2 - the heavy chain variable region of the second antigen-binding fragment, and the light chain variable region of the first antigen-binding fragment - linker peptide L2 - the light chain variable region of the second antigen-binding fragment. The specific linker peptide connections are shown in Table 1, and the molecular configuration of the anti-HER2×CCR4 bispecific antibody is shown in Figure 1.

[0054] Table 1

[0055] 1.2 Construction of plasmid for anti-HER2×CCR4 bispecific antibody

[0056] The plasmids used to construct the anti-HER2×CCR4 bispecific antibody include four types, and the plasmid names and corresponding vectors are shown in Table 2.

[0057] Table 2. Heavy chain and light chain plasmid names and corresponding vector names for anti-HER2×CCR4 bispecific antibodies.

[0058] The specific details of the antibody sequences inserted into the four plasmids used to construct the anti-HER2×CCR4 bispecific antibody are as follows:

[0059] H1-pAb20-hCHIgG1 plasmid: The three fragments of Trastuzumab monoclonal antibody heavy chain variable region, linker peptide H1, and Moglizumab monoclonal antibody heavy chain variable region were cloned into the heavy chain vector pAb20-hCHIgG1 (Suzhou Hongxun) containing the antibody heavy chain constant region, to obtain the H1-pAb20-hCHIgG1 plasmid expressing the heavy chain fragment of anti-HER2×CCR4 bispecific antibody linked by linker peptide H1. The base sequence of the H1-pAb20-hCHIgG1 plasmid is shown in SEQ ID NO.19.

[0060] H2-pAb20-hCHIgG1 plasmid: The three fragments of Trastuzumab monoclonal antibody heavy chain variable region, linker peptide H2, and Moglizumab monoclonal antibody heavy chain variable region were cloned into the Suzhou Hongxun heavy chain vector pAb20-hCHIgG1 containing the antibody heavy chain constant region, to obtain the H2-pAb20-hCHIgG1 plasmid expressing the heavy chain fragment of anti-HER2×CCR4 bispecific antibody linked by linker peptide H2. The base sequence of the H2-pAb20-hCHIgG1 plasmid is shown in SEQ ID NO.20.

[0061] L1-pAb20-hCK plasmid: The three fragments of Trastuzumab monoclonal antibody light chain variable region, linker peptide L1, and Moglizumab monoclonal antibody light chain variable region 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 anti-HER2×CCR4 bispecific antibody linked by linker peptide L1. The base sequence of the L1-pAb20-hCK plasmid is shown in SEQ ID NO.21.

[0062] L2-pAb20-hCK plasmid: The three fragments of Trastuzumab monoclonal antibody light chain variable region, linker peptide L2, and Moglizumab monoclonal antibody light chain variable region 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 anti-HER2×CCR4 bispecific antibody linked by linker peptide L2. The base sequence of the L2-pAb20-hCK plasmid is shown in SEQ ID NO.22.

[0063] 1.3 Construction of anti-HER2×CCR4 bispecific antibody expression vector

[0064] Four plasmids of the anti-HER2×CCR4 bispecific antibody were transfected into 293F cells using PEI transfection reagent via liposome transfection, with the heavy chain:light chain ratio being 1:3. The combination of bispecific antibody transfection plasmids is shown in Table 3.

[0065] Table 3. Combinations of anti-HER2×CCR4 bispecific antibody transfection plasmids

[0066] The culture conditions and transfection expression steps for 293F cells are as follows:

[0067] At 0.5×10 6 Cells were seeded at a rate of cells / ml into 200 ml of culture medium in a 1 L shake flask. The cells were incubated at 37°C, 160 rpm, and 5% CO2 in a shaker incubator, ensuring that the cells doubled in size every 24 hours without clumping. Pre-transfection passaging was performed to achieve a 293F cell density of 1 × 10⁻⁶ cells / ml. 6 Cells / ml were cultured at 37°C, 160 rpm, and 5% CO2 concentration in a shaker incubator for 4 h for acclimatization. 200 μg of plasmid (filtered and sterilized) was added to 10 ml of PBS and vortexed for 3 seconds to mix thoroughly. 600 μl of filtered and sterilized PEI solution was added to the PBS / DNA mixture. After the PEI-DNA mixture was allowed to stand at room temperature for 20 min, the DNA / PEI mixture was added to the cells. The cells were cultured at 37°C, 5% CO2, and 80% humidity for 5 days. Expression-promoting feed was added 24 h post-transfection, and then every 24 h thereafter until the cells were harvested. On day 5 post-transfection, the supernatant was collected, centrifuged at 4°C, 2000 rpm for 30 min, and filtered through a 0.22 μm syringe filter.

[0068] Purification of bispecific antibodies in supernatant using Protein A gravity column: Fix the Protein A gravity column on an iron stand, remove the lower and upper stoppers sequentially, and drain the column's protective buffer; add 5 column volumes of equilibration buffer (0.5M NaCl, 20mM Na2HPO4, pH=7.0) 2-3 times to equilibrate the packing material to the same buffer system as the target protein, thus protecting the protein; add the sample to the equilibrated gravity column, retaining each 1 ml of sample for 2 minutes to ensure sufficient contact between the target protein and the medium, improving the recovery rate of the target protein, and collect the eluent; add 10-15 column volumes of washing buffer (0.5M NaCl, 20mM Na2HPO4, pH=7.0) Use Na2HPO4 (pH=7.0) to remove non-specifically adsorbed proteins and collect the washings. Add elution buffer (0.1M citric acid) at pH=4 and allow it to bind to the packing material for 10 min. Elute the target protein (0.1M citric acid) with 5-10 column volumes of elution buffer (pH=2.5), collecting fractions, one tube per column volume, and analyze them separately. The eluted fraction must be immediately neutralized using 1 / 10 of the volume of neutralizing buffer. Equilibrate the packing material sequentially with 3 column volumes of equilibration buffer and 5 column volumes of deionized water. Store the gravity column in an equal volume of 20% ethanol at 2-8℃ to prevent bacterial contamination of the packing material.

[0069] The size and structure of the anti-HER2×CCR4 bispecific antibodies were detected by SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The results, shown in Figure 2, indicate that 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 all four HER2×CCR4 bispecific antibodies were ≥250 kDa. On reduced SDS-PAGE, the molecular weights of the heavy chains were ≈64 kDa and the light chains were ≈36 kDa. The purified antibodies exhibited high purity with no impurities observed.

[0070] Example 2: Identification of the binding activity of anti-HER2×CCR4 bispecific antibody to HER2 protein

[0071] Coating solution with pH 9.6 (0.035 mol / L NaHCO3, 0.015 mol / L...) Dilute HER2 protein to 10 μg / ml with Na2CO3, then add 100 μl / well to an ELISA plate; incubate overnight at 4°C; wash three times the next day with PBST buffer (pH=7.4); add 5% BSA buffer to each well and block at room temperature for 2 h; wash three times with PBST buffer (pH=7.4); add serially diluted PBST buffer to the antibody to be tested, the antibody starting at a concentration of 20000 ng / ml, and serially diluted 5-fold to 10 gradients, and incubate at room temperature for 1 h; wash three times with PBST buffer (pH=7.4), add secondary antibody HRP-labeled goat anti-human Fc antibody (purchased from Solarbio), and incubate at room temperature for 1 h; wash three times with PBST buffer (pH=7.4) and blot dry, add 100 μl TMB solution (purchased from Sigma) to each well, and incubate at 37°C in the dark for 20 minutes; add 50 μl 2M PBST solution to each well. The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450nm using an ELISA reader. Data analysis was performed using GraphPad Prism, and EC50 was calculated by plotting the data.

[0072] As shown in Figure 3, the four anti-HER2×CCR4 bispecific antibodies were able to bind to the HER2 antigen, with EC50 values ​​of 6.272 ng / ml, 10.58 ng / ml, 9.562 ng / ml, and 6.149 ng / ml, respectively. Compared with the Trastuzumab monoclonal antibody (EC50 = 3.996 ng / ml), the affinity of the four anti-HER2×CCR4 bispecific antibodies for the HER2 protein was not significantly reduced, and they still maintained a high affinity.

[0073] Example 3: Identification of the binding activity of anti-HER2×CCR4 bispecific antibody to CCR4 protein

[0074] CCR4 protein was diluted to 10 μg / ml with carbonate coating buffer (pH 9.6, 0.035 mol / L NaHCO3, 0.015 mol / L Na2CO3), and then 100 μl / well was added to each ELISA plate. The plate was incubated overnight at 4°C. The next day, the plate was washed three times with PBST buffer (pH 7.4). Each well was blocked with 5% BSA buffer for 2 hours at room temperature. The plate was washed three times with PBST buffer (pH 7.4). Then, the antibody to be tested was serially diluted with PBST buffer, starting at 20000 ng / ml and serially diluted 5-fold to 10 gradients, and incubated for 1 hour at room temperature. The plate was washed three times with PBST buffer (pH 7.4), and HRP-labeled goat anti-human Fc antibody (purchased from Solarbio) was added and incubated for 1 hour at room temperature. The plate was washed three times with PBST buffer (pH 7.4) and blotted dry. 100 μl of the antibody was added to each well. TMB solution (purchased from Sigma) was incubated 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. The OD value was read at 450 nm using a microplate reader. Data analysis was performed using GraphPad Prism, and EC50 was plotted and calculated.

[0075] As shown in Figure 4, the four anti-HER2×CCR4 bispecific antibodies were able to bind to the CCR4 antigen, with EC50 values ​​of 21.52 ng / ml, 102 ng / ml, 33.34 ng / ml, and 1438 ng / ml, respectively. These four anti-HER2×CCR4 bispecific antibodies still bound to the CCR4 protein with relatively strong affinity, but compared to the Moglizumab monoclonal antibody (EC50 = 9.593 ng / ml), the affinity of the four anti-HER2×CCR4 bispecific antibodies for CCR4 was reduced. Among them, XL-11, linked by the longer linker peptides L1 and H1, showed the strongest affinity for the CCR4 protein. XL-22, linked by the shorter linker peptides L2 and H2, showed the weakest affinity for the CCR4 protein among the four anti-HER2×CCR4 bispecific antibodies.

[0076] Example 4: Identification of the binding of anti-HER2×CCR4 bispecific antibody to SKBR3 cell line with high HER2 expression

[0077] After the SKBR3 cells in the T25 flask reached confluence, they were digested with 0.5% trypsin and counted using a hemocytometer. The cells were then evenly distributed into 1.5 EP tubes, with 0.2 × 10⁶ cells per tube. 6Cells were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). The antibody to be detected was diluted to 0.15 mg / ml with 1% FBS-PBS and added to the cells, 100 μl / tube, mixed thoroughly by repositioning, and incubated at 37°C for 1 h. The cells were then centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). Anti-human IgG Fc-FITC fluorescent secondary antibody (Solepro, 1:100) was diluted with 1% FBS-PBS and added to the cells, 100 μl / tube, mixed thoroughly by repositioning. The cells were incubated at 4°C in the dark for 1 h. The cells were then centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). Add 300 μl of 1% FBS-PBS, resuspend the cells by pipetting, filter through a 40 μm cell filter, and then perform analysis.

[0078] As shown in Figure 5, the anti-HER2×CCR4 bispecific antibody was able to bind to SKBR3 cells that highly express HER2. Compared with the Trastuzumab monoclonal antibody, the binding ability of the four anti-HER2×CCR4 bispecific antibodies to SKBR3 cells that highly express HER2 was not significantly reduced, and they still maintained a strong binding ability.

[0079] Example 5: Identification of the binding of anti-HER2×CCR4 bispecific antibody to NCI-N87 cell line with high HER2 expression.

[0080] After the NCI-N87 cells in the T25 flask reached confluence, they were digested with 0.5% trypsin and counted using a hemocytometer. The cells were then evenly distributed into 1.5 EP tubes, with 0.2 × 10⁶ cells per tube. 6Cells were centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). The antibody to be detected was diluted to 0.15 mg / ml with 1% FBS-PBS and added to the cells, 100 μl / tube, mixed thoroughly by repositioning, and incubated at 37°C for 1 h. The cells were then centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). Anti-human IgG Fc-FITC fluorescent secondary antibody (Solepro, 1:100) was diluted with 1% FBS-PBS and added to the cells, 100 μl / tube, mixed thoroughly by repositioning. The cells were incubated at 4°C in the dark for 1 h. The cells were then centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged twice (800 rpm, 5 min). Add 300 μl of 1% FBS-PBS, resuspend the cells by pipetting, filter through a 40 μm cell filter, and then perform analysis.

[0081] As shown in Figure 6, the anti-HER2×CCR4 bispecific antibody was able to bind to NCI-N87 cells that highly express HER2. Compared with the Trastuzumab monoclonal antibody, the binding ability of the four anti-HER2×CCR4 bispecific antibodies to NCI-N87 cells that highly express HER2 was not significantly reduced, and they still maintained a strong binding ability.

[0082] Example 6: Identification of the binding of anti-HER2×CCR4 bispecific antibody to CCR4-expressing Treg cells

[0083] The collected peripheral blood was placed in a 50 mL centrifuge tube and centrifuged at 650 g for 15 min at room temperature. The upper yellow plasma layer was transferred to a new 50 mL centrifuge tube (the lower layer contains blood cells, which will be used for subsequent mononuclear cell extraction). The plasma was then inactivated in a 56 °C water bath for 30 min. After inactivation, the plasma appeared turbid. It was then centrifuged at 900 g for 10 min, and the supernatant was collected and stored at 4 °C for later use.

[0084] Take the lower red cell precipitate from the plasma extraction step, add physiological saline or PBS to the original volume, and gently pipette to mix. Take a 50mL centrifuge tube, add 15mL of lymphocyte separation medium, and slowly add the diluted blood from the previous step to the upper layer of the lymphocyte separation medium. The speed of adding the blood sample should be moderate, especially at the beginning, to avoid disrupting the interface between the lymphocyte separation medium and the blood sample. Centrifuge at 800g for 20min at room temperature (without brake deceleration). After centrifugation, the sample in the centrifuge tube will separate into 4 layers from top to bottom: plasma layer – white membrane layer – human lymphocyte separation medium layer – precipitate of red blood cells and granulocytes. Carefully aspirate the white membrane 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 precipitate in 40mL of physiological saline or PBS, and centrifuge at 300g for 10min. For samples with a high platelet count, the washing step can be repeated 1-2 times. Discard the supernatant, resuspend the precipitate in a small amount of buffer, take a portion of the cell suspension for counting, and set aside.

[0085] Centrifuge the PBMC suspension to remove the supernatant, then add a certain amount of Treg serum-free medium to adjust the PBMC concentration to 1×10⁻⁶. 8 cells / mL; enrichment antibody incubation: incubate enrichment antibody at 200 μL antibody / mL - 1.5 × 10⁻¹⁰. 8 The initial proportion of PBMCs will be added to the cell suspension, and the mixture will be quickly and gently pipetted to mix thoroughly. Incubate at room temperature for 15 minutes. Washing for unbound antibodies: After incubation, add 1×10⁻⁶ serum-free Treg medium. 8 Centrifuge at 300g for 10 min (cells / 10-40 mL), discard the supernatant, and add a certain amount of Treg serum-free medium to restore the cell suspension volume to the initial level. When discarding the supernatant, a small amount of buffer solution often remains at the tube opening; remove this residue as much as possible using a pipette, and wash again if necessary to avoid interference from some antibody components in the residual solution on the subsequent binding of magnetic beads to cells. Transfer the cell suspension to a flow cytometry tube, adding 100 μL of magnetic beads / 1 × 10⁻⁴ mL. 8 To initially prepare PBMCs, add streptavidin magnetic beads to the PBMCs, gently pipette to mix thoroughly, and incubate at room temperature for 10 minutes. After incubation, magnetically aspirate for 3 minutes and collect the unabsorbed cell suspension. If the cell count is low during magnetic aspiration, resuspend the cells in 1 mL of serum-free Treg medium before magnetic aspiration. Add the same volume of serum-free Treg medium as in the previous step to the flow cytometry tube containing the magnetically aspirated cells, gently disperse the magnetic beads, and magnetically aspirate again for 3 minutes. Collect the unabsorbed liquid and combine it with the cell suspension collected in the previous step. Repeat the magnetic aspiration process several times with the combined cell suspension, collecting the unabsorbed cell suspension to more completely remove non-target cells. The resulting cells are the enriched seed cells.

[0086] On day 0, prepare 30 mL of complete T-cell conditioning medium: 26 mL of serum-free Treg medium, 3 mL of 10% heat-inactivated autologous plasma, 1 mL of Treg-II, and IL-2 (final IL-2 concentration in the complete medium is 500 IU / mL). After thoroughly suspending the seed cells in this complete medium, inoculate them into T75 flasks. On days 4-5, supplement with 30 mL of complete Treg cell conditioning medium, 27 mL of serum-free Treg medium, 3 mL of 10% heat-inactivated autologous plasma, and 500 U / mL IL-2. On days 6-7, supplement with 60 mL of complete Treg cell conditioning medium, 53 mL of serum-free Treg medium, and 6 mL of 10% heat-inactivated autologous plasma. 1 mL of Treg-III and 500 U / mL IL-2 were added; on days 8-9, 120 mL of complete Treg cell culture medium, 108 mL of serum-free Treg culture medium, 12 mL of 10% heat-inactivated autologous plasma, and 500 U / mL IL-2 were added; on days 10-11, 240 mL of complete Treg cell culture medium, 216 mL of serum-free Treg culture medium, 24 mL of 10% heat-inactivated autologous plasma, and 500 U / mL IL-2 were added; on days 12-13, the remaining serum-free Treg culture medium, the remaining heat-inactivated autologous plasma, and 500 U / mL IL-2 were added; on days 14-15, Treg cells were harvested for analysis or downstream applications.

[0087] The harvested Treg cells were evenly divided into 1.5 EP tubes, with 0.2 × 10⁶ cells per tube. 6 Cells were centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged again (300g, 10 min). The cells were washed twice. The antibody to be detected was diluted to 0.15 mg / ml with 1% FBS-PBS and added to the cells sequentially, 100 μl / tube. The mixture was then incubated at 37°C for 1 h, centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged again (300g, 10 min). The cells were washed twice. Anti-human IgG Fc-FITC fluorescent secondary antibody (Solepro, 1:100) was diluted with 1% FBS-PBS and added to the cells, 100 μl / tube. The mixture was then incubated at 4°C in the dark for 1 h, centrifuged at 300g for 10 min, the supernatant was discarded, and the cells were resuspended in 1% FBS-PBS by repositioning and centrifuged again (300g, 10 min). The cells were washed twice. Add 300 μl of 1% FBS-PBS, resuspend the cells by pipetting, filter through a 40 μm cell filter, and then perform analysis.

[0088] As shown in Figure 7, the anti-HER2×CCR4 bispecific antibody was able to bind to CCR4-expressing Treg cells. Compared to the Moglizumab monoclonal antibody, the binding ability of all four anti-HER2×CCR4 bispecific antibodies to CCR4-expressing Treg cells was reduced. This may have prevented peripheral blood Treg cell depletion caused by excessive binding of anti-HER2×CCR4 bispecific antibodies, suggesting that all four anti-HER2×CCR4 bispecific antibodies have potentially better safety. There was no significant difference between bispecific antibodies composed of different linker peptide combinations.

[0089] Example 7: Standard lactate dehydrogenase (LDH) assay to detect the killing effect of anti-HER2×CCR4 bispecific antibody on SKBR3 cells with high HER2 expression in vitro.

[0090] After the SKBR3 cells in the T25 flask reached confluence, they were digested with 0.5% trypsin and counted using a hemocytometer. Cells were then seeded into 96-well plates at 200 μL / well (10,000 cells / well), ensuring the cell density did not exceed 80-90% at the time of detection. Growth medium was aspirated from the 96-well plates, and the cells were washed once with PBS. The medium was then replaced with low-serum medium containing 1% serum, and cultured for 1 hour for acclimatization. The target antibody was then added in serially diluted doses of 1.28, 32, 160, 800, and 40,000 ng / mL and incubated at 37°C for 30 minutes. 250,000 PBMCs were added to each well as the experimental group and incubated at 37°C for 2 hours. The supernatant was aspirated, centrifuged at 400g for 10 minutes, and added to the corresponding 96-well plates. 20 μL of 10% Triton X-100 solution was added to the cells as a maximum release. Add 20 μL of Assay Buffer to the cells as a spontaneous release, and add 20 μL of Assay Buffer to the cell-free wells in 1% low-serum medium as a background control. Adjust the culture medium volume of the cells in the wells to a final volume of 200 μL; incubate at 37°C in a CO2 incubator 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.

[0091] Add 100 μL of LDH Reaction Solution to each well. Incubate the plate at 37°C for a maximum of 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate using the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100

[0092] As shown in Figure 8, the four anti-HER2×CCR4 bispecific antibodies were able to kill SKBR3 cells that highly express HER2 in vitro, and the killing efficiency increased with increasing antibody concentration. Compared with the Trastuzumab monoclonal antibody, the four anti-HER2×CCR4 bispecific antibodies did not show a significant decrease in the killing efficiency of SKBR3 cells that highly express HER2 in vitro, and still had a good ability to kill HER2-positive tumor cells.

[0093] Example 8: Standard lactate dehydrogenase (LDH) assay to detect the killing effect of anti-HER2×CCR4 bispecific antibody on NCI-N87 cells with high HER2 expression in vitro.

[0094] After the NCI-N87 cells in the T25 flask reached confluence, they were digested with 0.5% trypsin and counted using a hemocytometer. Cells were then seeded into 96-well plates at 200 μL / well (10,000 cells / well), ensuring the cell density did not exceed 80-90% at the time of detection. Growth medium was aspirated from the 96-well plates, and the cells were washed once with PBS. The medium was replaced with low-serum medium containing 1% serum, and cultured for 1 hour for acclimatization. The test antibody was then added in serially diluted doses of 1.28, 32, 160, 800, and 40,000 ng / mL and incubated at 37°C for 30 minutes. 250,000 PBMCs were added to each well as the experimental group and incubated at 37°C for 2 hours. The supernatant was aspirated, centrifuged at 400g for 10 minutes, and added to the corresponding 96-well plates. 20M 10% Triton X-100 solution was added to the cells for maximum release. Add 20 μL of Assay Buffer to the cells as a spontaneous release, and add 20 μL of Assay Buffer to the cell-free wells in 1% low-serum medium as a background control. Adjust the culture medium volume of the cells in the wells to a final volume of 200 μL; incubate at 37°C in a CO2 incubator 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.

[0095] Add 100 μL of LDH Reaction Solution to each well. Incubate the plate at 37°C for a maximum of 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate using the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100

[0096] As shown in Figure 9, the four anti-HER2×CCR4 bispecific antibodies were able to kill NCI-N87 cells that highly express HER2 in vitro, and the killing efficiency increased with increasing antibody concentration. Compared with the Trastuzumab monoclonal antibody, the four anti-HER2×CCR4 bispecific antibodies did not show a significant decrease in the killing efficiency of NCI-N87 cells that highly express HER2 in vitro, and still had a good ability to kill HER2-positive tumor cells.

[0097] Example 9: Standard lactate dehydrogenase (LDH) assay to detect the killing effect of anti-HER2×CCR4 bispecific antibody on CCR4-expressing Treg cells in vitro.

[0098] After the primary Treg cells induced in the T75 flasks reached confluence, they were counted using a hemocytometer. Cells were then seeded into 96-well plates at 200 μL / well (10,000 cells / well), ensuring the cell density did not exceed 80-90% at the time of detection. Growth medium was aspirated from the 96-well plates, and the cells were washed once with PBS. The medium was then replaced with low-serum medium containing 1% serum, and cultured for 1 hour for adaptation. The test antibody was added in serially diluted doses of 1.28, 32, 160, 800, and 40,000 ng / mL and incubated at 37°C for 30 minutes. 250,000 PBMCs were added to each well as the experimental group and incubated at 37°C for 2 hours. The supernatant was aspirated, centrifuged at 400g for 10 minutes, and added to the corresponding 96-well plates. 20 μL of 10% Triton X-100 solution was added to the cells as a maximum release. Add 20 μL of Assay Buffer to the cells as a spontaneous release, and add 20 μL of Assay Buffer to the cell-free wells in 1% low-serum medium as a background control. Adjust the culture medium volume of the cells in the wells to a final volume of 200 μL; incubate at 37°C in a CO2 incubator 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.

[0099] Add 100 μL of LDH Reaction Solution to each well. Incubate the plate at 37°C for a maximum of 30 min. Read the absorbance at 565 nm using a microplate reader. Subtract the background A565 level from all wells. Calculate the killing rate using the formula: Cytotoxicity (%) = (A sample - A target cell spontaneous - A effector cell spontaneous) / (A maximum release - A spontaneous release) × 100

[0100] As shown in Figure 10, the four anti-HER2×CCR4 bispecific antibodies were able to kill CCR4-expressing Treg cells in vitro, and the killing efficiency increased with increasing antibody concentration. Compared with the Moglizumab monoclonal antibody, the killing ability of the four anti-HER2×CCR4 bispecific antibodies against CCR4-expressing Treg cells was reduced. Among them, XL-11, which is linked by the longer linker peptides L1 and H1, showed the strongest killing ability against CCR4-expressing Treg cells, while XL-22, which is linked by the shorter linker peptides L2 and H2, showed the weakest killing ability against CCR4-expressing Treg cells.

[0101] Example 10: Chemotaxis assay to detect the effect of anti-HER2×CCR4 bispecific antibody on the in vitro inhibition of Treg cell chemotaxis

[0102] Add 600 μL of SKBR3 tumor cell culture supernatant and 100 ng / ml CCL22 mixture to a 24-well plate; pipette 150,000 Tregs into 200 μL of Treg-specific culture medium, add 0.1 mg / ml of penicillin-dextrose antibody, mix well by pipetting, and then drop the mixture into the chamber by swirling in the air; place the chamber in the well plate and check that there are no air bubbles between the culture medium and the chamber; let stand for 15 min to avoid cell aggregation during sedimentation, and then place in an incubator for 4 h; count the number of cells in the lower chamber using a hemocytometer.

[0103] As shown in Figure 11, all four anti-HER2×CCR4 bispecific antibodies inhibited the chemotaxis of Treg cells to CCL22. Compared with the Moglizumab monoclonal antibody, the ability of the four anti-HER2×CCR4 bispecific antibodies to inhibit the chemotaxis of Treg cells to CCL22 was reduced. Among them, XL-11, which is linked by the longer linker peptides L1 and H1, showed the strongest ability to inhibit the chemotaxis of Treg cells to CCL22, while XL-22, which is linked by the shorter linker peptides L2 and H2, showed the weakest ability to inhibit the chemotaxis of Treg cells to CCL22.

[0104] The experimental results above show that the four bispecific antibodies targeting HER2 and CCR4 provided by this invention all have good antigen-binding activity and are all high-affinity antibodies. They can reduce the binding of antibodies to peripheral blood Treg cells to a certain extent, and have good killing activity against tumor cells and Treg cells in the tumor microenvironment. At the same time, they inhibit the chemotaxis of Treg cells to the tumor microenvironment with high expression of CCL22, and have good anti-cancer activity and equally good safety. Among them, XL-11, which is linked by the longer linker peptides L1 and H1, has the best anti-cancer activity.

[0105] This invention provides a concept and method for anti-HER2 and CCR4 bispecific antibody and its application. There are many methods and approaches to implement this technical solution. The above description is only a preferred embodiment of this invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

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

2. The bispecific antibody against human HER2 / CCR4 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 bispecific antibody against human HER2 / CCR4 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 via a linker 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 linker peptide.

4. The bispecific antibody against human HER2 / CCR4 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 in SEQ ID NO.1; the amino acid sequence of the heavy chain variable region of the second antigen-binding region is shown in SEQ ID NO.2; the amino acid sequence of the light chain variable region of the first antigen-binding region is shown in SEQ ID NO.5; the amino acid sequence of the light chain variable region of the second antigen-binding region is shown in SEQ ID NO.6; the amino acid sequence of the linker peptide is 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 shown in SEQ ID NO.

9.

5. An engineered bacterium capable of expressing the bispecific antibody against human HER2 / CCR4 as described in any one of claims 1-4, characterized in that: A plasmid expressing the first antigen-binding fragment heavy chain variable region-linking peptide-second antigen-binding fragment heavy chain variable region was constructed, and a plasmid expressing the first antigen-binding fragment light chain variable region-linking peptide-second antigen-binding light chain variable region was also constructed. These plasmids were then transfected into expression vector strains to obtain engineered bacteria expressing bispecific antibodies.

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

7. The engineered bacteria according to claim 6, characterized in that: The plasmids expressing the heavy chain variable region and the plasmids expressing the light chain variable region were mixed 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 described in any one of claims 5-7 are cultured in a CO2 shaking incubator at 170 rpm and 37°C, with 5% CO2 introduced during the culture process. The protein expressed by the engineered bacteria is collected, and the bispecific antibody against human HER2 / CCR4 is isolated and purified.

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

10. The application according to claim 9, characterized in that: Used to prepare anti-tumor drugs; or used to prepare drugs for the treatment or prevention of immune diseases, namely graft-versus-host disease.

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